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Mechanisms of glucocorticoid type II receptor regulation and activation in brain

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Title:
Mechanisms of glucocorticoid type II receptor regulation and activation in brain
Creator:
Densmore, Charles L., 1954-
Publication Date:
Language:
English
Physical Description:
234 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Cytosol ( jstor )
Glucocorticoid receptors ( jstor )
Glucocorticoids ( jstor )
Incubation ( jstor )
Liver ( jstor )
Molybdates ( jstor )
Purification ( jstor )
Rats ( jstor )
Receptors ( jstor )
Steroids ( jstor )
Brain -- drug effects ( mesh )
Dissertations, Academic -- Neuroscience -- UF ( mesh )
Glucocorticoids -- physiology ( mesh )
Neuroscience thesis Ph.D ( mesh )
Receptors, Glucocorticoid -- isolation & purification ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 202-232.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Charles L. Densmore.

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University of Florida
Holding Location:
University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
023160667 ( ALEPH )
17945246 ( OCLC )
AEL4006 ( NOTIS )
AA00006107_00001 ( sobekcm )

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Full Text












MECHANISMS OF GLUCOCORTICOID TYPE II
RECEPTOR REGULATION AND ACTIVATION
IN BRAIN







By



CHARLES L. DENSMORE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1987




MECHANISMS OF GLUCOCORTICOID TYPE II
RECEPTOR REGULATION AND ACTIVATION
IN BRAIN
By
CHARLES L. DENSMORE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987


ACKNOWLEDGMENTS
I greatfully acknowledge the interest, support and patience of my
supervisory committee including Drs. Kimon Angelides, Steven Childers,
Robert Cohen, and Kathleen Shiverick. I am especially thankful for the
never-tiring, unselfish support of my advisor and friend, Dr. William G.
Luttge.
I am particularly indebted to Seyed M. Emadian and Yun-Chia (Jenny)
Chou who have helped me in more ways than they realize.
I would also like to thank Mary Rupp for always seeing to it that
my experiments ran smoothly and Kimmy Bloom for the painstaking artwork.
Finally, this work would never have been possible without the
constant love and support of my wife Anita.
ii


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT v
CHAPTERS
IGENERAL INTRODUCTION 1
IIHYDROPHOBIC INTERACTION CHROMATOGRAPHY
OF VARIOUS FORMS OF THE OCCUPIED
AND UNOCCUPIED GLUCOCORTICOID RECEPTOR 13
Introduction 13
Materials and Methods 15
Results./ 19
Discussion 43
IIISULFHYDRYL REGULATION OF GLUCOCORTICOID
BINDING CAPACITY 52
Introduction 52
Materials and Methods 62
Results 66
Discussion 108
IVTHE PURIFICATION AND SUBSEQUENT ACTIVATION OF
GLUCOCORTICOID RECEPTORS 127
Introduction 127
Materials and Methods 143
Results 148
Discussion 161
iii


V CHARACTERIZATION OF THE UNOCCUPIED
GLUCOCORTICOID RECEPTOR 171
Introduction 171
Materials and Methods 177
Results 180
Discussion 193
VI CONCLUDING REMARKS 196
BIBLIOGRAPHY 202
BIOGRAPHICAL SKETCH 233
IV


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MECHANISMS OF GLUCOCORTICOID TYPE II
RECEPTOR REGULATION AND ACTIVATION
IN BRAIN
By
Charles L. Densmore
August 1987
Chairman: William G. Luttge
Major Department: Neuroscience
Glucocorticoids have been shown to have profound effects on the
mammalian nervous system, most of which have been shown to result from a
receptor-mediated change in concentration or activity of key proteins.
This change is a consequence of an action of the glucocorticoid-
receptor complex on the molecular machinery that controls gene
expression. Upon binding of a steroid by the receptor, it undergoes a
temperature-dependent activation, correlated with changes in a number of
receptor properties, before it can effectively interact with its nuclear
acceptor sites.
The surface hydrophobicity of the glucocorticoid receptor in brain
cytosol was examined using hydrophobic interaction chromatography.
There was a clear increase in hydrophobicity upon activation, a finding
with important implications for the interactions between activated
receptors and numerous nuclear components.
v


The successful purification of the glucocorticoid receptor allowed
for an investigation of activation relatively free of cytosolic factors
that could interfere with, or contribute to, the process. Although
activation of unpurified receptors led to a 50-fold increase in
DNA-cellulose binding, there was no increase when purified receptors
from brain or liver were subjected to identical activation conditions.
However, other changes in purified receptors associated with activation,
including an increase in surface hydrophobicity, implied the possibility
of a multistep process.
Unoccupied glucocorticoid receptors can exist in a non-steroid-
binding or down-regulated state which can be up-regulated to a
steroid-binding state upon addition of sulfhydryl reducing reagents. An
investigation of the hydrodynamic and biochemical nature of the up- and
down-regulated receptors revealed that they exhibited the same
sedimentation properties, but differed in surface hydrophobicity. In
addition, the up-, but not the down-, regulated form proved susceptible
to irreversible inactivation by some sulfhydryl reactive reagents,
allowing for a determination of the respective populations of the two
forms in cytosol or homogenate.
Sedimentation of the unoccupied receptor was examined by post
labeling procedures and was found to differ in the presence (9-10 S
only) or absence (9-10 & 4-5 S) of molybdate. This effect of molybdate
was mimicked somewhat by heat-stable cytosolic factors.
vi


CHAPTER I
GENERAL INTRODUCTION
Glucocorticoid steroid hormones are known to have profound
metabolic, neuroendocrine and behavioral effects in the mammalian brain.
Glucocorticoids, in addition to regulating ACTH secretion (for review
see Keller-Wood and Dallman, 1984), produce alterations in mood, changes
in detection and recognition of sensory stimuli, changes in sleep and in
the extinction of previously acquired habits, and alterations of
electric activity of the brain (for reviews see Raynaud et al., 1980;
McEwen et al., 1982; Schraa and Dirks, 1982; Luttge, 1983; De Kloet,
1984; Reese and Gray, 1984; Meyer, 1985). Since most of these reviews
were written, many new reports concerning the role of glucocorticoids in
the nervous system have appeared. A number of studies have further
investigated the localization of glucocorticoid binding in different
regions of the central nervous system including the spinal cord (Clark
et al., 1981; Duncan and Stumpf, 1984; Orti et al., 1985), the locus
ceruleus (Krasuakaya, 1982), the hypothalamus (Kato, 1983;
Ribarac-Stepic et al., 1984), the caudate-putamen (Defiore and Turner,
1983), the hippocampus (De Kloet et al., 1984), or a variety of
different brain regions (Tornello et al., 1981; Dordevic-Markovic et
al., 1983; Alexis et al., 1983; Sarrieau et al., 1983, 1984; Birmingham
et al., 1984; Ribarac-Stepic et al., 1984).
Many recent studies have focused on the role of glucocorticoids in
the development of the nervous system. Receptor binding studies in the
1


2
developing brain imply an early sensitivity to glucocorticoids (Kitraki
et al., 1984), particularly in the cerebellum (Pavlik and Buresova,
1984). The general in vivo effect of glucocorticoids on brain growth
and development appears to be one of suppression (Slotkin et al., 1982;
Doerner, 1983; Meyer, 1983; DeKosky et al., 1984; Pavlik et al., 1984)
although adrenalectomy of young rats was shown to result in
hypomyelination of the central nervous system (Preston and McMorris,
1984) indicating that the effects of glucocorticoids on neural
development are complex. Using a brain cell culture system, Stephens
(1983) reported that glucocorticoids (cortisol) stimulate rather than
inhibit the processes of myelinogenesis and brain development.
Investigations of the development of synapses between cholinergic
neurons and retina muscles in a cell culture system led to the
hypothesis by Puro (1983) that glucocorticoids regulate the development
of mechanisms which couple neuronal depolarization with release of
neurotransmitter. Lending some potential support to this hypothesis are
the findings of Leeuwin et al. (1983) who reported that glucocorticoids
induced significant changes in the morphology of synaptic vesicles of
cholinergic (phrenic) nerve terminals. Smith and Fauquet (1984)
reported that glucocorticoids indirectly stimulate adrenergic
differentiation in cultures of migrating and premigratory neural crest
by selectively enhancing catecholaminergic properties in neural crest
cells that had already been exposed to an appropriate signal of another
kind. Finally, a variety of behavioral, biochemical and morphological
teratologies of the central nervous system are known to be induced by
glucocorticoids (Bohn, 1984; Pratt et al., 1984).


3
Glucocorticoids have profound and complex effects on brain
metabolism. Podvigina et al. (1983) reported that a variety of
glucocorticoids inhibited respiration and phosphorylation and increased
glycolysis in slices and homogenates of rat cerebral cortex, hippocampus
and hypothalamus. In agreement with glucocorticoid binding data, these
workers found hippocampus to be most sensitive and cortex to be least
sensitive. Interestingly, Kamenov (1981) has suggested that the degree
to which glucocorticoids affect respiration may itself be under the
indirect control of the nervous system and may vary seasonally in some
animals. These and many other metabolic functions in brain are
modulated by glucocorticoids via an impressive number of enzymes. Patel
et al. (1983) showed recently that corticosterone treatment resulted in
an increase in glutamine synthetase activity in rat brain. These
findings were confirmed in mouse brain cell culture by Stephens (1983)
who found that cortisol also increased the activity of 2',3'-cyclic
nucleotide phosphohydrolase while it caused a decrease in arylsulfatase
A activity (thought to be important in myelinogenesis). Earlier work
regarding the role of glucocorticoids in glia is reviewed in McEwen
(1984). The activity of glycerol 3-phosphate dehydrogenase, another
enzyme thought to be involved in myelination and present in high
concentrations in oligodendrocytes, was recently shown to be reduced in
rat brain after adrenalectomy. Rat brain citrate synthetase activity,
on the other hand, has been recently shown to undergo an actinomycin
D-reversible decrease upon administration of cortisol (Sharma and
Patnaik, 1984). These same workers reported earlier that cortisol
differentially regulates the activity of malate dehydrogenase isoenzymes
in rat brain (Sharma and Patnaik, 1982). Complex results have been


obtained for tyrosine aminotransferase activity in brain as well.
Mishunina and Babicheva (1981) reported that a single injection of
cortisol to rats caused a reduction of tyrosine aminotransferase
activity in the hypothalamus and medulla oblongata while causing an
increase in the activity in the hippocampus. Glucocorticoid injection
has also been shown to increase arginase activity in guinea pig brain
(Bjelakovic and Nikolic, 1983) and to "modulate" ornithine decarboxylase
activity in rat brain (De Kloet et al., 1983). Interestingly, tyrosine
hydroxylase activity in the superior cervical ganglion was shown to be
increased by injections of the synthetic glucocorticoid, dexamethasone,
but not by endogenous glucocorticoids (corticosterone or cortisol)
regardless of dose (Sze and Hedrick, 1983). Eranko et al. (1982), using
fluorescent histochemical techniques, reported that injection of newborn
rats with cortisol caused a great increase in the number of the small,
intensely fluorescent cells and the appearance of similar small cells
with intense immunohistochemical reactions for tyrosine hydroxylase
(TH), dopamine-B-hydroxylase (DBH) and phenylethanolamine (norepi
nephrine) N-methyltransferase (PNMT) in the superior cervical ganglion.
At the same time, the epinephrine content and the PNMT activity of the
ganglion greatly increased, while no significant changes were observed
in the dopamine or norepinephrine content or TH or DBH activity. PNMT
expression has also been shown to be influenced by glucocorticoids in
cells derived from the neural crest (Bohn, 1983).
Single acute doses of dexamethasone have been recently shown to
affect the levels of monoamines and their metabolites in rat brain
(Rothschild et al., 1985). These researchers reported significant
increases in dopamine levels in the hypothalamus and nucleus accumbens


5
of dexamethasone-treated rats when compared with saline-treated rats,
whereas no significant effect of dexamethasone on dopamine levels in
frontal or striatal brain areas was found. These workers also found
that dexamethasone led to a significant increase in serotonin in
hypothalamus, whereas dexamethasone caused a significant decrease in
serotonin in the frontal cortex. These findings may help to explain the
observations that corticosteroids lower prolactin levels and can induce
psychiatric disturbances as well as the finding that affectively ill
patients with high post dexamethasone cortisol levels are frequently
psychotic
Other recent findings of relevance to the effects of gluco
corticoids on neurotransmitter systems include the report by Mishunina
and Kononenko (1983) that a single injection of cortisol increased the
uptake of gamma-aminobutyric acid (GABA) by synaptosomes of hippocampus,
hypothalamus and cerebral cortex. However, in vitro studies by these
same workers resulted in a glucocorticoid-dependent increase in GABA
uptake by only the hypothalamus synaptosomes, indicating differential
mechanisms of glucocorticoid action in these different brain regions.
Haidarliu et al. (1983) reported that perfusion of the rat neostriatum
with dexamethasone after stereotaxic injections of [3H]-labeled dopamine
into the caudate nucleus increased basal but inhibited potassium-
stimulated [3H]dopamine release and yield of metabolites. Slotkin et
al. (1982) reported that neonatal exposure to dexamethasone resulted in
alterations in norepinephrine synthesis and turnover in both central and
peripheral sympathetic neurons. Further demonstration of the
relationship between glucocorticoids and central neurotransmitter
systems is evident from studies reporting a decrease in the


6
corticosterone binding capacity of hippocampus after administration of
6-hydroxydopamine (Weidenfeld et al., 1983) or 5,7-dihydroxytryptamine
(Siegel, 1983). Similarly, the possible role of medial forebrain bundle
catecholaminergic fibers in the modulation of glucocorticoid negative
feedback effects has been further elucidated by investigating the
effects of 6-hydroxydopamine treatments in rat brains (Feldman et al.,
1983). In addition to effects on transmitter uptake and synthetic and
degradative enzyme activity, some of the effects of glucocorticoids on
catecholaminergic function are known to be via catecholaminergic
receptors (see review by Davies and Lefkowitz, 1984).
Many of the actions of glucocorticoids outside the brain, such as
their effects on lipolysis, gluconeogenesis, glycogenesis and
glycogenolysis, are regulated primarily by cAMP or hormones acting
through cAMP leading to the notion that glucocorjticoids have
potentiating (permissive or synergistic) effects on the action of many
hormones that act through cAMP as the intracellular second messenger
(for review see Liu, 1984). Such wide-ranging effects of gluco
corticoids on the effects of these other hormone systems, thought to be
via the regulation of the autophosphorylation of cAMP-dependent protein
kinase, will likely be shown to play an important role in the
glucocorticoid modulation of neural function as well.
A recent finding of potential importance to the field of epilepsy
research involves the discovery that natural, but not synthetic, gluco
corticoids modulate the binding of [3H]muscimol, a GABA agonist, in
crude synaptosomal membranes and in brain sections of rats (Majewska et
al., 1985). In general, nanomolar concentrations concentrations of
corticosterone and pregnenolone-sulfate enhanced mucimol binding in


7
cerebral cortex, cerebellum, thalamus and hippocampus via an apparent
increase in the affinities of GABA receptors. More recently, two
metabolites of the steroid hormones progesterone and deoxycortico
sterone, were shown to be potent barbiturate-like ligands of the GABA
receptor-chloride ion channel complex (Majewska et al., 1986). At low
concentrations both steroids inhibited binding of the convulsant
t-butylbicyclophosphorothionate to the GABA-receptor complex and
increased binding of benzodiazepine flunitrazepam. In addition,
chloride uptake into isolated brain vesicles was stimulated and the
inhibitory actions of GABA in cultured rat hippocampal and spinal cord
neurons were potentiated. These findings may help to explain the
ability of certain steroid hormones to rapidly alter neuronal excit
ability (see below) and may provide a mechanism for the anesthetic and
hypnotic actions of naturally occurring and synthetic anesthetic
steroids.
The effects of glucocorticoids on the electrical activity of
neurons has been studied at the level of single neurons by the method of
microiontophoresis. This technique has been used to study glucocort
icoid-induced changes in activity of neurons in a variety of brain
regions including hypothalamus (Mandelbrod et al., 1981), medial septal
area (Papir-Kricheli and Feldman, 1981), reticular formation (Avanzino
et al., 1983) and hippocampus (Belyi et al., 1982). In most of these
studies glucocorticoids were found to inhibit some neurons, excite some
neurons and have no effect on still other neurons in the same brain
region, although the ratios of the three groups varied between regions.
Peripherally, the effects of glucocorticoids on neuromuscular function
have also been investigated. These effects are generally facilitatory


8
(see review by Hall, 1983), although there have been reports to the
contrary (Korneeva and Emelyanov, 1981).
The role of glucocorticoids in the regulation of physiological
reactions to stress has been widely studied (for review see Munck et
al., 1984; Sapolsky et al., 1986) and at least some of these stress-
related functions of glucocorticoids are known to involve the central
nervous system. One example that has received a good deal of attention
recently involves the effects of glucocorticoids on stress-induced
analgesia. Adrenalectomy, hypophysectomy and/or glucocorticoid
treatment have all been shown to have significant effects on the
stress-induced (cold water, footshock, etc.) inhibition of pain
responsiveness. Markel et al. (1984) reported that adrenalectomy
significantly reduced the threshold for footshock sensitivity in rats
and that steroid replacement restored, or even potentiated, this pain
sensitivity. Similarly, Mousa et al. (1983) reported that analgesia
induced in rats by cold-water swim stress and measured by the tail-flick
and hot-plate methods was significantly antagonized after IP pretreat
ment for three days with dexamethasone. These investigators go on to
suggest that since naloxone also attenuated the analgesia developed by
the cold-water swim stressor, corticosteroids may have a role in
modulating stress-induced analgesia via the endogenous opiate system.
Evidence of this was presented recently by Terman et al. (1984) who used
inescapable foot shock as a stressor and the tail-flick test as a
measure of analgesia. These workers, using multiple doses of gluco
corticoids, found that lower doses potentiated and higher doses
significantly reduced stress analgesia in both sham and hypophysect-
omized animals. Despite some evidence to the contrary (Baron and


9
Gintzler, 1984), these results, along with the finding that nonopiod
analgesia is unaffected by dexamethasone (Lewis et al., 1980) and that
corticosterone exerts the same biphasic dose-dependent effects on
morphine analgesia (Terman et al., 1985), suggest an opiod action for
glucocorticoids, perhaps at the receptor level. Of some potential
relevance to this topic is a report that adrenalectomy significantly
potentiated and corticosterone administration attenuated stress-induced
decreases in norepinephrine contents in the hypothalamus and thalamus,
and the increases in 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (a
principle metabolite of norepinephrine) levels in the hypothalamus,
amygdala and thalamus (Nakagawa et al., 1983).
The idea that glucocorticoids, via the adrenocortical axis, may
have an important role in the process of aging has been examined in
depth recently (Sapolsky et al., 1986). It has been shown that the aged
male rat is impaired in terminating the secretion of glucocorticoids at
the end of stress. This hormonal excess is thought to be due to
degenerative changes in a region of the brain which normally inhibits
glucocorticoid release; the degeneration, in turn, appears to be caused
by cumulative exposure to glucocorticoids (Sapolsky and McEwen, 1985;
Sapolsky, 1986). Together, these effects form a feed-forward cascade in
the aging subject with potentially serious pathophysiological
consequences.
In addition to the above findings regarding the role of
glucocorticoids in the nervous system, glucocorticoids are clinically
important in the field of neurosurgery, most notably for the treatment
of various kinds of brain edema related to intracranial tumors, cerebral
infarctions and head injuries (Bouzarth and Shenkin, 1974;


10
Sugiura et al., 1980; Yu et al., 1981).
Although there is limited evidence of glucocorticoid binding
directly to synaptic (Towle and Sze, 1983) and other (Duval et al.,
1983) membranes, most of the glucocorticoid effects amenable to
experimentation have been shown to result from a receptor-mediated
change in concentration or activity of key proteins. This change is a
consequence of an action of the glucocorticoid-receptor complex on the
molecular machinery that controls gene expression. Recent advances such
as recombinant-DNA and monoclonal-antibody techniques have offered new
ways of exploring this machinery. Recently, a monoclonal antibody
against the rat liver glucocorticoid receptor in combination with the
indirect immunoperoxidase technique has made it possible to demonstrate
glucocorticoid receptor-immunoreactive nerve and glial cell nuclei all
over the tel- and diencephalon of the male rat (Fuxe et al., 1985).
Since the glucocorticoid receptor qualifies as one of the few proteins
known to control gene expression in eukaryotes, it is not surprizing
that glucocorticoids have become a fashionable tool for the molecular
biologist (for reviews see Ringold et al., 1983; Lan et al., 1984; Hager
et al., 1984; Rousseau, 1984a; Scheidereit et al., 1986). A major step
in the understanding of the structure and function of the glucocorticoid
receptor was made recently when the primary structure of a functional
human glucocorticoid receptor cDNA was identified and subsequently
cloned (Govindan et al., 1985) and expressed in vitro (Hollenberg et
al., 1985). The focus of the following review and the proposed
research, however, will be the receptor molecule itself and the changes
in this molecule brought about by the binding of a steroid ligand which
enable it to interact with the machinery that controls gene expression.


11
Although little has been said in this brief review regarding the
structure or function of the receptors in brain for other classes of
steroids (i.e. estrogens, progestins, androgens), it is particularly
relevant to briefly note recent findings regarding the so called
mineralocorticoid, or "type I", receptor in neural tissues. The
relevance of mineralocorticoid receptors to glucocorticoid actions in
neural tissues stems first from the fact that [3H]aldosterone has been
shown to bind to glucocorticoid, or "type II", receptors as well as type
I receptors in brain cytosol (Anderson and Fanestil, 1976) by virtue of
biphasic Scatchard plots for [3H]aldosterone, but not [3H]dexamethasone.
Later reports indicated that inclusion of the new synthetic gluco
corticoid RU 26988 (unlabeled), which binds to type II but not type I
receptors or corticosteroid binding globulin (Mogiulewsky and Raynaud,
1980), linearizes the [3H]aldosterone Scatchard plott(Beaumont and
Fanestil, 1983; Emadian et al., 1986), leaving only a high-affinity
component assumed to be the type I receptor. A second point of
relevance involves the fact that in addition to aldosterone,
corticosterone has a high affinity for the type I receptor (Krozowski
and Funder, 1983), which has led some to believe that corticosterone may
be a major, if not the primary, ligand for type I receptors in brain.
This topic is of great concern to those investigating glucocorticoid
actions in the nervous system and is currently the subject of
investigation by others in this lab. It should be noted, however, that
synthetic glucocorticoids, such as dexamethasone or triamcinolone
acetonide, have a much lower affinity for the type I receptor and
usually possess "purer" glucocorticoid properties than corticosterone as
determined by their physiological actions in peripheral tissues


12
(Meyer, 1985). In addition to their high specificity for type II
glucocorticoid receptors, other advantages of using these synthetic
glucocorticoids include the fact that their rate of dissociation from
the receptor is considerably slower than that of natural glucocorticoids
and have an insignificant affinity for corticosteroid binding globulin.
For these and other reasons, the research reported here was carried out
using dexamethasone and/or triamcinolone acetonide exclusively, and
therefore all further references to "glucocorticoid receptors" will
imply type II receptors for adrenal steroids unless otherwise indicated.
Major areas of glucocorticoid receptor research that are of
particular relevance to the glucocorticoid modulation of nervous system
function include the following: 1) The stabilization and potential
mechanisms of up- and down-regulation of unoccupied glucocorticoid
receptors. 2) Activation of the glucocorticoid-receptor complex to the
nuclear-binding form and subsequent interaction with the genome. 3)
Purification of the glucocorticoid receptor and subsequent detailed
physicochemical analysis.


CHAPTER II
HYDROPHOBIC INTERACTION CHROMATOGRAPHY OF VARIOUS FORMS OF THE OCCUPIED
AND UNOCCUPIED GLUCOCORTICOID RECEPTOR
Introduction
Hydrophobic interactions basically result from the adherence of
non-polar solutes or groups in aqueous solution, resulting,
consequently, from their repulsion from the strong polar-polar
interactions of water molecules. Like its ionic properties, a
molecule's surface hydrophobicity depends upon its primary structure.
A change in the surface hydrophobicity of glucocorticoid-receptor
complexes associated with activation has been suggested by workers
reporting an increase in the partitioning of these complexes after
activation into the polyethylene glycol (PEG) layer in an aqueous
dextran-PEG two phase system (Andreasen, 1978 and 1982; Andreasen and
Mainwaring, 1980) and by Luttge et al. (1984d) who, in addition to an
increase in partitioning into PEG, found a large increase in the
fractional adsorption of [3H]triamcinolone acetonide-receptor complexes
to glass fiber (Whatman GF/C) filters following activation. This
putative increase in hydrophobicity does not appear to be unique to the
glucocorticoid receptor, since similar transformations following
activation have been reported recently for estrogen (Gschwendt and
Kittstein, 1980; Murayama and Fukai, 1983), androgen (Bruchovsky et al.,
1981) and progesterone (Lamb and Bullock, 1983) receptors. Although the
functional significance of this increase in hydrophobicity is unknown,
13


14
it may contribute to the increased binding affinity of the activated
receptor for DNA. Increasing the hydrophobicity of the receptor may
also increase its rate of permeation through the nuclear membrane.
Hydrophobic interactions have also been reported to account for the
high affinity binding of glucocorticoids to their receptors (Wolf et
al., 1978; Alsen, 1983; Eliard and Rousseau, 1984). According to these
workers, binding requires both sides of the steroid to be enveloped by
the receptor.
The influence of hydrophobic interactions on the structure and
steroid-binding properties of glucocorticoid receptors has recently been
investigated by means of chromatography on Sephacryl S-300 or Lipidex
1000 or by incubation with charcoal or phospholipase C (Bell et al.,
1986). These workers suggest that receptor activation is preceded by
structural changes associated with the loss of a lipid factor from the
complex. They also reported that non-polar steroid antagonists, and
lipophilic compounds such as phenothiazines, bound to secondary,
hydrophilic sites on the receptor and exerted allosteric effects on the
primary steroid-binding site implying that hydrophobic interactions may
be important determinants of the structure and properties of
glucocorticoid receptors.
One particularly useful method for investigating the hydrophobic
characteristics of steroid receptors is hydrophobic interaction
chromatography. Typically this involves characterizing the chromato
graphic behavior of the receptor in question on a series of hydrophobic
matrices of decreasing polarity. Bruchovsky et al. (1981) investigated
the hydrophobic properties of the rat prostatic nuclear androgen
receptor using gamma-amino-alkyl derivatives of agarose with varying


15
alkyl substituent lengths. These workers found the adsorption of
receptor to the modified agarose gels increased with the length of the
alkyl substituent. However, the presence of the terminal amino group in
these gels introduces the possibility of ionic effects which could
complicate the interpretation of the hydrophobic interactions (Shaltiel,
1974). More recently, Lamb and Bullock (1983) investigated the hydro-
phobic properties of the rabbit uterine progesterone receptor using a
series of alkyl agarose columns of increasing alkyl chain length (no
terminal amino group). No such studies have been conducted on
glucocortcoid receptors.
In addition to gaining further knowledge regarding the process of
receptor activation, more detailed hydrophobic interaction studies of
both activated and unactivated forms of the glucocorticoid-receptor
complex, as well as the non-steroid bound form of the receptor, are
likely to provide valuable information concerning the physicochemical
properties of each of these receptor forms. Such studies might also
provide potentially useful means by which partial purification of these
different receptor forms can be achieved.
Materials and Methods
Chemicals, Steroids and Isotopes
[6,7-3H]Triamcinolone acetonide, or 9a-fluoro-llb,16a,17a,21-tetra-
hydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide, ([3H]TA, specific
activity = 37 Ci/mmol) was purchased from New England Nuclear (Boston,
MA). Sephadex G-25 (fine) was obtained from Pharmacia Fine Chemicals
(Piscataway, NJ). Dithiothreitol (DTT) and 4-(2-hydroxyethyl)-l-piper-
azineethane-sulfonic acid were courtesy of Research Organics (Cleveland,
OH). Sodium molybdate (Na2Mo04), calf thymus DNA-cellulose, glycerol,


16
PPO (2,5-diphenyloxazole) and dimethyl POPOP (l,4-bis[2(4-methyl-5-
phenyloxazoyl)]benzene), phenyl agarose and alkyl agaroses were all
purchased from Sigma Chemical Co. (St. Louis, MO). Scinti Verse II was
purchased from Fisher, Inc. (Fair Lawn, NJ). All other chemicals and
solvents were reagent grade.
Animals
All studies used female CD-I mice (Charles River Laboratories,
Wilmington, MA) that were subjected to combined ovariectomy and
adrenalectomy approximately 1 week prior to each experiment in order to
remove known sources of endogenous steroids. Both operations were
performed bilaterally via a lateral, subcostal approach under barbi
turate anesthesia, and mice were given 0.9% NaCl (w:v) in place of
drinking water. On the day of the experiment mice were anesthetized
with ether and perfused slowly through the heart with ice-cold HEPES-
buffered saline (20-30 ml, isotonic, pH 7.6).
Cytosol Preparation and Steroid Binding
Brains were removed from the perfused animals and homogenized (2 x
10 strokes at 1000 rpm) in 4 volumes of ice cold Buffer A (20 mM HEPES,
2 mM DTT and 20 mM Na2Mo04, pH 7.6 at 0 C) in a glass homogenizer with
a Teflon pestle milled to a clearance between the pestle and homogeniz
ation tube of .125 mm on the radius (to minimize rupture of the brain
cell nuclei (McEwen and Zigmond, 1972). The crude homogenate was
centrifuged at 100,000 g for 20 min and the supernatant recentrifuged at
100,000 g for an additional 60 min to yield cytosol. During these
centrifuge runs, and during all other procedures, unless otherwise
indicated, careful attention was paid to maintaining the cytosol at
0-2 C. Final protein concentrations were typically in the 6-8 mg/ml


17
range. For experiments involving occupied (activated or unactivated)
receptors, cytosol samples were incubated with 20 nM [3H]TA for 24 to 40
hours at 0 C with or without a 200-fold excess of unlabeled TA.
Experiments involving hydrophobic interaction chromatography of
unoccupied receptors required that the individual fractions collected
from the hydrophobic and control columns be postlabeled with 20 nM
[3H]TA +/- 4 um [1H]TA. Prior to hydrophobic interaction chromatography
of unlabeled receptors, cytosol was run on Sephadex G-25 columns equili
brated and eluted with the appropriate buffer to be used for
subsequently eluting the hydrophobic columns.
Activation and Removal of Unbound Steroid
[3H]TA-labeled cytosol samples to be activated were first run on
Sephadex G-25 columns (0.6 x 14 cm) equilibrated with buffer containing
20 mM HEPES and 2 mM DTT only. This column run resulted in the removal
of both free steroid and molybdate, allowing for uninhibited activation
(Luttge and Densmore, 1984; Luttge et al., 1984a,b,d). The bound
fraction was then incubated at 22 C for variable periods of time to
determine the minimum period of time required to achieve total
activation. The degree of activation was determined by DNA-cellulose
binding assay. For the purpose of hydrophobic interaction chromato
graphy of activated receptors, the maximally activated receptor
preparation was again run on a second Sephadex G-25 column equilibrated
with the appropriate buffer (the same buffer used to equilibrate and
elute the subsequent alkyl agarose columns). For unactivated receptors,
the cytosol was treated in an identical fasion except that the first
Sephadex G-25 column was equilibrated and eluted with HEPES buffer
containing 20mM molybdate and 2 mM DTT and the bound fraction was


18
subjected to a 0 C (rather than a 22 C) incubation prior to running on
the second Sephadex G-25 column (identical to the second column run for
the activated preparation).
In the case of postlabeling of unoccupied receptor fractions or
monitoring of dissociation occurring during the hydrophobic column runs
of prelabeled receptors, all or part of each hydrophobic column fraction
was again subjected to Sephadex G-25 chromatography to remove free
steroid.
DNA-cellulose Binding Assay
Calf thymus DNA-cellulose, prepared originally by the method of
Alberts and Herrick (1971), was added to HEPES buffer containing 2 mM
DTT to yield a final concentration of 10 mg/ml (4.1 mg DNA/g
DNA-cellulose). In a typical assay (run as duplicates) a 100 ul aliquot
of [3H]TA- labeled cytosol was added to 300 ul of the DNA-cellulose
slurry. The mixture was then vortexed gently and incubated at 0 C for
60 min. Assay tubes were oscillated at 150 rpm throughout the
incubation and vortexed gently every 10 min. The DNA-cellulose was
collected by centrifugation (2000 x g for 5 min), the supernatant
discarded and the DNA-cellulose pellet washed 3 times with 1 ml of HEPES
buffer plus 2 mM DTT. The entire pellet was resuspended in deionized
water and transferred to scintillation vials for determination of bound
radioactivity. DNA- cellulose was prepared in buffer not containing
molybdate in order to reduce the concentration of molybdate during the
binding assay, since high concentrations of molybdate were found to
reduce the efficacy of receptor binding to DNA-cellulose (Luttge et al.,
1984a).


19
Receptor Stability Determinations
Prelabeled receptor preparations (both activated and unactivated),
were run on Sephadex G-25 columns equilibrated and eluted with 50 mM
molybdate and varying concentrations of KC1 (0 2,000 mM) in order to
determine the effects of ionic strength on receptor stability. A
fraction of the bound fraction was immediately run on a second Sephadex
G-25 column prior to specific binding determination, while the remainder
was incubated for 4 hr at 0 C prior to being run on a second Sephadex
G-25 column and specific binding determined.
Results
The initial experiment in the investigation of glucocorticoid
receptor hydrophobicity involved hydrophobic interaction chromatography
of activated and unactivated glucocorticoid receptor complexes on a
limited Shaltiel series using a series of elution buffers of decreasing
hydrophobic character. This experiment used a series of hydrophobic
gels created by covalently attaching alkyl groups of increasing chain
length to agarose including propyl (n = 3 carbons), hexyl (n = 6), octyl
(n = 8), decyl (n = 10) and dodecyl (n = 12) agarose, along with
unmodified agarose as a control. Activated and unactivated receptor
preparations equilibrated in 600 mM KC1 and 50 mM molybdate were applied
to each of the columns (containing 1 ml of gel) and then eluted step
wise with the following buffers (each of which contained 50 mM molybdate
in HEPES buffer): 600 mM KC1, 300 mM KC1, 0 mM KC1, 10% glycerol and 30%
glycerol. Results (not shown) indicated that activated preparations
were retained longer than unactivated and that this difference was the
largest on the hexyl agarose columns. Both receptor forms appeared to
be eluted completely with higher salt concentrations on all gels except


20
octyl, decyl and dodecyl which exhibited variable retention of both
forms of the receptor in the presence of the more hydrophobic elution
buffers.
Further refinement of the procedure to investigate receptor
hydrophobicity next involved hydrophobic interaction chromatography of
activated and unactivated glucocorticoid receptor complexes on a more
complete Shaltiel series of alkyl agarose columns using a series of
elution buffers of decreasing hydrophobic character. In addition to the
original gels used, butyl (n=4), pentyl (n=5) and phenyl (n=P) agarose
were added and the gel volume was increased to 1.5 ml. In addition,
smaller fractions (0.5 ml) were collected to increase resolution and a
different series of step wise buffer elutions were used including: 600
mM KC1, 0 mM KC1 and 10% ethylene glycol (all in KEPES buffer plus 50 mM
molybdate). A final elution of 6 M urea was used to remove those
receptors (and other proteins) that were strongly retained throughout
all of the elution steps. With the higher resolution resulting from
smaller fraction size, it was obvious that activated receptors were
retained longer than unactivated receptors run on butyl, pentyl and
hexyl agarose columns. On all three columns, however, both forms of the
receptor were eluted completely, though at different rates, by 600 mM
KC1. Phenyl agarose strongly retained the activated receptor, whereas
most of the unactivated form of the receptor was eluted rapidly under
the high ionic strength conditions. Octyl, decyl and dodecyl agarose
retained the receptors more than the other gels except that there were
no significant differences between the activated and unactivated forms
on these columns. Overall, the pentyl agarose exhibited the greatest
differences in retention between the activated and unactivated forms


21
while still allowing for both forms to be eluted completely under the
same buffer conditions.
Since the results of the previous experiments indicated that any
binding retained by any of the hydrophobic columns after adequately
washing with 600 mM KC1 could not be eluted with anything tested short
of denaturing solutions such as 6 M urea, hydrophobic interaction
chromatography of activated and unactivated glucocorticoid receptor
complexes on a Shaltiel series using only 600 mM KC1 was performed.
This experiment was similar to the previously described experiment
except that only one elution buffer was used instead of a series of
different buffers. The results basically replicated those of the
previous experiment in that the activated form of the receptor
consistently shows a greater degree of retardation on most of the
columns with pentyl, hexyl and phenyl agarose providing the greatest
discrimination between the two forms (Figure 2-1).
Since high ionic strength elution buffers (HEPES buffer plus 50 mM
molybdate and 600 mM KC1) were able to discriminate between the
activated and unactivated forms of the receptor on virtually all of the
columns tested while allowing all of the binding to be eluted, the
effects of using lower or higher ionic strength buffers to equilibrate
and elute the hydrophobic columns were investigated. Because of
uncertainties regarding the activation of unactivated glucocorticoid
receptors and the stability of glucocorticoid binding under high salt
conditions, hydrophobic interaction chromatography of activated and
unactivated glucocorticoid receptor complexes on a Shaltiel series
equilibrated and eluted with only HEPES buffer and 50 mM molybdate (no
KC1) was first attempted (Figure 2-2). Proply, butyl, pentyl and hexyl


Figure 2-1. Hydrophobic interaction chromatography of unactivated and activated Type II glucocorticoid
receptor complexes on a Shaltiel series equilibrated and eluted with HEPES buffer containing 50 mM
molybdate and 600 mM KC1. Brain cytosol prepared with HEPES buffer containing 20 mM molybdate and labeled
with 20 nM [3H]TA for 40 hr at 0 C, was run on Sephadex G-25 columns equilibrated and eluted with HEPES
buffer plus either 0 or 20 mM molybdate to remove the molybdate and/or free steroid. The molybdate-free
cytosol was activated by a 24 min incubation at 22 C (solid triangle), whereas the molybdate-containing,
unactivated cytosol was left at 0 C (solid circles). Both groups were subsequently run on Sephadex G-25
columns equilibrated and eluted with HEPES buffer containing 50 mM molybdate and 600 mM KC1 prior to being
run on each of the alkyl, phenyl and control agarose columns equilibrated and eluted with the same buffer.
Cytosol (0.5 ml) was applied to the columns and 10 fractions (0.5 ml) were collected. Binding is expressed
as percent of the total counts (> 15,500 cpm) applied to each column. Results presented here are
representative of 3 independent replications. 0 = unmodified agarose (control), 3 = propyl agarose, 4 =
butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose, 10 = decyl agarose and 12 =
dodecyl agarose.


Percent of Total
ro
LO
Fraction Number


Figure 2-2. Hydrophobic interaction chromatography of unactivated and activated Type II glucocorticoid
receptor complexes on a Shaltiel series equilibrated and eluted under low salt conditions (HEPES buffer
containing 50 mM molybdate and no KC1). Brain cytosol was prepared, labeled and activated (solid
triangles) or left unactivated (solid circles) as described in Figure A. Both groups were subsequently run
on Sephadex G-25 columns equilibrated and eluted with 50 mM molybdate, but no KC1, prior to being run on
each of the alkyl, phenyl and control agarose columns equilibrated and eluted with the same buffer.
Cytosol (0.5 ml) was applied to each column and 10 fractions (0.5 ml) were collected. Binding is expressed
as percent of the total counts ( > 15,000 cpm) applied to each column. Results presented here are
represetative of 3 independent replications. 0 = unmodified agarose (control), 3 = propyl agarose, 4 =
butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose, 10 = decyl agarose and 12 =
dodecyl agarose.


Percent of Totol
60
40
20
60
40
20
60
40
20
2 4 6 8 10
2 4 6 8 10
2 4 6 8 10
Fraction Number


26
agaroses all showed an increased affinity for the unactivated receptor,
resulting in varying degrees of increased retention when compared to
elution by higher salt. Interestingly, pentyl agarose showed the
greatest retention of the unoccupied receptor of any of the gels. In
contrast, the unactivated receptor exhibited a decrease in retention on
the phenyl agarose. There was no appreciable differences in the elution
profiles obtained from octyl, decyl or dodecyl agarose in high or low
salt. As was the case for unactivated receptors, propyl, butyl, pentyl
and hexyl agaroses all showed an increased affinity for the activated
receptor under low salt conditions, resulting in varying degrees of
increased retention when compared to elution by higher salt. Interest
ingly, pentyl agarose exhibited the greatest retention of any of the
gels examined for both the activated and the unactivated forms of the
receptor. There was no appreciable differences in the elution profiles
obtained from octyl, decyl, dodecyl or phenyl in high or low salt.
The finding that octyl agarose exhibited greater retention of both
forms of the receptor than decyl agarose, which exhibited greater
retention than dodecyl agarose regardless of ionic strength (one would
have expected the reverse if these interactions were truly hydrophobic),
led to a more extensive investigation of the interactions between the
glucocorticoid receptor and these highly hydrophobic gels. Although
free steroid is removed from the cytosolic preparations immediately
prior to applying these preparations on the hydrophobic columns and
binding with [3H]TA has been shown to exhibit high stability and very
slow dissociation even under high salt conditions, the possibility that
the hydrophobic alkyls might themselves be destabilizing the receptors
via strong hydrophobic interactions had not been examined. This


27
involved replicating the previously described experiment whereby
activated and unactivated receptors where applied to the complete
Shaltiel series of columns equilibrated and eluted with HEPES buffer
plus 50 mM molybdate and 600 mM KC1 except that each fraction eluted
from the column was divided into two equal halves. One half was counted
directly as before while the other half was subjected to an additional
bound/free separation on Sephadex G-25 columns. When the profiles
determined by each of these methods for each of the gels were compared,
there were no significant differences observed except in the case of the
octyl, decyl and dodecyl agaroses. None of the counts eluting from
these three columns eluted in the bound fraction during the subsequent
Sephadex G-25 bound/free separation.
Since the results from the preceding experiment indicated how
receptor instability and/or dissociation could complicate the
interpretation of the results from hydrophobic interaction chromato
graphy of these receptors, and since many of these experiments required
higher concentrations of molybdate and potassium chloride than are
generally encountered throughout most of the literature regarding
glucocorticoid receptors, effects of these parameters on receptor
binding properties obviously warranted further study. In this
experiment, cytosol was prepared in the usual fashion in the presence of
20 mM molybdate and 2 mM DTT and labeled with 40 nM [3H]TA plus or minus
unlabeled TA for 24 hours to achieve maximal binding. After this
initial incubation, aliquots were taken to determine total and non
specific binding. Next, aliquots of prelabeled cytosol were applied to
Sephadex G-25 columns preequilibrated with every possible combination of
the following concentrations of sodium molybdate (0, 20 or 50 mM) and


28
KC1 (O, 50, 150, 300, 600, 1200 or 1800 mM). Of the bound fraction
collected from each of the 42 columns (21 total and 21 nonspecific), an
aliquot was counted to represent the "zero hour" binding for each buffer
condition. Each of the samples representing total binding was then
further subdivided into two tubes, one containing 20 nM [3H]TA (for the
determination of destabilization) and one containing 4 uM unlabeled TA
(for the determination of destabilization plus dissociation). Each of
the samples representing nonspecific binding was also subdivided into
two tubes, one containing 20 nM [3H]TA and 4 uM unlabeled TA and the
other containing only 4 uM unlabeled TA. After an 8 hour incubation at
0 C, aliquots of each of these experimental groups was again subjected
to bound/free separation on Sephadex G-25 columns. Rates of destabil
ization and dissociation (which rarely exceeded a few percent) were very
low for all groups, to the point that neither was likely to have been a
significant factor in any of the hydrophobic interaction chromatography
studies involving high concentrations of molybdate and/or KC1. It must
be emphasized, however, that these results cannot necessarily be applied
to the binding of this receptor to other ligands, such as DEX or
corticosterone, or to the binding of this receptor under different
temperature or pH conditions. However, since the conditions of this
experiment matched very closely the conditions of the hydrophobic
studies, any significant loss in binding seen during these studies is
likely to be the result of destabilizing effects of the hydrophobic
matrix instead of the buffer itself.
Since potassium chloride concentrations of up to 1200 mM with and
without molybdate were shown to have little effect on the stability or
rate of steroid (TA) dissociation of glucocorticoid receptors maintained


29
at low temperatures, and since retention of both activated and
unactivated receptors was shown to increase dramatically on alkyl
(particularly pentyl) agarose gels when ionic strength was lowered, the
effects of ionic strength and molybdate concentration on hydrophobic
interaction chromatography of both activated and unactivated receptors
was investigated in greater detail. Unactivated receptor preparation
was applied to pentyl agarose columns equilibrated in, and then eluted
with, one of the following: HEPES only, 10 mM molybdate, 50 mM
molybdate, 150 mM KC1 and 50 mM molybdate, 300 mM KC1 and 50 mM
molybdate, 600 mM KC1 and 50 mM molybdate, 900 mM KC1 and 50 mM
molybdate or 1,200 mM KC1 and 50 mM molybdate (Figure 2-3). Despite the
finding that high ionic strength had little effect on TA dissociation
from glucocorticoid receptors, the eluted fractions were subjected to
another bound/free separation step as an extra precaution. Pentyl
agarose was chosen as the gel to study the effects of ionic strength
since it was pentyl agarose that displayed total retention of both forms
of the receptor under low salt conditions. Since the effect of
molybdate (which had been present in all prior hydrophobic interaction
chromatography experiments in this study) was unknown, the concentration
of both molybdate and KC1 were varied in this experiment. Columns
eluted with HEPES only, 10 mM molybdate or 50 mM molybdate displayed
total retention by the column. The elution profiles of all the
KCl-containing buffers were very similar except that a very slight
decrease in retention was observed going from 150 to 300 to 600 mM KC1.
The differences between these three concentrations, though possibly
significant, were not very dramatic. Elution profiles resulting from
600, 900 and 1,200 mM KC1 showed virtually no changes. In a parallel


Figure 2-3. Hydrophobic interaction chromatography of unactivated and activated Type II glucocorticoid
receptor complexes on pentyl agarose columns equilibrated and eluted with increasing ionic strength and
molybdate concentrations. Brain cytosol was prepared, labeled and activated (solid triangles) or left
unactivated (solid circles) as described in Figure 2-1. Both groups were subsequently run on Sephadex G-25
columns equilibrated and eluted with HEPES buffer plus one of the following: no molybdate or KC1, 10 mM
molybdate, 50 mM molybdate, 50 mM molybdate and 150 mM KC1, 50 mM molybdate and 300 mM KC1, 50 mM molybdate
and 600 mM KC1, 50 mM molybdate and 900 mM KC1, 50 mM molybdate and 1200 mM KC1 prior to being run on
pentyl agarose columns equilibrated and eluted with the same buffer. Cytosol (0.5 ml) was applied to each
column and 10 fractions (0.5 ml) were collected. Binding is expressed as percent of the total counts ( >
13,700 cpm) applied to each column. Results presented here are representative of 3 independent
replications. 0 = unmodified agarose (control) run only with 50 mM molybdate and 600 mM KC1, all other
columns are pentyl agarose.


60
40
20
60
40
20
60
40
20
Fraction Number


32
experiment, an activated receptor preparation was run on identical
columns under indentical conditions. Unlike the unactivated form,
however, the activated form was also very strongly (but not completely)
retained when eluted with 150 mM KC1 and again (but to a slightly lesser
degree) when eluted with 300 mM KC1. Elution profiles with 600, 900 and
1,200 mM KC1 were all rather similar.
For the purpose of comparing the hydrophobic properties of the
different forms of the glucocorticoid receptor to those of other well
studied proteins, the next two experiments examined the hydrophobicity
of two purified proteins, bovine serum albumin (BSA) and immuno-gamma-
globulin (IgG). Each of the two proteins were first radiolabeled with
[14C]formaldehyde (Rice and Means, 1971) and then run on a Shaltiel
series using 600 mM KC1 and 50 mM molybdate. All other conditions were
identical to the previously described experiments for glucocorticoid
receptors being run on a Shaltiel series under these same salt
conditions. Like the unactivated glucocorticoid receptor, BSA exhibited
some slight retention by the propyl, butyl, pentyl, hexyl and phenyl
agaroses and was totally retained by the octyl, decyl and dodecyl gels
(Figure 2-4). Like the unactivated receptor, BSA exhibited some slight
retention by the propyl, butyl, pentyl, hexyl and phenyl agaroses and
was totally retained by the octyl, decyl and dodecyl gels. The chromat
ographic behavior of IgG was significantly different from that of BSA on
several of the gels in the series. Of particular interest were the
differences between the two proteins on pentyl, octyl, decyl and dodecyl
agaroses. The pentyl agarose exhibited a higher affinity for the IgG
than the BSA although both proteins did elute from the column. The
biggest difference occurred on the decyl agarose where the BSA was


Figure 2-4. Hydrophobic interaction chromatography of bovine serum albumen (solid circle) and
immuno-gamma-globulin (solid triangle) on a Shaltiel series equilibrated and eluted with HEPES buffer
containing 50 mM molybdate and 600 mM KC1. Both proteins were [14C]methylated (for detection) to low
specific activity with [14C]formaldehyde and then diluted into HEPES buffer plus 50 mM molybdate and 600 mM
KC1 prior to being run on each of the alkyl, phenyl and control agarose columns equilibrated and eluted
with the same buffer. Cytosol (0.5 ml) was applied to each columns and 10 fractions (0.5 ml) were
collected. Results are expressed as percent of the total counts ( > 20,000 cpm) applied to column. The
profiles presented here are representative of 2 independent replications. 0 = unmodified agarose
(control), 3 = propyl agarose, 4 = butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose,
10 = decyl agarose and 12 = dodecyl agarose.


60
40
20
60
40
20
60
40
20
Fraction Number


35
totally retained but the IgG eluted in a pattern similar to the propyl
and butyl agarose profiles. Also of interest was the unexpected finding
that IgG exhibited a greater affinity for hexyl agarose than for pentyl
agarose and a greater affinity for decyl agarose than for octyl agarose.
The effects of changing ionic strength on these profiles was not
determined.
In an effort to improve resolution of the chromatographic profiles
of the activated and unactivated forms of the glucocorticoid receptor as
well as to increase separation between the 2 forms, these preparations
were run individually on pentyl agarose columns much longer than those
used for the experiments involving the complete Shaltiel series (7 ml as
opposed to 1.5 ml). These preparations were run on columns equilibrated
and eluted with 600 mM KC1 and 50 mM molybdate and fractions of 0.5 ml
were collected so that fraction size was smaller relative to gel volume,
thereby increasing resolution further. As the results illustrate
(Figure 2-5), the unactivated receptors were represented by the sharper,
faster eluting peak, whereas the activated receptors were represented by
a broader, more slowly eluting peak. Although a much high resolution
was attained, the two different profiles didn't change much relative to
one another when compared to their relative shapes and positions when
run under identical buffer conditions on the smaller pentyl agarose
columns. In other words, the separation between the two peaks was not
significantly increased by increasing column length and there was still
a significant amount of overlap between them. In an attempt to increase
the separation between the two peaks, the experiment was repeated under
identical conditions except that the KC1 concentration was lowered from
600 mM to 400 mM since the affinity of activated receptors for pentyl


Figure 2-5. Hydrophobic interaction chromatography of activated and unactivated Type II glucocorticoid
receptors on long (7 ml) pentyl agarose columns. Brain cytosol was prepared in HEPES buffer plus 20 mM
molybdate and 2 mM DTT and incubated with 20 nM [3H]TA at 0 C for 24 hr. Aliquots were then either run on
Sephadex G-25 columns equilibrated and eluted with 600 mM KC1 and 50 mM molybdate (unactivated, solid
circles) or run first on Sephadex G-25 columns equilibrated and eluted with only HEPES buffer, incubated at
22 C for 24 min to activate the receptor complexes and then run on a Sephadex G-25 column equilibrated and
eluted with 600 mM KC1 and 50 mM molybdate (activated, solid triangles). Aliquots (0.5 ml) of each
treatment were run on pentyl agarose columns equilibrated and eluted with 600 mM KC1 and 50 mM molybdate
and 0.5 ml fractions were collected directly into scintillation vials for counting. Binding is expressed
as percent of the total counts applied to the column eluting with each fraction. Each condition was run in
triplicate and the profiles shown are representative.




38
agarose had previously been shown to increase to a greater extent than
the affinity of pentyl agarose for unactivated receptors when lowering
the KC1 concentration from 600 mM to 300 mM. The results were that
under the 400 mM KC1 elution conditions, the activated peak was broader
than with 600 mM KC1 elution while the unactivated receptor peak did not
change significantly (data not shown). Overlap of peaks was still
substantial.
To more closely investigate the relationship between column shape
and elution profile on hydrophobic interaction chromatography columns,
the next experiment examined the profiles for both the activated and
unactivated forms of the glucocorticoid receptor on two dramatically
different shaped pentyl agarose columns with the same gel volume.
Although two different sizes of columns had been used previously, the
shape difference (ratio of gel length to width) were not great and
differences in gel volume, relative sample size and relative fraction
size made comparisons of relative chromatographic resolution difficult.
In this experiment, the gel volume for both columns was 7 ml, but the
length to width ratio for one column was 18.6 (this column was identical
in dimensions to the 7 ml column described in the previous experiment)
while the length to width ratio for the second column, which was
significantly shorter and rounder, was 0.5, but, as previously
mentioned, contained an identical volumn of gel. There was therefore a
37.2-fold difference in the length to width ratio of the two columns.
The size of the sample applied to each column as well as the size of the
fractions collected from each column were identical and the elution
buffer contained 600 mM KC1 and 50 mM molybdate. The results of this
experiment (data not shown) indicate that there were no major


39
differences in the overall chromatographic profiles (position of peak
fraction, etc.) obtained from the two columns for either the activated
or unactivated forms of the receptor although there appeared to be a
very slight decrease in resolution (sharpness of peak) on the short wide
column for the unactivated receptor. This slight decrease in resolution
may have been related to technical problems in evenly applying such a
small sample volume to such a large gel surface.
Since most of the work to date characterizing the glucocorticoid
receptor has involved steroid labeled forms, little is known about the
physicochemical properties of the unoccupied glucocorticoid receptor.
Since hydrophobic interactions are thought to account for the high
affinity binding of glucocorticoids to their receptors (Wolff et al.,
1978; Alsen, 1983; Eliard and Rousseau, 1984), the empty hydrophobic
pocket exposed on a receptor whose binding site is unoccupied by a
steroid ligand may have an effect on the overall hydrophobicity of the
receptor that could have functional implications in vivo. This
experiment examined the hydrophobicity of the unoccupied glucocorticoid
receptor using a Shaltiel series of alkyl agarose and phenyl agarose
columns under experimental conditions identical to those previously
described except post-, instead of pre-, labeling was required (Figure
2-6) for analysis of activated and unactivated receptors when 600 mM KC1
and 50 mM molybdate was used. This allowed for direct comparison of the
results between all three forms of the receptor. Basically, the
unoccupied receptor behaved almost exactly like the unactivated occupied
receptor. Only slight differences between the two were detected on
profiles from the pentyl agarose columns. To determine if the slight
difference seen on this column was meaningful, unoccupied receptor


Figure 2-6. Hydrophobic interaction chromatography of unoccupied Type II glucocorticoid receptors and free
[3H]TA on a Shaltiel series equilibrated and eluted with HEPES buffer containing 50 mM molybdate and 600 mM
KC1. For unoccupied receptor runs (open circles), brain cytosol prepared in HEPES buffer plus 20 mM
molybdate and 2 mM DTT was run on Sephadex G-25 columns equilibrated and eluted with HEPES buffer plus 50
mM molybdate and 600 mM KC1 prior to being run on each of the alkyl, phenyl and control agarose columns
equilibrated and eluted with the same buffer. Cytosol (0.5 ml) was applied to each column and 10 fractions
(0.5 ml) were collected and postlabeled with 20 nM [3H]triamcinolone acetonide for 40 hr at 0 C. For free
steroid runs (open triangles), steroid was dried in a tube and resolubilized in HEPES buffer plus 50 mM
molybdate and 600 mM KC1 before 0.5 ml aliquots were applied to each column and fractions collected as
above. Binding is expressed as percent of the total counts ( > 14,050 cpm) applied to each column.
Results presented here are representative of 3 independent replications. 0 = unmodified agarose (control),
3 = propyl agarose, 4 = butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose, 10 = decyl
agarose and 12 = dodecyl agarose.


60
40
20
60
40
20
60
40
20
Fraction Number


42
preparation was run on the long (7 ml) pentyl agarose column previously
described for higher resolution analysis of the activated and
unactivated forms. The chromatographic profile obtained for the
unoccupied receptor turned out to be virtually identical to that
obtained for the unactivated steroid-labeled form (data not shown).
The possibility of using hydrophobic interaction chromatography as
a means of separating bound from free steroid for glucocorticoid
receptor binding assays was investigated. The results of this
experiment may also prove useful in interpreting data from experiments
where some degree of dissociation was known to occur on the hydrophobic
columns. This experiment examined the chromatographic profiles of free
[3H]TA on a Shaltiel series of columns equilibrated and eluted with 600
mM KC1 and 50 mM molybdate under conditions identical to those used for
the hydrophobic interaction chromatography of various forms of the
glucocorticoid receptor. Results indicate (Fig. 2-6) that the chromato
graphic profile of the free steroid on propyl, butyl, pentyl and hexyl
agarose was identical to that for the free steroid on unmodified
agarose. There was a slight increase in the affinity of [3H]TA for
octyl and decyl agarose and an even greater retention by dodecyl and
phenyl agarose. It should be noted that all of the steroid applied to
each of the columns was eluted by the seventh or eighth fraction. When
compared to the profiles for glucocorticoid receptors, the free steroid
profile overlaped these profiles in virtually every case. When free
steroid was run on a long 7 ml column identical to that previously
described, it eluted about midway between the peaks for the unactivated
and activated receptor and overlapped both, demonstrating that
hydrophobic interaction chromatography under the conditions used in


43
these experiments is less suitable for bound/free steroid separations
than chromatography on unmodified agarose.
Discussion
The results of this study indicate the presence of hydrophobic
regions on the different forms of the glucocorticoid receptor in brain.
This is in agreement with the findings of Lamb and Bullock (1983) for
progesterone receptors in uterus and Bruchovsky et al. (1981) for
androgen receptors in prostate. Even more important is the fact that
the degree of surface hydrophobicity of the glucocorticoid receptor
complex (assumed to be directly related to the degree of retention by
the hydrophobic gels) appears to be dramatically increased upon heat
activation of the receptor to the nuclear binding form. While such a
change in the surface hydrophobicity of glucocorticoid receptor
complexes associated with activation has been suggested by other workers
reporting an increase in the partitioning of these complexes after
activation into the less polar polyethylene glycol (PEG) layer in an
aqueous dextran-PEG two phase system (Andreasen, 1978 and 1982;
Andreasen and Mainwaring, 1980) and by this lab (Luttge et al., 1984d),
which reported a large increase in the fractional adsorption of
glucocorticoid receptor complexes to glass fiber (Whatman GF/C) filters
following heat activation, hydrophobic interaction chromatography has
never been used to study this phenomenon in glucocorticoid receptors.
Furthermore, although it is possible that the dramatic increase in
surface hydrophobicity associated with heat-induced activation could
represent an artefact of the nonphysiological conditions existing during
the in vitro activation process, this increased hydrophobicity is


nevertheless consistent with the presumed function of glucocorticoid
receptors in general. It is certainly possible that the increased
hydrophobicity of the activated receptor could account, at least in
part, for the increased affinity for a number of different nuclear
components including chromatin (Sakaue and Thompson, 1977; Simons et
al., 1976), nucleosomes (Climent et al., 1977), DNA (Baxter et al.,
1972; Rousseau et al., 1975; Sluyser, 1983), RNA (Tymoczko and Phillips,
1983), the nuclear matrix (Buttyan et al., 1983; Kirsch et al., 1986)
and the nuclear membrane (Smith and von Holt, 1981). If the unactivated
receptor resides in the cytosolic/cytoplasmic compartment, as originally
thought (Szego, 1974), activation-induced increases in hydrophobicity
might facilitate the permeation of the receptor through the nuclear
membrane. If, on the other hand, the current controversial theory
stating that unoccupied and occupied unactivated receptors actually
reside in the nucleus in vivo (for review see Walters, 1985) is true,
then increased hydrophobicity could decrease the likelyhood of "leakage"
of activated receptors from nuclei by virtue of their greater affinity
for nuclear components.
A Shaltiel series of alkyl agarose, as well as phenyl agarose,
columns were used since the ideal gel(s) to study the surface hydropho
bicity of the various receptor forms under different buffer conditions
was unpredictable initially. Although it has been shown that the
hydrophobicity of a linear aliphatic carbon increases linearly with
carbon chain length (Tanford, 1972), it has been argued by others
(Shanbhag and Axelsson, 1975), that the effective hydrophobicity depends
also on the flexibility of the hydrocarbon chain and consequently on the
degree of interaction within such chains, particularly for long chains.


45
The mechanisms by which proteins are adsorbed to hydrophobic matrices
are no doubt complex and this problem can be exemplified in the present
study by the case of the adsorption of IGG on a series of alkyl agarose
columns, in which case a decrease in adsorption between pentyl and hexyl
and between octyl and decyl agarose. This is similar to the case of the
adsorption of erthrocytes on a series of alkyl agarose derivatives,
wherein a decrease in adsorption between hexyl and octyl agarose was
noticed to occur without an obvious explanation. The present study
found that bovine serum albumen had a higher affinity for pentyl agarose
than did IGG, whereas the reverse was true for octyl, decyl and dodecyl
agaroses.
Another problem adding to the unpredictable nature of hydrophobic
interactions stems from the fact that some hydrophobic ligands denature
proteins through a "detergent-like" action (Hofstee, 1973). This was
evidenced by the fact that what initially appeared to be a partial
elution of both activated and unactivated receptors from octyl, decyl
and dodecyl agaroses, later proved to be dissociated free steroid
resulting from the denaturation of the receptors. The degree of
denaturation increased with alkyl chain length with dodecyl agarose
producing the greatest effect. In addition, the degree of denaturation
for activated and unactivated forms appeared to be roughly equivalent,
implying the possibility that the disruptive hydrophobic interaction
leading to loss of steroid binding involved a region or regions common
to both forms of the receptor. This finding may be relevant to the
interpretation of other studies involving hydrophobic interaction
chromatography. For instance, Lamb and Bullock (1983) reported that a
large volume of buffer was required for elution of the progesterone


46
receptor from decyl agarose, which they claimed was probably due to slow
equilibrium of the column with elution buffer. Another possibility is
that the progesterone receptors tightly bound to the decyl agarose may
have been undergoing a slow denaturation resulting in broad profiles of
eluted radioactivity representing free steroid only. When dissociation
was taken into account in the present study, no steroid receptor
complexes could be eluted intact from octyl decyl or dodecyl agarose
using any of a number of buffers including some with increased "hydro-
phobic character" such as glycerol or ethylene glycol. Thurow and
Geisen (1984) have reported that polypropylene glycol/polyethylene
glycol block polymers prevent both the adsorption of dissolved proteins
to hydrophobic interfaces and the resultant aggregation and denatur
ation. However, it is not known whether or not these polymers can
successfully elute the intact receptors from highly hydrophobic
interfaces if they are already attached and the presence of such
polymers prior to any hydrophobic interactions would reduce or eliminate
any such interactions, rendering the whole procedure useless.
Overall, the most promising results were obtained with the pentyl,
hexyl and phenyl agarose gels. Phenyl-agarose has reportedly been
effective in hydrophobic studies of the progesterone receptor (Logeat et
al., 1981; Lamb and Bullock, 1983) and in differentiating between the
activated and unactivated forms of the estrogen receptor (Gschwendt and
Kittstein, 1980). The activated form of the glucocorticoid receptor
bound more tightly to phenyl agarose than to alkyl agarose (excluding
octyl or longer alkyl agaroses). When calculated as the change in
standard free energy on transfer from an aqueous solution to a solution
of pure hydrocarbon, a phenyl group is less hydrophobic than a pentyl


47
group (Rosengren et al., 1975; Lamb and Bullock, 1983). A portion of
the unactivated receptor was retained by phenyl agarose under high salt
conditions, while the same receptor preparation was only slightly
retarded on the pentyl and hexyl agarose columns. While the activated
form of the receptor eluted much more slowly from the pentyl and hexyl
agaroses than did the unactivated form, the activated form was
completely retained on the phenyl agarose under low and high salt
conditions. Shaltiel (1974) has proposed that adsorption requires the
interaction of an alkyl residue of sufficient length with a hydrophobic
"pocket" within the protein. It has been suggested that the aromatic
ring on phenyl agarose may be more complementary than alkyl groups to
the contours of the hydrophobic "pocket on the progesterone receptor
(Lamb and Bullock, 1983). Because of similarities in amino acid
sequence between the two receptor types (Conneely et al., 1986), such a
pocket may also exist in or on the glucocorticoid receptor and may be
made more accessible for hydrophobic interactions by heat activation.
Alternatively, the receptor may form a charge transfer complex with the
phenyl group, thus increasing the interaction. One might therefore
conclude that phenyl agarose is the best gel for differentiating between
the activated and unactivated forms of the glucocorticoid receptor based
on differences in surface hydrophobicity. Unfortunately, the affinity
of the activated receptor for this particular matrix prevents removal of
the receptor from the matrix in an intact form. In addition, minor
changes in hydrophobic interactions between the activated receptor and
the matrix (i.e., as caused by changes in ionic strength, temp., etc.)
cannot be readily monitored by phenyl agarose and therefore alkyl
agaroses are best suited for such studies.


48
A number of factors in addition to the hydrophobicity of the
attached groups influence the adsorption of receptors and other proteins
to hydrophobic matrices. Jennissen (1976) has presented evidence in
favor of the idea that the adsorption of proteins to alkyl agarose
derivatives takes place at a critical alkyl group density. In other
words, this hypothesis considers that the protein needs to present
multiple attachment points in order to be adsorbed by a determinate
member of the series of alkyl agarose derivatives. Another factor
affecting the chromatographic behavior of a given protein on a
particular hydrophobic matrix is the overall protein concentration
applied to the column. Small amounts of protein bind more homogeneously
on a particular column than a large amount. This should not have been a
factor, however, in the differences in hydrophobicity exhibited by
activated and unactivated receptors in this study since great care was
taken to maintain the same protein concentration in all treatment groups
and throughout all experiments. On the other hand, this could very well
have been a factor in the chromatographic differences seen between the
unpurified activated receptor and the purified activated receptor since
there were hundreds- to thousands-fold differences in protein
concentration (discussed in more detail in a later section). Another
way in which protein concentration, especially in impure, crude
preparations such as cytosol, could potentially affect the chromato
graphic profile of glucocorticoid receptors on these columns is through
indirect, or secondary, interactions with other proteins bound tightly
to the columns.
The influence of salt on the retention of receptors and other
proteins to hydrophobic gels is probably due to a number of factors


49
acting on the protein as well as on the hydrophobic matrix. Pahlman et
al. (1977) concluded that salting-out ions, such as potassium chloride,
cause conformational, but not structural, changes in biomolecules. The
"structure forming" properties of salting-out ions enhance intra
molecular, as well as intermolecular, hydrophobic bonding as reflected
by a stabilization of the hydrophobic core of the biomolecule (Hofstee,
1975). High concentrations of salting-out ions can adversely affect the
solubility of a protein by decreasing the availability of water
molecules in the bulk and increasing the surface tension of water,
resulting in an inhancement of the hydrophobic interactions (Tanford,
1968; Melander and Horvath, 1977). It has been recently shown that the
free energy of a protein is increased by addition of salting-out ions
and this unfavorable free energy is smaller for the proteins bound to
columns because of their smaller surface area exposed to solvent
(Arakawa, 1986). The present study used, for the most part, a
combination of two salts: sodium molybdate and potassium chloride. The
50 mM concentration of molybdate was required to prevent activation and
to increase stability of all forms of the receptor under conditions
requiring high concentrations of potassium chloride since lower concen
trations of molybdate have been shown to be inadequate in preventing
activation under high salt conditions (Weatherill and Bell, 1985). It
was therefore not feasible to run meaningful experiments in the presence
of potassium chloride only, although the possibility exists that the
presence of molybdate may have influenced the effects that potassium
chloride would have had (aside from increasing activation) on protein
conformation and therefore, possibly, hydrophobic interactions. When
the effects of salt concentration on the chromatographic profiles of


50
both activated and unactivated receptor complexes on pentyl agarose were
examined, it was clear that all of the gels tested except phenyl agarose
exhibited a greater affinity for both forms of the receptor under low
salt conditions than under high salt conditions. Even the unmodified
agarose exhibited a slight affinity for the activated form of the
receptor under these conditions which may be due to low level
electrostatic or other interactions with the gel matrix itself (Janson,
1967). Since this was opposite of what was expected, a more detailed
study was carried out using only pentyl agarose and increasing
concentrations of both molybdate and potassium chloride. Elution of
activated receptors from the pentyl agarose required a higher
concentration of potassium chloride than was required to elute the
unactivated receptors. Although electrostatic interactions with the
matrix (the alkyl groups are supposedly uncharged after attachment to an
agarose matrix (Rosengren et al., 1975)) probably contributes to the
higher affinity at lower salt concentrations, the effects observed are
likely to involve a combination of electrostatic and hydrophobic
interactions since the activated receptor elutes from ion-exchange
columns at a lower salt concentration than the unactivated form (Sakaue
and Thompson, 1977). Once the electrostatic interactions are overcome
by using 600 mM concentrations of potassium chloride or greater, the
chromatographic differences between the two forms are probably purely
hydrophobic. It was reported by Peale et al. (1985) that the assayed
levels of charged estrogen receptors, using both Sephadex gel filtration
or dextran-coated charcoal treatments, in buffers containing 150-200 mM
ionic strength (roughly physiologic) was approximately one half that of
estrogen receptor levels assayed in buffers either at 0-50 or 400-450 mM


51
ionic strength. However, molybdate was shown to eliminate this
reduction.
Although Wolf et al. (1978) have shown that the high affinity
binding of a glucocorticoid by its receptor is largely due to hydro-
phobic interactions, the present study found little evidence that the
primary site of the hydrophobic interactions with alkyl and phenyl
agarose involved the steroid binding site. In fact, the chromatographic
behavior of the unoccupied receptor differed little from that of the
occupied unactivated receptor on practically all of the gels examined.
These findings are in agreement with those of Lamb and Bullock (1983)
who found that progesterone receptors bound to, and eluted from, alkyl
agarose equally well in both the free and complexed form. This, in
fact, might be expected in light of the high degree of specificity
normally associated with steroid-receptor interactions. It has been
implied, however, that steroid binding might be involved with some
hydrophobic interactions between the rat estrogen receptor and Cibacron
Blue (Tenenbaum and Leclerq, 1980).
In an attempt to improve the chromatographic resolution of the
activated and unactivated glucocorticoid receptors eluting from alkyl
agarose columns, pentyl agarose columns with various shapes (but with
the same gel volume) were compared. In addition, elution flow rates
were varied. None of these changes, however, resulted in any
significant improvement in chromatographic resolution providing further
support to the early observation by Frank and Evans (1945) that the
hydrophobic interaction is a spontaneous process.


CHAPTER III
SULFHYDRYL REGULATION OF GLUCOCORTICOID BINDING CAPACITY
Introduction
The concentration of glucocorticoid receptors is subject to
fluctuations resulting from several factors (De Nicola et al., 1982;
Svec, 1985a), including autoregulation by the ligands themselves
(Tornello et al., 1982; Ho-Kim et al., 1983; Muramatsu et al., 1983;
Sapolsky et al., 1984; Svec, 1985b; Luttge et al., unpublished). The
glucocorticoid-induced depletion of glucocorticoid receptors is
reportedly due to an increased degradation of receptors and not due to a
decreased rate of receptor synthesis (Svec and Rudis, 1981; McIntyre and
Samuels, 1985). Androgens, on the other hand, have been associated with
an augmentation of their receptors, apparently resulting from an
increase both in receptor half-life and in rates of receptor synthesis
(Syms et al., 1985). Glucocorticoid receptors may require an
energy-dependent up-regulation or transformation before they are able to
bind the steroid. Evidence for such an up-regulation in brain was
presented by Turner and McEwen (1980), who reported a lack of depletion
of hippocampal cytosol glucocorticoid receptors after multiple
injections of [3H]corticosterone. These results could not be explained
entirely by receptor synthesis and suggested that in the hippocampus an
"excess of glucocorticoid binding proteins exist, and that the
availability of functional cytosol receptors may be regulated to
maintain a relatively constant cellular level. Evidence of a rapid
52


53
down-regulation has also been observed. Recently, the loss of a
biological response associated with such a down-regulation of
glucocorticoid receptor levels was demonstrated in a mouse cell line
(Danielsen and Stallcup, 1984). In addition to circulating levels of
steroids, other factors may play a role in the regulation of steroid
receptor concentration. Of particular relevance to the role of
glucocorticoids in the nervous system is recent evidence that neural
input influences significantly the levels of both androgen (Bernard et
al., 1984) and glucocorticoid (DuBois and Almon, 1981) receptor levels
in striated muscle as determined from denervation studies. Similarly,
Cardinali et al. (1983) reported that pineal cytoplasmic and nuclear
estrogen and androgen receptors are modulated by norepinephrine released
from nerve endings at the pinealocyte level. These findings suggest that
target cells may utilize a variety of homeostatic mechanisms to regulate
the amount of receptor capable of interaction with the free steroid.
Little is known, however, about these regulatory mechanisms and even
less is known about the quantification, production and turnover of these
cryptic (downregulated) receptors. In addition, factors which
stabilize or destabilize unoccupied receptors under in vitro conditions
may also provide valuable information concerning the receptor and its
regulation.
One potential mechanism of receptor up- and down-regulation for
which there is fairly substantial evidence involves the reduction and
oxidation of sulfhydryl groups on the receptor. Oxidation of receptor
sulfhydryl groups (probably located in or near the steroid binding site)
has been shown to reversibly inactivate glucocorticoid receptors in a
number of tissues (Rees and Bell, 1975; Granberg and Ballard, 1977;


54
Sando et al., 1979; McBlain and Shyamala, 1980; Housley et al., 1982;
Harrison et al., 1983; Densmore et al., 1984d; Izawa et al., 1984).
These sulfhydryl groups are probably located in or near the steroid
binding site as evidenced by the fact that mesylated derivatives of
glucocorticoids have been shown to bind covalently to the receptor,
presumably by means of an interaction between the mesylate group and a
binding-site sulfhydryl group (Simons et al., 1980). The affinity
labeling of the estrogen receptor has similarly indicated the presence
of a thiol group at the binding site that appears to be directly
involved in estradiol-receptor binding (Ikeda, 1982). Addition of
sulfhydryl reducing agents such as dithiothreitol (DTT) or
2-mercaptoethanol leads to a total recovery of lost binding under
appropriate conditions. Further evidence that the sulfhydryl-disulfide
balance may be important in the regulation of glucocorticoid binding
capacity was presented recently when it was shown that endogenous
thioredoxin was responsible for up-regulation of glucocorticoid
receptors in liver cytosol (Grippo et al., 1983). In addition to
effects on binding capacity, oxidation of yet other sulfhydryl groups on
the activated glucocorticoid-receptor complex by various sulfhydryl
reactive agents has been shown to interfere with the DNA-binding ability
of receptors from thymus (Bodwell et al., 1984) and most recently
Kaufmann et al. (1986) has presented evidence suggesting that disulfide
bonds mediate the association between the activated glucocorticoid
receptor and the nuclear matrix, representing yet another possibility
for the regulation of glucocorticoid action. Much work in
this area remains to be done, however, before it can be determined
whether these mechanisms are likely to be of significance in vivo.


55
Despite the growing body of evidence that ligand binding to the
glucocorticoid receptor as well as binding of the activated gluco
corticoid-receptor complex to nuclei or DNA are dependent upon the
reduction of certain sulfhydryl groups on the receptor surface, there
has been surprizingly little speculation concerning the possibility of
thiol/disulfide exchange as a control mechanism for glucocorticoid
action in vivo. The specific modulation of enzyme activity by post-
translational modification of specific amino acid side chains is well
established as a mechanism of metabolic regulation. Essentially all the
amino acids possessing chemically reactive side groups are capable of
undergoing some sort of posttranslational modification: the carboxyl
groups of glutamate and aspartate, the imidazole side chain of
histidine, the hydroxyl groups of serine, threonine and tyrosine, the
amine group of lysine and even the guanidino group of arginine.
However, the sulfhydryl group of cysteine is potentially the most
reactive nucleophile in proteins. The process of thiol/disulfide
exchange could provide a mechanism for the equilibration of the
sulfhydryl oxidation state of proteins with the thiol/disulfide status
of the surrounding environment. Although the regulation of enzyme
activity by thiol/disulfide exchange was proposed some time ago (Barron,
1951), it has received less attention than other posttranslational
modifications. According to Gilbert (1984), thiol/disulfide exchange as
a biological control mechanism must exhibit at least the following six
properties:
1. The intracellular thiol/disulfide status should vary in vivo in
response to some metabolic signal. Isaacs and Binkley (1977a) reported
that the disulfide-sulfhydryl ratios of subcellular (nuclear, microsomal


56
and cytosolic) fractions of rat hepatic tissue varied diurnally with the
ratio lowest in the early morning (after feeding) and highest in the
early evening (after fasting). The primary reaction was the reversible
formation of mixed disulfides of glutathione with proteins and the
change in the overall disulfide-sulfhydryl ratio was approximately
4-fold. These same workers (Isaacs and Binkley, 1977b) also reported
changes in the disulfide-sulfhydryl ratio associated with cyclic AMP
levels. Intaperitoneal injections of dibutyryl cyclic AMP induced an
increase in hepatic glutathione protein mixed disulfides combined with a
corresponding decrease in reduced glutathione and protein sulfhydryl.
2. Oxidation of protein sulfhydryl groups by thiol/disulfide exchange
should activate some enzymes or receptors, inactivate others and not
affect the activity of still others. Gilbert (1984) has compiled a
large number of enzyme activities that have been suggested to be
modulated by reversible thiol/disulfide exchange. Like some steroid
receptors, many enzymes are reversibly inactivated by oxidation with a
number of biologically occurring disulfides. One example is
phosphofructokinase from either rabbit muscle (Gilbert, 1982) or mouse
liver (Binkley and Richardson, 1982). Fructose-1,6-biphosphatase,
however, undergoes a 2- to 4-fold activation in the presence of such
disulfides (Gilbert, 1984).
Many enzymes have sulfydryl groups that can be chemically modified
by a variety of sulfhydryl-specific reagents such as iodoacetamide,
N-ethylmaleimide or Ellman's reagent with a resultant effect on enzyme
activity. Most of these reagents are highly reactive toward sulfhydryl
groups and somewhat less specific as a result. In addition, these
reactive disulfides are much better oxidizing agents than most


57
biologically occurring disulfides. The enzymes aldolase and thiolase,
for example, both have sulfhydryl groups that react with iodoacetamide,
N-ethylmaleimide or reactive disulfides, however, incubation with lOmM
glutathione disulfide at pH 8.0 for 24 hr (very harsh oxidizing
conditions) had only a minimal effect on their activity (Gilbert, 1984).
A similar situation has been observed with steroid receptors. Tashima
et al. (1984) reported that monoiodoacetamide, N-ethylmaleimide and
5,5'-dithiobis(2-nitrobenzoate) all inactivated the binding capacity of
unoccupied mineralocorticoid ([3H]aldosterone) receptors, while the
binding capacity of these receptors was unaffected by endogenous
oxidizing factors that lead to the temperature-dependent, reversible
inactivation of glucocorticoid ([3H]dexamethasone) receptors under
identical in vitro conditions (Emadian et al., 1985). Therefore, the
presence of a chemically reactive sulfhydryl group is not in itself
sufficient evidence to suggest regulation of any enzyme or receptor by
thiol/disulfide exchange.
3. The thiol/disulfide redox potential of a regulated protein
should be near the observed thiol/disulfide ratio in vivo. If the
thiol/disulfide status of a regulated protein is in equilibrium with the
thiol/disulfide status of the cell, the equilibrium constant for the
thiol/disulfide exchange reaction
Ered + RSSR = Eox + RSH
Keq = [Eox][RSH]/[Ered][RSSR]
must lie near the intracellular thiol/disulfide ratio (Gilbert, 1984).
According to this equilibrium model, changes in the ratio of oxidized to
reduced receptor ([Eox]/[Ered]), and hence, the binding capacity, would
result from changes in the thiol/disulfide ratio ([RSH]/[RSSR]). Such a


58
relationship has been observed, although the literature concerning the
thiol/disulfide redox potentials of individual proteins is very limited.
Two enzymes that do equilibrate with glutathione/glutathione-disulfide
(GSH/GSSG) redox buffers in vitro are rabbit muscle phosphofructokinase
(Gilbert, 1982) and chicken liver fatty acid synthetase (Walters and
Gilbert, 1984). Unfortunately, there are no reports concerning redox
potential measurement in steroid receptor systems. However,
intracellular ratios of GSH/GSSG, the most predominant low molecular
weight thiol/disulfide pair, typically decrease during in vitro
procedures if precautions are not taken to "trap thiols and disulfides
at their original in vivo levels. This is because, at neutral pH, GSH
is oxidized in air to GSSG; and due to the large excess of GSH over
GSSG, postmortem oxidation of a small amount of the total GSH will
result in a large artifactual increase in the observed GSSG
concentration. One study reported an increase in GSSG concentration of
about 50% in 100 min in acid-soluble extracts of rat liver adjusted to
pH 7.4 (Vina et al., 1978). Such an increase would lead to changes in
the GSH/GSSG ratio that would still be within the range of change
observed to occur diurnally in rats (Isaacs and Binkley, 1977),
indicating that changes seen in receptor binding activity due to
postmortem oxidation of intracellular thiols might possibly reflect a
receptor thiol/disulfide redox potential reasonably near the
physiologically significant range. However, much work remains to be
done before such a question can be answered.
4. Thiol/disulfide exchange reactions must be kinetically competent
under physiological conditions. In order for the thiol/disulfide
exchange between protein sulfhydryl groups and biological disulfides to


59
be considered a viable regulatory mechanism, the rate of this process in
vivo must be at least as fast as the changes in the intracellular
thiol/disulfide ratio. With some exceptions, most enzymes thus far
studied would probably require catalysis of in vivo thiol/disulfide
exchange in order for the process to be considered a viable mechanism of
regulation (Gilbert, 1984). Since precise rates of thiol/disulfide
exchange for steroid receptors have not been determined under defined
conditions, it is currently unknown whether such a system would be
kinetically competent in the absence of catalysis. However, recent
reports have provided direct evidence for the existence of an endogenous
thioredoxin-mediated reducing system in rat liver cytosol that maintains
the glucocorticoid receptor in a reduced state in vitro in the presence
of NADPH (Grippo et al., 1983, 1985). As for kinetic competence of this
reaction, it should be noted that E. coli thioredoxin-(SH)2 reduces
bovine insulin at least 10,000 times more rapidly than dithiotreitol at
pH 7 (Holmgren, 1979). Endogenous mammalian thioredoxin activity has
probably been undetected in many glucocorticoid receptor studies because
of its easy inactivation by oxidation of structural SH groups (Luthman
and Holmgren, 1982). Therefore, the same in vitro conditions (oxidation
of reactive thiols) that inactivate unoccupied glucocorticoid receptors
also simultaneously inactivate the enzymes that catalyze rapid reduction
of receptor disulfides. The possible role of other enzymes that
catalyze the reverse reaction (disulfide formation) have not been
investigated with regards to steroid receptor systems. One such
possibility, protein disulfide-isomerase, has been shown to have a
rather broad substrate specificity, is widely distributed and has been
detected in most vertebrate tissues (Hillson et al., 1984).


60
5. For regulated proteins, the oxidized and reduced forms should
both be observable in vivo. The relative levels of the oxidized and
reduced forms must change in response to changes in the cellular
thiol/disulfide ratio. Although there is evidence that some enzymes,
such as phosphofructokinase, exist in vivo in both an oxidized and
reduced form, it is currently unknown whether both forms of the
glucocorticoid, or any other steroid, receptor exist in vivo. This is
for several reasons. First, in vitro preparation of cytosol for steroid
receptor binding studies is not an instantaneous process and oxidation,
as stated earlier, is most likely occurring if sulfhydryl group reducing
agents are not present. However, in the presence of such reducing
agents (such as dithiothreitol or 2-mercaptoethanol), the overall
thiol/disulfide ratio of the cytosol is being shifted in favor of thiol
formation. Even though nonenzymatic reduction of disulfides is
relatively slow, dithiothreitol and other exogenously added reductants
may reactivating or "up-regulating" enzymes such as thioredoxin that
rapidly catalyze dithiol-disulfide oxidoreductions (Holmgren, 1977,
1979). These problems are magnified by the fact that, like cytosol
preparation, the measurement of steroid binding capacity itself is very
time consuming. At saturating concentrations of DEX or TA, low
temperature incubations of 24 hours or longer are often required to
reach equilibrium (or near equilibrium) binding. Finally, ratios of
oxidized versus reduced receptors can not be assumed from receptor
binding studies alone. The possibility exists that the oxidation or
reduction of nonreceptor cytosolic components may indirectly modulate
receptor binding capacity via nonthiol/disulfide exchange mechanisms.


61
6. The response of particular enzyme or receptor activities to
oxidation by thiol/disulfide exchange should be consistent with the
metabolic function of the enzyme or receptor. If changes in the
thiol/disulfide ratio are coupled to changes in the activities of some
regulated enzymes and receptors, and if one assumes that such ratios are
based entirely on the feeding state of the animal (fasting leads to a
decrease in the ratio (Isaacs and Binkley, 1977a)), three general
consequences might follow (Gilbert, 1984). (1) Enzymes or receptors
that should be active only in the fed state should lose activity on
oxidation or gain activity on reduction. (2) Enzymes or receptors that
should be active only in the fasted state should either gain activity on
oxidation or lose activity on reduction. (3) Enzymes or receptors that
must function in both the fed and fasted state should either not undergo
thiol/disulfide exchange or the thiol/disulfide exchange should not
affect the enzyme activity. Although, as stated previously, it has been
reported that a decrease in the cellular thiol/disulfide ratio can be
induced by starvation or the administration of agents that increase the
intracellular concentration of cAMP, such as glucagon or epinephrine
(Isaacs and Binkley, 1977a,b), the effects of many other factors
(disease, stress, other hormonal systems) on cellular thiol/disulfide
ratios simply has not been investigated in great detail. Therefore,
while the relationship between some enzymes and changes in the cellular
thiol/disulfide ratio might appear to be consistent with the metabolic
function of the enzyme, the apparent absence or inconsistency of such a
relationship based merely on the feeding status of an animal should not
be taken as an absolute disqualification of redox control of a receptor
system whose functions are as vastly complex as that of the


62
glucocorticoid receptor. In addition, tissue differences in cellular
thiol/disulfide ratio, as well as levels of enzymes catalyzing
thiol/disulfide exchange, are known to exist. This may, in part,
explain the tissue differences reported in the dithiothreitol-inhibited
instability of unoccupied glucocorticoid receptors (Granberg and
Ballard, 1977), and may indicate tissue differences in the degree of
redox control.
Materials and Methods
Chemicals, Steroids and Isotopes
[6,7-3H]Triamcinolone acetonide, or 9a-fluoro-llb,16a,17a,21-tetra-
hydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide, ([3H]TA, specific
activity = 37 Ci/mmol) and [6,7-3H]dexamethasone, or 9a-fluoro-16a-
methylprednisolone ([3H]DEX, specific activity = 44.1-48.9 Ci/mmol) were
purchased from New England Nuclear (Boston, MA). Sephadex G-25 (fine)
and Dextran T70 were obtained from Pharmacia Fine Chemicals (Piscataway,
NJ). Dinitrobenzoic acid (DTNB), N-ethylmaleimide NEM), iodoacetamide,
iodoacetic acid, cystamine, glutathione disulfide (GSSG) and para-
chloro-mercurisulfonate (PCMS), sodium-m-periodate, sodium-p-periodate,
6,6'-dithionicotinic acid (DTNT), methyl methane thiosulfonate (MMTS),
mercuric chloride, sodium molybdate (Na2Mo04), calf thymus DNA-
cellulose, glycerol, sucrose, activated charcoal, PPO (2,5-diphenyl-
oxazole) and dimethyl POPOP (1,4-bis[2(4-methyl-5-phenyloxazoyl)]-
benzene) and pentyl agarose were all purchased from Sigma Chemical Co.
(St. Louis, MO). Dithiothreitol (DTT) and
4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid (HEPES) were
courtesy of Research Organics (Cleveland, OH). Scinti Verse II was


63
purchased from Fisher, Inc. (Fair Lawn, NJ). All other chemicals and
solvents were reagent grade.
Animals
All studies used female CD-I mice (Charles River Laboratories,
Wilmington, MA) that were subjected to combined ovariectomy and
adrenalectomy approximately 1 week prior to each experiment in order to
remove known sources of endogenous steroids. Both operations were
performed bilaterally via a lateral, subcostal approach under barbi
turate anesthesia, and mice were given 0.9% NaCl (w:v) in place of
drinking water. On the day of the experiment mice were anesthetized
with ether and perfused slowly through the heart with ice-cold
HEPES-buffered saline (20-30 ml, isotonic, pH 7.6).
Cytosol Preparation and Steroid Binding
Brains were removed from the perfused animals and homogenized (2x10
strokes at 1000 rpm) in 4 volumes of ice cold buffer containing 20 mM
HEPES and 20 mM Na2Mo04, pH 7.6 at 0 C) in a glass homogenizer with a
Teflon pestle milled to a clearance between the pestle and homogeniz
ation tube of 0.125 mm on the radius (to minimize rupture of the brain
cell nuclei (McEwen and Zigmond, 1972)). The crude homogenate was
centrifuged at 100,000 g for 20 min and the supernatant recentrifuged at
100,000 g for an additional 60 min to yield cytosol. During these
centrifuge runs, and during all other procedures, unless otherwise
indicated, careful attention was paid to maintaining the cytosol at 0-2
C. Final protein concentrations were typically in the 6-8 mg/ml range.
After the appropriate experimental manipulations, cytosol samples were
incubated with 20 nM [3H]TA or [3H]DEX for 24 to 40 hours at 0 C with or
without a 200-fold excess of unlabeled steroid.


64
Charcoal Pretreatment
The dextran-coated charcoal (DCC) mixture was prepared overnight by
adding 1.25% (w/v) activated charcoal and 0.625% dextran T70 to buffers
identical in composition to that in which cytosol was prepared. Just
prior to use, the desired volume of DCC suspension was centrifuged at
2,230 g for 10 min, the supernatant discarded and an equivalent volume
of cytosol added to the pellet. The DCC was resuspended by gentle
vortexing and the mixture incubated for 20 min at 0 C with 30 sec
intermittent vortexing every 5 min. At the end of the incubation
period, the dispersed DCC was pelletted by centrifugation at 2,230 g for
10 min and the resulting supernatant (i.e. the DCC-pretreated cytosol)
carefully aspirated for subsequent steps. Control groups underwent
identical procedural steps (in the absence of DCC) in parallel with
their DCC-treated counterparts. The thermal stability of unoccupied
receptors in each of these groups was tested by incubating the cytosol
in a water bath maintained at the desired temperature for the period of
time designated for each experiment. Following this "aging, the cytosol
preparations were returned immediately to an ice bath. The protein
concentration in such preparations ranged from 3.1 to 3.9 mg/ml (Lowry
et al., 1951).
Hydrophobic Interaction Chromatography
Unlabeled cytosol was either up- or down-regulated by an
appropriate incubation in the presence or absence of DTT, respectively
(described in greater detail later). The different cytosolic
preparations were then run on Sephadex G-25 columns equilibrated and
eluted with HEPES buffer containing 600 mM KC1 and 50 mM molybdate at pH
7.6. Of the macromolecular fraction collected from these columns, 0.5 ml


65
was then run on a 7 ml pentyl agarose column equilibrated and eluted
with HEPES buffer containing 600 mM KC1 and 50 mM molybdate at pH 7.6.
Fractions (0.5 ml) were collected and incubated with 20 nM [3H]DEX +/- 4
uM [1H]DEX for 40 hr at 0 C. The fractions were then assayed for
specific binding.
Sucrose Density Gradient Sedimentation
Unlabeled cytosol was either up- or down-regulated by an
appropriate incubation in the presence or absence of DTT, respectively.
Aliquots (400 ul) were layered onto linear 5-20% sucrose density
gradients (5 ml; prepared with HEPES buffer containing 20 mM molybdate
and either with (for up-regulated receptors) or without (for down-
regulated receptors) 2 mM DTT and centrifuged at 0 C for 19-20 hr at
200-234,000 g (average) in a Beckman SW 50.1 rotor. The cellulose
nitrate tubes were punctured and 28-30 fractions (180 ul) collected and
incubated with 20 nM [3H]DEX +/- 4 uM [1H]DEX in the presence of 2 mM
DTT at 0 C for 24 hr. The individual fractions were then assayed for
specific binding. Sedimentation coefficients (S20,w) were calculated
from the linear regression of S20,w vs sedimentation distance (Martin
and Ames, 1961) for the following standard proteins run in parrallel
tubes: chicken ovalbumin (0VALB, 3.6 S), bovine serum albumin (BSA, 4.3
S), bovine gamma globulin (IgG, 7.4 S) and catalase (CAT, 11.3 S). The
standard proteins were [14C]methylated (for detection) to low specific
activity with [14C]formaldehyde by the method of Rice and Means (1971).
Steroid Binding Determination
In all experiments, bound [3H]-steroid was separated from free on
Sephadex G-25 columns (0.6 x 14 cm) pre-equilibrated in buffer identical
to that in which the cytosol was prepared. Duplicate aliquots from each


66
assay tube (BT and BNS) were layered onto separate columns, allowed to
penetrate the gel and the macromolecular (bound) fraction eluted (with
homologous buffer) directly into scintillation vials for liquid
scintillation spectrometry.
Results
The purpose of this investigation was to determine the role of
sulfhydryl oxidation-reduction in the regulation of steroid binding
capacity of the unoccupied glucocorticoid receptor. It was therefore of
importance to first characterize the time- and temperature-dependent
changes in binding capacity occurring in cytosol in the absence of
exogenously-added sulfhydryl reducing or oxidizing reagents. The first
experiment investigated the temperature-dependent reversible loss of
glucocorticoid binding capacity in the presence of molybdate, but in the
absence of sulfhydryl reducing reagents such as DTT. Unlabeled cytosol
was preincubated or "aged" at 22 C for 0, 2 and 4 hr, followed by
addition of buffer with or without DTT prior to the steroid incubation.
The experiment also examined the effects of aging of cytosol after the
addition of DTT. Results (Figure 3-1) indicate that the binding
capacity lost at 22 C in the presence of molybdate is completely
restorable upon addition of DTT. Aging of the unlabeled cytosol after
DTT addition had little effect on the binding capacity. Under these
conditions, half of the maximal binding capacity (that measured after
DTT addition) was reversibly inactivated after less than 4 hr of 22 C
aging, although approximately 25% of the maximal binding capacity
appeared to have already been lost prior to any 22 C aging whatsoever.
The next experiment was designed to more carefully investigate the
phenomenon studied in the previous experiment, except the period of


Figure 3-1. Temperature-dependent reversible loss of glucocorticoid
binding capacity of unoccupied Type II glucocorticoid receptors in the
presence of molybdate and absence of DTT. Solid circles represent brain
cytosol prepared in HEPES buffer containing 20 mM molybdate that has
been incubated at 22 C in the absence of steroid prior to a 24 hr
incubation with 20 nM [3H]DEX +/- 4 uM [1H]DEX at 0 C. Solid squares
represent the addition of DTT (2 mM final concentration) to the aged
cytosol just prior to the initiation of steroid incubations. Open
symbols represent one additional hr of 22 C aging after the addition of
HEPES buffer containing either 20 mM molybdate only (open circle) or 20
mM molybdate plus 20 mM DTT (open squares, 2 mM final DTT
concentration), but prior to steroid incubations. Bound-free steroid
separations were performed on Sephadex G-25 columns. Specific binding
is expressed as percent of the 0 hr control group (250 fmole/mg cytosol
protein).


T
T
T
T


69
preincubation aging was greatly extended to determine if the reversible
down-regulation of glucocorticoid binding capacity could reach 100%
under in vitro conditions. This experiment also sought to determine if
unlabeled glucocorticoid receptors in the sulfhydryl down-regulated
state were more or less stable for extended periods at 22 C than
up-regulated unlabeled receptors. Like the previous experiment,
unlabeled cytosol prepared with 20 mM molybdate was subjected to a
preincubation aging at 22 C, but this time for 0, 2, 5, 9, 21 and 30 hr
(Figure 3-2). In addition to the nonDTT-containing groups that received
HEPES buffer with or without DTT after the 22 C aging, another group was
included that contained DTT throughout the aging step. As in the
previous experiment, suboptimal binding was encountered even prior to 22
C aging when DTT was absent from the cytosol. Down regulation appeared
to reach virtual completion at 22 C, but preincubation on the order of
30 hr was required. Maximum binding capacity (i.e., after addition of
DTT) diminished only slightly (about 30%) during the 30 hr period.
There were clearly no significant differences in binding between the
groups provided with DTT after the preincubation and those provided with
DTT prior to the preincubation.
In an attempt to further characterize the endogenous factor(s)
involved in the reversible in vitro down-regulation of unoccupied
glucocorticoid receptors, cytosol preparations were subjected to a
dextran-coated charcoal (DCC) pretreatment with or without DTT addition
(before and/or after DCC). The effect of temperature on this
DTT-reversible binding loss was also examined under each of the possible
conditions by aging half of the cytosol from each group for an
additional 4 hr at 22 C prior to incubation with labeled steroid.


Figure 3-2. Temperature-dependent reversible loss of glucocorticoid binding capacity of unoccupied Type II
glucocorticoid receptors in the presence of molybdate and absence of DTT after extended periods of
incubation at 22 C. Open bars represent brain cytosol prepared in HEPES buffer containing 20 mM molybdate
and incubated at 22 C in the absence of steroid prior to a 24 hr incubation with 20 nM [3H]DEX +/- 4 uM
[1H]DEX at 0 C. Hatched bars represent the addition of DTT (2 mM final concentration) to the preincubated
cytosol just prior to the initiation of steroid incubations. Solid bars represent the addition of 2 mM DTT
prior to the incubation at 22 C in the absence of steroid followed by the steroid incubations. Bound-free
steroid separations were performed on Sephadex G-25 columns. Specific binding is expressed as percent of
the 0 hr control group (310 fmole/mg cytosol protein) and represents the mean +/- S.E.M. of 3 independent
replications.


100
75
50
25
5 9
Duration of 22C Aging (hours)
0
2
21
30


72
Finally, liver and kidney cytosol preparations were treated in an
identical manner for comparative purposes since these tissues reportedly
have a higher endogenous reducing potential (Granberg and Ballard,
1977). For brain, the DCC pretreatment in the absence of DTT additions
or subsequent 22 C aging led to a significant decrease (approximately
40%) in the binding capacity for [3H]DEX (Figure 3-3). There was also
an increase in the rate of loss of binding capacity at 22 C. When DTT
was added back to cytosol after DCC pretreatment there was virtually a
100% recovery of binding capacity seen in cytosol treated with DTT but
not DCC. When DTT was added to cytosol prior to DCC treatment, there
was still a loss of binding capacity to a level similar to that seen if
no DTT was present before DCC. However, there was no significant
additional loss of binding capacity after a subsequent 22 C aging as was
the case for the DTT-, then DCC-treated group. Interestingly, when DTT
was added both before and after the DCC pretreatment, there was only a
partial restoration of the total binding capacity although there was
again very little loss of binding capacity associated with aging.
Results for liver and kidney were clearly different from those obtained
for brain and surprizingly different from what was expected based on the
findings of other workers regarding the endogenous reducing potential
for these tissues. The degree of loss of binding capacity seen in
groups not pretreated with DCC, DTT or 22 C aging was greater in liver
and much greater in kidney than that observed for brain. In addition,
the rate of loss of binding capacity for these same groups during 22 C
aging was greatly enhanced for both tissues when compared to brain.
However, similar to brain cytosol, DCC pretreatment led to a significant
decrease in binding capacity that was fully reversible upon addition of


Figure 3-3. Effects of dextran-coated charcoal pretreatment and DTT on
glucocorticoid binding capacity and thermal stability of unoccupied Type
II glucocorticoid receptors from brain, liver and kidney. Concentrated
cytosol was prepared in HEPES buffer plus 20 mM molybdate and then
diluted with either additional molybdate-containing buffer or molybdate-
containing buffer plus 20 mM DTT (2 mM final concentration) prior to a
20 min incubation in the presence or absence of dextran- coated charcoal
at 0 C. Cytosols were then further diluted with either additional
molybdate-containing buffer or molybdate-containing buffer plus 20 mM
DTT (2 mM final concentration) and then incubated at 22 C for either 0
(solid bars) or 4 (hatched bars) hr in the absence of steroid prior to
incubation with 20 nM [3H]DEX +/- 4 uM [1H]DEX at 0 C for 24 hr.
Bound-free steroid separations were performed on Sephadex G-25 columns.
Specific binding is expressed as percent of the 0 hr control groups
(300, 281, and 341 fmole/mg protein for brain, liver and kidney
cytosols, respectively) and represents the mean + S.E.M. of 5
independent replications and 3 independent replications for liver and
kidney.


120
100
80
60
40
20
100
80
60
40
20
00
80
60
40
20
DT
DC
74
BRAIN
0-HR AGING
4-HR AGING
LIVER
KIDNEY
- + + +
+ + + -f
+ - +


75
DTT. Unlike brain cytosol, however, both liver and kidney cytosols
exhibited a significant degree of protection from DCC-induced binding
loss when pretreated with DTT. However, DTT pretreatment preceding DCC
pretreatment of liver and kidney cytosol appeared to be less effective
in preventing further binding loss during 22 C aging than was found to
be the case for brain cytosol treated with DTT before and after DCC
pretreatment was significantly higher for liver and kidney than it was
for brain.
Since the chemical nature of the sulfhydryl up- and down-regulation
process was unknown, an investigation was made to determine if the
process involved any major, easily measurable changes in the molecular
structure of the receptor (i.e. receptor subunit association or
dissociation or major conformational changes). The aim of this
experiment was to compare the sedimentation properties of up- and
down-regulated unoccupied glucocorticoid receptors from brain using
sucrose density gradient analysis. Cytosol was up-regulated by
incubating with DTT at 0 C prior to the gradient run on sucrose
gradients prepared and DTT. Cytosol was down-regulated by incubating
without DTT at 22 C for 4 hr prior to the gradient run on sucrose
gradients prepared without DTT. All cytosol groups and sucrose
gradients contained molybdate. After an 18 hr gradient run, gradients
of both treatment groups were subjected to fractionation followed by
postlabeling of the individual fractions with [3H]DEX. Steroid
incubation conditions were identical for both groups except that DTT had
to be added back to each fraction of the down-regulated group to allow
for up-regulation so that binding could be determined. The profiles
representing the specific binding determined for each fraction indicated


76
that both forms sediment in one major peak of approximately 9.2 S
(Figure 3-4). The results therefore provide little evidence for any
major conformational changes in the receptor associated with sulfhydryl
regulation of binding capacity under these conditions.
To determine if the changes in glucocorticoid binding capacity
associated with sulfhydryl down-regulation involve merely a decrease in
the maximal binding or, instead, a modification of the receptor binding
affinity, Scatchard analysis of DEX binding was performed for cytosol
either containing or not containing DTT (Figure 3-5). The addition of
DTT led to an apparent increase in both the affinity and maximal
binding. Equilibrium binding constants were also determined after 4 hr
of 22 C aging of the cytosol with and without DTT prior to steroid
incubation. This aging only had a significant effect on the parameters
determined for non-DTT-containing cytosol, leading to a further decrease
in both maximal binding and apparent affinity.
If the apparent reduction in affinity associated with sulfhydryl
down-regulation as determined by Scatchard analysis is real, it is
likely to be due to either a decrease in the rate of steroid
association, an increase in the rate of steroid dissociation or both.
Each of these kinetic parameters were investigated. To study the effect
of reducing reagents on the rate of steroid dissociation from the
glucocorticoid receptor, cytosol was first prelabeled with [3H]DEX in
the presence of molybdate and DTT prior to being run on a Sephadex G-25
column equilibrated in non-DTT-containing buffer to remove both the free
steroid and the DTT. DTT was then added back to half of the preparation
while the other half was appropriately diluted with non-DTT-containing
buffer. As illustrated in Figure 3-6, there was no significant


Figure 3-4. Sucrose density gradient analysis of sulfhydryl-reduced and sulfhydryl-oxidized unoccupied
Type II glucocorticoid receptors. Brain cytosol prepared in the absence of DTT (HEPES buffer plus 20 mM
molybdate) was either incubated in the absence of steroid for 4 hr at 22 C (solid squares) or supplemented
with DTT (solid triangles, 2 mM final concentration) and stored at 0 C for 4 hr. Aliquots (400 ul) from
both cytosol samples were then sedimented through 5-20% linear sucrose gradients in a SW 50.1 rotor at
200,000 x g for 18 hr. Samples containing DTT (solid triangles) were run on gradients prepared with HEPES
buffer plus 20 mM molybdate and 2 mM DTT, whereas samples not containing DTT (solid squares) were run on
gradients prepared with HEPES buffer containing only 20 mM molybdate. Fractions were collected directly
into ice-cold tubes containing either [3H]DEX in HEPES buffer plus 20 mM molybdate and 2 mM DTT (solid
triangles) or [3HJDEX plus HEPES buffer plus 20 mM molybdate and 20 mM DTT (solid squares, 2 mM DTT final
concentration). The individual fractions were then incubated for 24 hr at 0 C prior to bound-free steroid
separations on LH-20 columns. Results presented here represent the mean of 2 independent replications.


PERCENT OF THE TOTAL BSP


Figure 3-5. Scatchard analysis of [3H]DEX binding to Type II
glucocorticoid receptors. Brain cytosol was prepared in HEPES buffer
containing 20 mM molybdate plus (circles) or minus (triangles) 2 mM DTT
and with (solid symbols) or without (open symbols) a 4 hr incubation at
22 C in the absence of steroid prior to incubation with [3H]DEX +/-
200-fold [1H]DEX at 0 C for 24 hr. Bound-free steroid separations were
performed on Sephadex G-25 columns.


Bsp / F
80
0-4
02-

+ DTT
4 HRS
AT
22C
o
+ DTT
0 HRS
AT
oc
A
DTT
4 HRS
AT
22 C
A
-DTT
0 HRS
AT
o
0
O
06 08 1-0
Bsp (nM)


Figure 3-6. Effect of DTT on the rate of dissociation at 22 C of [3H]DEX from Type II glucocorticoid
receptors. Brain cytosol prepared in HEPES buffer plus 20 mM molybdate and 2 mM DTT was incubated with 40
nM [3H]DEX for 50 hr at 0 C. Labeled cytosol was then run on a jacketed Sephadex G-25 column (1 x 50 cm)
at -4 C to remove both the free steroid and the DTT. Cytosol was then incubated in the presence of 10 uM
[1H]DEX at 22 C either with (circles) or without (squares) 2 mM DTT. Aliquots were removed periodically
for bound-free steroid separations on Sephadex LH-20 columns.


HOURS
PERCENT OF ZERO HR CONTROL
Z8
IOO


83
differences in the rate of dissociation between receptors in the
presence or absence of DTT. Although an attempt was made to study the
effect of DTT on the rate of association of [3H]DEX to glucocorticoid
receptors, the results of this experiment were confounded by the fact
that the binding capacity of cytosol lacking DTT was undergoing a
time-dependent loss that did not occur in the presence of DTT.
A series of experiments were next performed to further characterize
the sulfhydryl up and down-regulation of glucocorticoid binding capacity
by studying the reactivity of the essential sulfhydryl group(s) with a
variety of known reversible and irreversible sulfhydryl-reactive agents.
The first experiment examined the effects of a 1 hr incubation of
unlabeled cytosol with 1 mM dithionitrobenzoic acid (DTNB),
n-ethylmaleimide (NEM), iodoacetamide, cystamine, glutathione disulfide
(GSSG) and para-chloromercurisulfonate (PCMS) with or without the

subsequent addition of 10 mM DTT on glucocorticoid binding capacity as
measured by [3HJDEX binding (Figure 3-7). DTNB and cystamine led to a
virtual total loss in binding capacity which was completely reversible
upon DTT addition. GSSG resulted in only a partial loss in binding
capacity, but this loss was also completely reversible upon DTT
addition. NEM, iodoacetamide and PCMS led to varying degrees of loss
which were not completely reversible with DTT. In a follow-up
experiment, conditions identical to those described in the previous
experiment were used to investigate the effects on glucocorticoid
binding capacity of several additional sulfhydryl reactive reagents
including iodoacetic acid, sodium-m-periodate, sodium-p-periodate,
6,6'-dithionicotinic acid (DTNT), methyl methane thiosulfonate (MMTS)
and mercuric chloride (Figure 3-8). Iodoacetic acid was relatively


Figure 3-7. Effects of O C incubation of cytosol with various known reversible and irreversible
sulfhydryl-reactive reagents on Type II glucocorticoid receptor binding capacity with and without
subsequent addition of excess DTT. Brain cytosol prepared in HEPES buffer plus 20 mM molybdate was
incubated at 0 C for 1 hr with either additional HEPES buffer dilution (BUFF) or a 1 mM final concentration
of dithionitro- benzoic acid (DTNB), n-ethyl maleimide (NEM), iodoacetamide (IAM), cystamine (CYS),
glutathione dissulfide (GSSG) and para-chloro- mercurisulfonate (PCMS). Cytosol samples were then
supplemented with (solid bars) or without (open bars) and excess of DTT (10 mM final concentration) prior
to incubation with 20 nM [3H]DEX +/- 4 um [1H]DEX at 0 C for 40 hr. Bound-free steroid separations were
performed on Sephadex G-25 columns. Specific binding is expressed as percent of the BUFF control group
after addition of DTT (317 fmol/mg protein) and represents the mean +/- S.E.M. of 3 independent
replications.


100
75
50
25
BUFF DTNB CYS GSSG I AM NEM PCMS
00
i_n


Figure 3-8. Effects of O C incubation of cytosol with additional known reversible and irreversible
sulfhydryl-reactive reagents on Type II glucocorticoid receptor binding capacity with and without
subsequent addition of excess DTT. Brain cytosol prepared in HEPES buffer plus 20 mM molybdate was
incubated at 0 C for 1 hr with either additional HEPES buffer (BUFF) or a 1 mM final concentration of
6,6'-dithionicotinic acid (DTDN), methyl methane thiosulfonate (MMTS), sodium-m-periodate (SMP),
sodium-p-periodate (SPP), iodoacetic acid (IAA) or mercuric chloride (HgC12). Cytosol samples were then
supplemented with (solid bars) or without (open bars) an excess of DTT (10 mM final concentration) prior to
incubation with 20 nM [3H]DEX +/- 4 um [1H]DEX at 0 C for 40 hr. Bound-free steroid separations were
performed on Sephadex G-25 minicolumns. Specfic binding is expressed as percent of the BUFF control group
after addition of DTT ( 347 fmol/mg protein) and represents the mean +/- S.E.M. of 3 independent
replications.


Percent of Control
BUFF DTDN MMTS SMP SPP IAA HgCI2


88
ineffective in reducing the binding capacity, possibly due to the pH of
the system or the low concentration used, but it did produce some
irreversible losses. There was no difference between the two isomers of
sodium periodate which were intermediate in reducing binding capacity
which was not completely DTT-reversible. DTNT resulted in nearly a
total loss of binding capacity which was completely reversible while
MMTS was much less effective in reducing binding capacity. However,
total reversibility with DTT in the case of MMTS was less certain than
for DTNT. Finally, exposure to mercuric chloride resulted in a complete
loss of binding capacity which was partially reversible.
The next step was to investigate the ability of DTT-reversible
sulfhydryl reactive reagents to protect unoccupied receptors from
irreversible inactivation due to non-DTT-reversible sulfhydryl reactive
reagents. This experiment was designed to determine if unoccupied
receptors, down-regulated by endogenous sulfhydryl oxidizing factors,
were more or less protected from irreversible sulfhydryl reactive
reagents than were receptors down-regulated by the DTT-reversible
reagents used in the previous experiments. This was an effort to
possibly shed light on the nature of the down-regulated form in vivo as
well as to provide information on a possible means of "trapping" up-
and/or down-regulated forms of the receptor so that the true status of
up- and down-regulated receptors in vivo may someday be determined.
This experiment involved first, a 1 hr incubation of unlabeled cytosol
at 0 C with DTNB, cystamine, GSSG or no reagent for the DTT-reversible
down-regulation of the unoccupied receptors. This was followed by an
additional 1 hr incubation of each of the above groups with either NEM,
iodoacetamide, mercuric chloride or no reagent. This second incubation


89
with DTT-irreversible sulfhydryl reactive reagents was followed by
further dilution with or without DTT prior to [3H]DEX binding. Binding
data obtained for all 32 possible groups are illustrated in Figure 3-9.
DTNB and cystamine provided almost complete protection against
irreversible losses caused by either NEM or iodoacetamide. GSSG, on the
other hand, provided only a slight protection against the NEM- and
iodoacetamide-induced losses in binding capacity. Interestingly, none
of the reversible reagents provided any significant degree of protection
against the irreversible effects of mercuric chloride when compared to
the control that subsequently received mercuric chloride. These later
findings imply that either mercuric chloride is ineffective against the
endogenous down-regulated form, while being effective against the forms
down-regulated via reaction with DTNB, cystamine or GSSG, or that
mercuric chloride's effects are relatively nonspecific against both up-
and down-regulated receptors.
If one makes the assumption that mercuric chloride is acting
specifically on the sulfhydryl up-regulated form of the receptor, a
possibility implied by the results of the previous experiment, then one
would expect the incubation of cytosol containing quantitatively known
populations of both up- and down-regulated glucocorticoid receptors with
mercuric chloride, followed by addition of excess DTT (to neutralize the
reaction) at various time intervals, to result in a decreasing binding
capacity that approaches asymptotically the level of down-regulated
receptors. The results, shown in Figure 3-10, basically confirm this.
In the presence of 2 mM mercuric chloride, the binding capacity was
reduced by around 35% of maximum, a level approximating that of the
down-regulated receptors determined under similar conditions.


Figure 3-9 The ability of DTT-reversible sulfhydryl reagents to
protect unoccupied Type II glucocorticoid receptors from irreversible
inactivation due to non DTT-reversible sulfhydryl-reactive reagents.
Brain cytosol prepared with HEPES buffer plus 25 mM molybdate was
incubated at 0 C with either additional HEPES buffer (BUFF) or a 1 mM
final concentration of dithionitrobenzoic acid (DTNB), cystamine (CYS)
or glutathione dissulfide (GSSG). Cytosol samples were then incubated
at 0 C for a second hr with either additional HEPES buffer (BUFF) or a 2
mM final concentration of n-ethyl maleimide (NEM), iodoacetamide (IAM)
or mercuric chloride (HgC12). Each subset of cytosol was then incubated
at 0 C for an additional 1 hr prior to the addition of either HEPES
buffer (open bars) or HEPES buffer containing excess DTT (20 mM final
concentration, solid bars) followed by incubation at 0 C for 40 hr with
20 nM [3HJDEX +/- 4 um [1H]DEX. Bound-free steroid separations were
performed on Sephadex G-25 columns. Specific binding is expressed as
percent of the BUFF-BUFF plus DTT control group (302 fmol/mg protein)
and represents the mean +/- S.E.M. of 3 independent replications.


Percent of Control Percent of Control
91
BUFF BUFF BUFF BUFF DTNB DTNB DTNB DTNB
BUFF NEM 1AM HgCI2 BUFF NEM 1AM HgCI2
CYS CYS CYS CYS GSSG GSSG GSSG GSSG
BUFF NEM I AM HgCI2 BUFF NEM I AM HgCI2


Figure 3-10. Time course of the effects of 1 mM mercuric chloride on Type II glucocorticoid receptor
binding capacity in the absence of steroid and with the subsequent addition of DTT. Brain cytosol prepared
with HEPES buffer plus 20 mM molybdate was either brought to a final concentration of 1 mM mercuric
chloride (triangles) or was diluted with an appropriate volume of HEPES buffer (circles). Cytosols were
then incubated at 0 C for the times indicated prior to addition of excess DTT (10 mM final concentration)
followed by incubation at 0 C for 40 hr with 20 nM [3H]DEX +/- 4 urn [1H]DEX. Bound-free steroid
separations were performed on Sephadex G-25 columns. Specific binding is expressed as the percent of the 0
hr control (no mercuric chloride) (297 fmol/mg protein) and represents the mean +/- S.E.M. of 3 independent
replications.


Percent of Control
Duration of 0C Aging (Hours)


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MECHANISMS OF GLUCOCORTICOID TYPE II
RECEPTOR REGULATION AND ACTIVATION
IN BRAIN
By
CHARLES L. DENSMORE
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1987

ACKNOWLEDGMENTS
I greatfully acknowledge the interest, support and patience of my
supervisory committee including Drs. Kimon Angelides, Steven Childers,
Robert Cohen, and Kathleen Shiverick. I am especially thankful for the
never-tiring, unselfish support of my advisor and friend, Dr. William G.
Luttge.
I am particularly indebted to Seyed M. Emadian and Yun-Chia (Jenny)
Chou who have helped me in more ways than they realize.
I would also like to thank Mary Rupp for always seeing to it that
my experiments ran smoothly and Kimmy Bloom for the painstaking artwork.
Finally, this work would never have been possible without the
constant love and support of my wife Anita.
ii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
ABSTRACT v
CHAPTERS
IGENERAL INTRODUCTION 1
IIHYDROPHOBIC INTERACTION CHROMATOGRAPHY
OF VARIOUS FORMS OF THE OCCUPIED
AND UNOCCUPIED GLUCOCORTICOID RECEPTOR 13
Introduction . 13
Materials and Methods 15
Results..' 19
Discussion 43
IIISULFHYDRYL REGULATION OF GLUCOCORTICOID
BINDING CAPACITY 52
Introduction 52
Materials and Methods 62
Results 66
Discussion 108
IVTHE PURIFICATION AND SUBSEQUENT ACTIVATION OF
GLUCOCORTICOID RECEPTORS 127
Introduction 127
Materials and Methods 143
Results 148
Discussion 161
iii

V CHARACTERIZATION OF THE UNOCCUPIED
GLUCOCORTICOID RECEPTOR 171
Introduction 171
Materials and Methods 177
Results 180
Discussion • ••• 193
VI CONCLUDING REMARKS 196
BIBLIOGRAPHY 202
BIOGRAPHICAL SKETCH 233
IV

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MECHANISMS OF GLUCOCORTICOID TYPE II
RECEPTOR REGULATION AND ACTIVATION
IN BRAIN
By
Charles L. Densmore
August 1987
Chairman: William G. Luttge
Major Department: Neuroscience
Glucocorticoids have been shown to have profound effects on the
mammalian nervous system, most of which have been shown to result from a
receptor-mediated change in concentration or activity of key proteins.
This change is a consequence of an action of the glucocorticoid-
receptor complex on the molecular machinery that controls gene
expression. Upon binding of a steroid by the receptor, it undergoes a
temperature-dependent activation, correlated with changes in a number of
receptor properties, before it can effectively interact with its nuclear
acceptor sites.
The surface hydrophobicity of the glucocorticoid receptor in brain
cytosol was examined using hydrophobic interaction chromatography.
There was a clear increase in hydrophobicity upon activation, a finding
with important implications for the interactions between activated
receptors and numerous nuclear components.
v

The successful purification of the glucocorticoid receptor allowed
for an investigation of activation relatively free of cytosolic factors
that could interfere with, or contribute to, the process. Although
activation of unpurified receptors led to a 50-fold increase in
DNA-cellulose binding, there was no increase when purified receptors
from brain or liver were subjected to identical activation conditions.
However, other changes in purified receptors associated with activation,
including an increase in surface hydrophobicity, implied the possibility
of a multistep process.
Unoccupied glucocorticoid receptors can exist in a non-steroid-
binding or down-regulated state which can be up-regulated to a
steroid-binding state upon addition of sulfhydryl reducing reagents. An
investigation of the hydrodynamic and biochemical nature of the up- and
down-regulated receptors revealed that they exhibited the same
sedimentation properties, but differed in surface hydrophobicity. In
addition, the up-, but not the down-, regulated form proved susceptible
to irreversible inactivation by some sulfhydryl reactive reagents,
allowing for a determination of the respective populations of the two
forms in cytosol or homogenate.
Sedimentation of the unoccupied receptor was examined by post¬
labeling procedures and was found to differ in the presence (9-10 S
only) or absence (9-10 & 4-5 S) of molybdate. This effect of molybdate
was mimicked somewhat by heat-stable cytosolic factors.
vi

CHAPTER I
GENERAL INTRODUCTION
Glucocorticoid steroid hormones are known to have profound
metabolic, neuroendocrine and behavioral effects in the mammalian brain.
Glucocorticoids, in addition to regulating ACTH secretion (for review
see Keller-Wood and Dallman, 1984), produce alterations in mood, changes
in detection and recognition of sensory stimuli, changes in sleep and in
the extinction of previously acquired habits, and alterations of
electric activity of the brain (for reviews see Raynaud et al., 1980;
McEwen et al., 1982; Schraa and Dirks, 1982; Luttge, 1983; De Kloet,
1984; Reese and Gray, 1984; Meyer, 1985). Since most of these reviews
were written, many new reports concerning the role of glucocorticoids in
the nervous system have appeared. A number of studies have further
investigated the localization of glucocorticoid binding in different
regions of the central nervous system including the spinal cord (Clark
et al., 1981; Duncan and Stumpf, 1984; Orti et al., 1985), the locus
ceruleus (Krasuakaya, 1982), the hypothalamus (Kato, 1983;
Ribarac-Stepic et al., 1984), the caudate-putamen (Defiore and Turner,
1983), the hippocampus (De Kloet et al., 1984), or a variety of
different brain regions (Tornello et al., 1981; Dordevic-Markovic et
al., 1983; Alexis et al., 1983; Sarrieau et al., 1983, 1984; Birmingham
et al., 1984; Ribarac-Stepic et al., 1984).
Many recent studies have focused on the role of glucocorticoids in
the development of the nervous system. Receptor binding studies in the
1

2
developing brain imply an early sensitivity to glucocorticoids (Kitraki
et al., 1984), particularly in the cerebellum (Pavlik and Buresova,
1984). The general in vivo effect of glucocorticoids on brain growth
and development appears to be one of suppression (Slotkin et al., 1982;
Doerner, 1983; Meyer, 1983; DeKosky et al., 1984; Pavlik et al., 1984)
although adrenalectomy of young rats was shown to result in
hypomyelination of the central nervous system (Preston and McMorris,
1984) indicating that the effects of glucocorticoids on neural
development are complex. Using a brain cell culture system, Stephens
(1983) reported that glucocorticoids (cortisol) stimulate rather than
inhibit the processes of myelinogenesis and brain development.
Investigations of the development of synapses between cholinergic
neurons and retina muscles in a cell culture system led to the
hypothesis by Puro (1983) that glucocorticoids regulate the development
of mechanisms which couple neuronal depolarization with release of
neurotransmitter. Lending some potential support to this hypothesis are
the findings of Leeuwin et al. (1983) who reported that glucocorticoids
induced significant changes in the morphology of synaptic vesicles of
cholinergic (phrenic) nerve terminals. Smith and Fauquet (1984)
reported that glucocorticoids indirectly stimulate adrenergic
differentiation in cultures of migrating and premigratory neural crest
by selectively enhancing catecholaminergic properties in neural crest
cells that had already been exposed to an appropriate signal of another
kind. Finally, a variety of behavioral, biochemical and morphological
teratologies of the central nervous system are known to be induced by
glucocorticoids (Bohn, 1984; Pratt et al., 1984).

3
Glucocorticoids have profound and complex effects on brain
metabolism. Podvigina et al. (1983) reported that a variety of
glucocorticoids inhibited respiration and phosphorylation and increased
glycolysis in slices and homogenates of rat cerebral cortex, hippocampus
and hypothalamus. In agreement with glucocorticoid binding data, these
workers found hippocampus to be most sensitive and cortex to be least
sensitive. Interestingly, Kamenov (1981) has suggested that the degree
to which glucocorticoids affect respiration may itself be under the
indirect control of the nervous system and may vary seasonally in some
animals. These and many other metabolic functions in brain are
modulated by glucocorticoids via an impressive number of enzymes. Patel
et al. (1983) showed recently that corticosterone treatment resulted in
an increase in glutamine synthetase activity in rat brain. These
findings were confirmed in mouse brain cell culture by Stephens (1983)
who found that cortisol also increased the activity of 2',3'-cyclic
nucleotide phosphohydrolase while it caused a decrease in arylsulfatase
A activity (thought to be important in myelinogenesis). Earlier work
regarding the role of glucocorticoids in glia is reviewed in McEwen
(1984). The activity of glycerol 3-phosphate dehydrogenase, another
enzyme thought to be involved in myelination and present in high
concentrations in oligodendrocytes, was recently shown to be reduced in
rat brain after adrenalectomy. Rat brain citrate synthetase activity,
on the other hand, has been recently shown to undergo an actinomycin
D-reversible decrease upon administration of cortisol (Sharma and
Patnaik, 1984). These same workers reported earlier that cortisol
differentially regulates the activity of malate dehydrogenase isoenzymes
in rat brain (Sharma and Patnaik, 1982). Complex results have been

obtained for tyrosine aminotransferase activity in brain as well.
Mishunina and Babicheva (1981) reported that a single injection of
cortisol to rats caused a reduction of tyrosine aminotransferase
activity in the hypothalamus and medulla oblongata while causing an
increase in the activity in the hippocampus. Glucocorticoid injection
has also been shown to increase arginase activity in guinea pig brain
(Bjelakovic and Nikolic, 1983) and to "modulate" ornithine decarboxylase
activity in rat brain (De Kloet et al., 1983). Interestingly, tyrosine
hydroxylase activity in the superior cervical ganglion was shown to be
increased by injections of the synthetic glucocorticoid, dexamethasone,
but not by endogenous glucocorticoids (corticosterone or cortisol)
regardless of dose (Sze and Hedrick, 1983). Eranko et al. (1982), using
fluorescent histochemical techniques, reported that injection of newborn
rats with cortisol caused a great increase in the number of the small,
intensely fluorescent cells and the appearance of similar small cells
with intense immunohistochemical reactions for tyrosine hydroxylase
(TH), dopamine-B-hydroxylase (DBH) and phenylethanolamine (norepi¬
nephrine) N-methyltransferase (PNMT) in the superior cervical ganglion.
At the same time, the epinephrine content and the PNMT activity of the
ganglion greatly increased, while no significant changes were observed
in the dopamine or norepinephrine content or TH or DBH activity. PNMT
expression has also been shown to be influenced by glucocorticoids in
cells derived from the neural crest (Bohn, 1983).
Single acute doses of dexamethasone have been recently shown to
affect the levels of monoamines and their metabolites in rat brain
(Rothschild et al., 1985). These researchers reported significant
increases in dopamine levels in the hypothalamus and nucleus accumbens

5
of dexamethasone-treated rats when compared with saline-treated rats,
whereas no significant effect of dexamethasone on dopamine levels in
frontal or striatal brain areas was found. These workers also found
that dexamethasone led to a significant increase in serotonin in
hypothalamus, whereas dexamethasone caused a significant decrease in
serotonin in the frontal cortex. These findings may help to explain the
observations that corticosteroids lower prolactin levels and can induce
psychiatric disturbances as well as the finding that affectively ill
patients with high post dexamethasone cortisol levels are frequently
psychotic•
Other recent findings of relevance to the effects of gluco¬
corticoids on neurotransmitter systems include the report by Mishunina
and Kononenko (1983) that a single injection of cortisol increased the
uptake of gamma-aminobutyric acid (GABA) by synaptosomes of hippocampus,
hypothalamus and cerebral cortex. However, in vitro studies by these
same workers resulted in a glucocorticoid-dependent increase in GABA
uptake by only the hypothalamus synaptosomes, indicating differential
mechanisms of glucocorticoid action in these different brain regions.
Haidarliu et al. (1983) reported that perfusion of the rat neostriatum
with dexamethasone after stereotaxic injections of [3H]-labeled dopamine
into the caudate nucleus increased basal but inhibited potassium-
stimulated [3H]dopamine release and yield of metabolites. Slotkin et
al. (1982) reported that neonatal exposure to dexamethasone resulted in
alterations in norepinephrine synthesis and turnover in both central and
peripheral sympathetic neurons. Further demonstration of the
relationship between glucocorticoids and central neurotransmitter
systems is evident from studies reporting a decrease in the

6
corticosterone binding capacity of hippocampus after administration of
6-hydroxydopamine (Weidenfeld et al., 1983) or 5,7-dihydroxytryptamine
(Siegel, 1983). Similarly, the possible role of medial forebrain bundle
catecholaminergic fibers in the modulation of glucocorticoid negative
feedback effects has been further elucidated by investigating the
effects of 6-hydroxydopamine treatments in rat brains (Feldman et al.,
1983). In addition to effects on transmitter uptake and synthetic and
degradative enzyme activity, some of the effects of glucocorticoids on
catecholaminergic function are known to be via catecholaminergic
receptors (see review by Davies and Lefkowitz, 1984).
Many of the actions of glucocorticoids outside the brain, such as
their effects on lipolysis, gluconeogenesis, glycogenesis and
glycogenolysis, are regulated primarily by cAMP or hormones acting
through cAMP leading to the notion that glucocorjticoids have
potentiating (permissive or synergistic) effects on the action of many
hormones that act through cAMP as the intracellular second messenger
(for review see Liu, 1984). Such wide-ranging effects of gluco¬
corticoids on the effects of these other hormone systems, thought to be
via the regulation of the autophosphorylation of cAMP-dependent protein
kinase, will likely be shown to play an important role in the
glucocorticoid modulation of neural function as well.
A recent finding of potential importance to the field of epilepsy
research involves the discovery that natural, but not synthetic, gluco¬
corticoids modulate the binding of [3H]muscimol, a GABA agonist, in
crude synaptosomal membranes and in brain sections of rats (Majewska et
al., 1985). In general, nanomolar concentrations concentrations of
corticosterone and pregnenolone-sulfate enhanced mucimol binding in

7
cerebral cortex, cerebellum, thalamus and hippocampus via an apparent
increase in the affinities of GABA receptors. More recently, two
metabolites of the steroid hormones progesterone and deoxycortico¬
sterone, were shown to be potent barbiturate-like ligands of the GABA
receptor-chloride ion channel complex (Majewska et al., 1986). At low
concentrations both steroids inhibited binding of the convulsant
t-butylbicyclophosphorothionate to the GABA-receptor complex and
increased binding of benzodiazepine flunitrazepam. In addition,
chloride uptake into isolated brain vesicles was stimulated and the
inhibitory actions of GABA in cultured rat hippocampal and spinal cord
neurons were potentiated. These findings may help to explain the
ability of certain steroid hormones to rapidly alter neuronal excit¬
ability (see below) and may provide a mechanism for the anesthetic and
hypnotic actions of naturally occurring and synthetic anesthetic
steroids.
The effects of glucocorticoids on the electrical activity of
neurons has been studied at the level of single neurons by the method of
microiontophoresis. This technique has been used to study glucocort¬
icoid-induced changes in activity of neurons in a variety of brain
regions including hypothalamus (Mandelbrod et al., 1981), medial septal
area (Papir-Kricheli and Feldman, 1981), reticular formation (Avanzino
et al., 1983) and hippocampus (Belyi et al., 1982). In most of these
studies glucocorticoids were found to inhibit some neurons, excite some
neurons and have no effect on still other neurons in the same brain
region, although the ratios of the three groups varied between regions.
Peripherally, the effects of glucocorticoids on neuromuscular function
have also been investigated. These effects are generally facilitatory

8
(see review by Hall, 1983), although there have been reports to the
contrary (Korneeva and Emelyanov, 1981).
The role of glucocorticoids in the regulation of physiological
reactions to stress has been widely studied (for review see Munck et
al., 1984; Sapolsky et al., 1986) and at least some of these stress-
related functions of glucocorticoids are known to involve the central
nervous system. One example that has received a good deal of attention
recently involves the effects of glucocorticoids on stress-induced
analgesia. Adrenalectomy, hypophysectomy and/or glucocorticoid
treatment have all been shown to have significant effects on the
stress-induced (cold water, footshock, etc.) inhibition of pain
responsiveness. Markel et al. (1984) reported that adrenalectomy
significantly reduced the threshold for footshock sensitivity in rats
and that steroid replacement restored, or even potentiated, this pain
sensitivity. Similarly, Mousa et al. (1983) reported that analgesia
induced in rats by cold-water swim stress and measured by the tail-flick
and hot-plate methods was significantly antagonized after IP pretreat¬
ment for three days with dexamethasone. These investigators go on to
suggest that since naloxone also attenuated the analgesia developed by
the cold-water swim stressor, corticosteroids may have a role in
modulating stress-induced analgesia via the endogenous opiate system.
Evidence of this was presented recently by Terman et al. (1984) who used
inescapable foot shock as a stressor and the tail-flick test as a
measure of analgesia. These workers, using multiple doses of gluco¬
corticoids, found that lower doses potentiated and higher doses
significantly reduced stress analgesia in both sham and hypophysect-
omized animals. Despite some evidence to the contrary (Baron and

9
Gintzler, 1984), these results, along with the finding that nonopiod
analgesia is unaffected by dexamethasone (Lewis et al., 1980) and that
corticosterone exerts the same biphasic dose-dependent effects on
morphine analgesia (Terman et al., 1985), suggest an opiod action for
glucocorticoids, perhaps at the receptor level. Of some potential
relevance to this topic is a report that adrenalectomy significantly
potentiated and corticosterone administration attenuated stress-induced
decreases in norepinephrine contents in the hypothalamus and thalamus,
and the increases in 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (a
principle metabolite of norepinephrine) levels in the hypothalamus,
amygdala and thalamus (Nakagawa et al., 1983).
The idea that glucocorticoids, via the adrenocortical axis, may
have an important role in the process of aging has been examined in
depth recently (Sapolsky et al., 1986). It has been shown that the aged
male rat is impaired in terminating the secretion of glucocorticoids at
the end of stress. This hormonal excess is thought to be due to
degenerative changes in a region of the brain which normally inhibits
glucocorticoid release; the degeneration, in turn, appears to be caused
by cumulative exposure to glucocorticoids (Sapolsky and McEwen, 1985;
Sapolsky, 1986). Together, these effects form a feed-forward cascade in
the aging subject with potentially serious pathophysiological
consequences.
In addition to the above findings regarding the role of
glucocorticoids in the nervous system, glucocorticoids are clinically
important in the field of neurosurgery, most notably for the treatment
of various kinds of brain edema related to intracranial tumors, cerebral
infarctions and head injuries (Bouzarth and Shenkin, 1974;

10
Sugiura et al., 1980; Yu et al., 1981).
Although there is limited evidence of glucocorticoid binding
directly to synaptic (Towle and Sze, 1983) and other (Duval et al.,
1983) membranes, most of the glucocorticoid effects amenable to
experimentation have been shown to result from a receptor-mediated
change in concentration or activity of key proteins. This change is a
consequence of an action of the glucocorticoid-receptor complex on the
molecular machinery that controls gene expression. Recent advances such
as recombinant-DNA and monoclonal-antibody techniques have offered new
ways of exploring this machinery. Recently, a monoclonal antibody
against the rat liver glucocorticoid receptor in combination with the
indirect immunoperoxidase technique has made it possible to demonstrate
glucocorticoid receptor-immunoreactive nerve and glial cell nuclei all
over the tel- and diencephalon of the male rat (Fuxe et al., 1985).
Since the glucocorticoid receptor qualifies as one of the few proteins
known to control gene expression in eukaryotes, it is not surprizing
that glucocorticoids have become a fashionable tool for the molecular
biologist (for reviews see Ringold et al., 1983; Lan et al., 1984; Hager
et al., 1984; Rousseau, 1984a; Scheidereit et al., 1986). A major step
in the understanding of the structure and function of the glucocorticoid
receptor was made recently when the primary structure of a functional
human glucocorticoid receptor cDNA was identified and subsequently
cloned (Govindan et al., 1985) and expressed in vitro (Hollenberg et
al., 1985). The focus of the following review and the proposed
research, however, will be the receptor molecule itself and the changes
in this molecule brought about by the binding of a steroid ligand which
enable it to interact with the machinery that controls gene expression.

11
Although little has been said in this brief review regarding the
structure or function of the receptors in brain for other classes of
steroids (i.e. estrogens, progestins, androgens), it is particularly
relevant to briefly note recent findings regarding the so called
mineralocorticoid, or "type I", receptor in neural tissues. The
relevance of mineralocorticoid receptors to glucocorticoid actions in
neural tissues stems first from the fact that [3H]aldosterone has been
shown to bind to glucocorticoid, or "type II", receptors as well as type
I receptors in brain cytosol (Anderson and Fanestil, 1976) by virtue of
biphasic Scatchard plots for [3H]aldosterone, but not [3H]dexamethasone.
Later reports indicated that inclusion of the new synthetic gluco¬
corticoid RU 26988 (unlabeled), which binds to type II but not type I
receptors or corticosteroid binding globulin (Mogiulewsky and Raynaud,
1980), linearizes the [3H]aldosterone Scatchard plott(Beaumont and
Fanestil, 1983; Emadian et al., 1986), leaving only a high-affinity
component assumed to be the type I receptor. A second point of
relevance involves the fact that in addition to aldosterone,
corticosterone has a high affinity for the type I receptor (Krozowski
and Funder, 1983), which has led some to believe that corticosterone may
be a major, if not the primary, ligand for type I receptors in brain.
This topic is of great concern to those investigating glucocorticoid
actions in the nervous system and is currently the subject of
investigation by others in this lab. It should be noted, however, that
synthetic glucocorticoids, such as dexamethasone or triamcinolone
acetonide, have a much lower affinity for the type I receptor and
usually possess "purer" glucocorticoid properties than corticosterone as
determined by their physiological actions in peripheral tissues

12
(Meyer, 1985). In addition to their high specificity for type II
glucocorticoid receptors, other advantages of using these synthetic
glucocorticoids include the fact that their rate of dissociation from
the receptor is considerably slower than that of natural glucocorticoids
and have an insignificant affinity for corticosteroid binding globulin.
For these and other reasons, the research reported here was carried out
using dexamethasone and/or triamcinolone acetonide exclusively, and
therefore all further references to "glucocorticoid receptors" will
imply type II receptors for adrenal steroids unless otherwise indicated.
Major areas of glucocorticoid receptor research that are of
particular relevance to the glucocorticoid modulation of nervous system
function include the following: 1) The stabilization and potential
mechanisms of up- and down-regulation of unoccupied glucocorticoid
receptors. 2) Activation of the glucocorticoid-receptor complex to the
nuclear-binding form and subsequent interaction with the genome. 3)
Purification of the glucocorticoid receptor and subsequent detailed
physicochemical analysis.

CHAPTER II
HYDROPHOBIC INTERACTION CHROMATOGRAPHY OF VARIOUS FORMS OF THE OCCUPIED
AND UNOCCUPIED GLUCOCORTICOID RECEPTOR
Introduction
Hydrophobic interactions basically result from the adherence of
non-polar solutes or groups in aqueous solution, resulting,
consequently, from their repulsion from the strong polar-polar
interactions of water molecules. Like its ionic properties, a
molecule's surface hydrophobicity depends upon its primary structure.
A change in the surface hydrophobicity of glucocorticoid-receptor
complexes associated with activation has been suggested by workers
reporting an increase in the partitioning of these complexes after
activation into the polyethylene glycol (PEG) layer in an aqueous
dextran-PEG two phase system (Andreasen, 1978 and 1982; Andreasen and
Mainwaring, 1980) and by Luttge et al. (1984d) who, in addition to an
increase in partitioning into PEG, found a large increase in the
fractional adsorption of [3H]triamcinolone acetonide-receptor complexes
to glass fiber (Whatman GF/C) filters following activation. This
putative increase in hydrophobicity does not appear to be unique to the
glucocorticoid receptor, since similar transformations following
activation have been reported recently for estrogen (Gschwendt and
Kittstein, 1980; Murayama and Fukai, 1983), androgen (Bruchovsky et al.,
1981) and progesterone (Lamb and Bullock, 1983) receptors. Although the
functional significance of this increase in hydrophobicity is unknown,
13

14
it may contribute to the increased binding affinity of the activated
receptor for DNA. Increasing the hydrophobicity of the receptor may
also increase its rate of permeation through the nuclear membrane.
Hydrophobic interactions have also been reported to account for the
high affinity binding of glucocorticoids to their receptors (Wolf et
al., 1978; Alísen, 1983; Eliard and Rousseau, 1984). According to these
workers, binding requires both sides of the steroid to be enveloped by
the receptor.
The influence of hydrophobic interactions on the structure and
steroid-binding properties of glucocorticoid receptors has recently been
investigated by means of chromatography on Sephacryl S-300 or Lipidex
1000 or by incubation with charcoal or phospholipase C (Bell et al.,
1986). These workers suggest that receptor activation is preceded by
structural changes associated with the loss of a lipid factor from the
complex. They also reported that non-polar steroid antagonists, and
lipophilic compounds such as phenothiazines, bound to secondary,
hydrophilic sites on the receptor and exerted allosteric effects on the
primary steroid-binding site implying that hydrophobic interactions may
be important determinants of the structure and properties of
glucocorticoid receptors.
One particularly useful method for investigating the hydrophobic
characteristics of steroid receptors is hydrophobic interaction
chromatography. Typically this involves characterizing the chromato¬
graphic behavior of the receptor in question on a series of hydrophobic
matrices of decreasing polarity. Bruchovsky et al. (1981) investigated
the hydrophobic properties of the rat prostatic nuclear androgen
receptor using gamma-amino-alkyl derivatives of agarose with varying

15
alkyl substituent lengths. These workers found the adsorption of
receptor to the modified agarose gels increased with the length of the
alkyl substituent. However, the presence of the terminal amino group in
these gels introduces the possibility of ionic effects which could
complicate the interpretation of the hydrophobic interactions (Shaltiel,
1974). More recently, Lamb and Bullock (1983) investigated the hydro-
phobic properties of the rabbit uterine progesterone receptor using a
series of alkyl agarose columns of increasing alkyl chain length (no
terminal amino group). No such studies have been conducted on
glucocortcoid receptors.
In addition to gaining further knowledge regarding the process of
receptor activation, more detailed hydrophobic interaction studies of
both activated and unactivated forms of the glucocorticoid-receptor
complex, as well as the non-steroid bound form of the receptor, are
likely to provide valuable information concerning the physicochemical
properties of each of these receptor forms. Such studies might also
provide potentially useful means by which partial purification of these
different receptor forms can be achieved.
Materials and Methods
Chemicals, Steroids and Isotopes
[6,7-3H]Triamcinolone acetonide, or 9a-fluoro-llb,16a,17a,21-tetra-
hydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide, ([3H]TA, specific
activity = 37 Ci/mmol) was purchased from New England Nuclear (Boston,
MA). Sephadex G-25 (fine) was obtained from Pharmacia Fine Chemicals
(Piscataway, NJ). Dithiothreitol (DTT) and 4-(2-hydroxyethyl)-l-piper-
azineethane-sulfonic acid were courtesy of Research Organics (Cleveland,
OH). Sodium molybdate (Na2Mo04), calf thymus DNA-cellulose, glycerol,

16
PPO (2,5-diphenyloxazole) and dimethyl POPOP (l,4-bis[2(4-methyl-5-
phenyloxazoyl)]benzene), phenyl agarose and alkyl agaroses were all
purchased from Sigma Chemical Co. (St. Louis, MO). Scinti Verse II was
purchased from Fisher, Inc. (Fair Lawn, NJ). All other chemicals and
solvents were reagent grade.
Animals
All studies used female CD-I mice (Charles River Laboratories,
Wilmington, MA) that were subjected to combined ovariectomy and
adrenalectomy approximately 1 week prior to each experiment in order to
remove known sources of endogenous steroids. Both operations were
performed bilaterally via a lateral, subcostal approach under barbi¬
turate anesthesia, and mice were given 0.9% NaCl (w:v) in place of
drinking water. On the day of the experiment mice were anesthetized
with ether and perfused slowly through the heart with ice-cold HEPES-
buffered saline (20-30 ml, isotonic, pH 7.6).
Cytosol Preparation and Steroid Binding
Brains were removed from the perfused animals and homogenized (2 x
10 strokes at 1000 rpm) in 4 volumes of ice cold Buffer A (20 mM HEPES,
2 mM DTT and 20 mM Na2Mo04, pH 7.6 at 0 C) in a glass homogenizer with
a Teflon pestle milled to a clearance between the pestle and homogeniz¬
ation tube of .125 mm on the radius (to minimize rupture of the brain
cell nuclei (McEwen and Zigmond, 1972). The crude homogenate was
centrifuged at 100,000 g for 20 min and the supernatant recentrifuged at
100,000 g for an additional 60 min to yield cytosol. During these
centrifuge runs, and during all other procedures, unless otherwise
indicated, careful attention was paid to maintaining the cytosol at
0-2 C. Final protein concentrations were typically in the 6-8 mg/ml

17
range. For experiments involving occupied (activated or unactivated)
receptors, cytosol samples were incubated with 20 nM [3H]TA for 24 to 40
hours at 0 C with or without a 200-fold excess of unlabeled TA.
Experiments involving hydrophobic interaction chromatography of
unoccupied receptors required that the individual fractions collected
from the hydrophobic and control columns be postlabeled with 20 nM
[3H]TA +/- 4 um [1H]TA. Prior to hydrophobic interaction chromatography
of unlabeled receptors, cytosol was run on Sephadex G-25 columns equili¬
brated and eluted with the appropriate buffer to be used for
subsequently eluting the hydrophobic columns.
Activation and Removal of Unbound Steroid
[3H]TA-labeled cytosol samples to be activated were first run on
Sephadex G-25 columns (0.6 x 14 cm) equilibrated with buffer containing
20 mM HEPES and 2 mM DTT only. This column run resulted in the removal
of both free steroid and molybdate, allowing for uninhibited activation
(Luttge and Densmore, 1984; Luttge et al., 1984a,b,d). The bound
fraction was then incubated at 22 C for variable periods of time to
determine the minimum period of time required to achieve total
activation. The degree of activation was determined by DNA-cellulose
binding assay. For the purpose of hydrophobic interaction chromato¬
graphy of activated receptors, the maximally activated receptor
preparation was again run on a second Sephadex G-25 column equilibrated
with the appropriate buffer (the same buffer used to equilibrate and
elute the subsequent alkyl agarose columns). For unactivated receptors,
the cytosol was treated in an identical fasion except that the first
Sephadex G-25 column was equilibrated and eluted with HEPES buffer
containing 20mM molybdate and 2 mM DTT and the bound fraction was

18
subjected to a 0 C (rather than a 22 C) incubation prior to running on
the second Sephadex G-25 column (identical to the second column run for
the activated preparation).
In the case of postlabeling of unoccupied receptor fractions or
monitoring of dissociation occurring during the hydrophobic column runs
of prelabeled receptors, all or part of each hydrophobic column fraction
was again subjected to Sephadex G-25 chromatography to remove free
steroid.
DNA-cellulose Binding Assay
Calf thymus DNA-cellulose, prepared originally by the method of
Alberts and Herrick (1971), was added to HEPES buffer containing 2 mM
DTT to yield a final concentration of 10 mg/ml (4.1 mg DNA/g
DNA-cellulose). In a typical assay (run as duplicates) a 100 ul aliquot
of [3H]TA- labeled cytosol was added to 300 ul of the DNA-cellulose
slurry. The mixture was then vortexed gently and incubated at 0 C for
60 min. Assay tubes were oscillated at 150 rpm throughout the
incubation and vortexed gently every 10 min. The DNA-cellulose was
collected by centrifugation (2000 x g for 5 min), the supernatant
discarded and the DNA-cellulose pellet washed 3 times with 1 ml of HEPES
buffer plus 2 mM DTT. The entire pellet was resuspended in deionized
water and transferred to scintillation vials for determination of bound
radioactivity. DNA- cellulose was prepared in buffer not containing
molybdate in order to reduce the concentration of molybdate during the
binding assay, since high concentrations of molybdate were found to
reduce the efficacy of receptor binding to DNA-cellulose (Luttge et al.,
1984a).

19
Receptor Stability Determinations
Prelabeled receptor preparations (both activated and unactivated),
were run on Sephadex G-25 columns equilibrated and eluted with 50 mM
molybdate and varying concentrations of KC1 (0 - 2,000 mM) in order to
determine the effects of ionic strength on receptor stability. A
fraction of the bound fraction was immediately run on a second Sephadex
G-25 column prior to specific binding determination, while the remainder
was incubated for 4 hr at 0 C prior to being run on a second Sephadex
G-25 column and specific binding determined.
Results
The initial experiment in the investigation of glucocorticoid
receptor hydrophobicity involved hydrophobic interaction chromatography
of activated and unactivated glucocorticoid receptor complexes on a
limited Shaltiel series using a series of elution buffers of decreasing
hydrophobic character. This experiment used a series of hydrophobic
gels created by covalently attaching alkyl groups of increasing chain
length to agarose including propyl (n = 3 carbons), hexyl (n = 6), octyl
(n = 8), decyl (n = 10) and dodecyl (n = 12) agarose, along with
unmodified agarose as a control. Activated and unactivated receptor
preparations equilibrated in 600 mM KC1 and 50 mM molybdate were applied
to each of the columns (containing 1 ml of gel) and then eluted step
wise with the following buffers (each of which contained 50 mM molybdate
in HEPES buffer): 600 mM KC1, 300 mM KC1, 0 mM KC1, 10% glycerol and 30%
glycerol. Results (not shown) indicated that activated preparations
were retained longer than unactivated and that this difference was the
largest on the hexyl agarose columns. Both receptor forms appeared to
be eluted completely with higher salt concentrations on all gels except

20
octyl, decyl and dodecyl which exhibited variable retention of both
forms of the receptor in the presence of the more hydrophobic elution
buffers•
Further refinement of the procedure to investigate receptor
hydrophobicity next involved hydrophobic interaction chromatography of
activated and unactivated glucocorticoid receptor complexes on a more
complete Shaltiel series of alkyl agarose columns using a series of
elution buffers of decreasing hydrophobic character. In addition to the
original gels used, butyl (n=4), pentyl (n=5) and phenyl (n=P) agarose
were added and the gel volume was increased to 1.5 ml. In addition,
smaller fractions (0.5 ml) were collected to increase resolution and a
different series of step wise buffer elutions were used including: 600
mM KC1, 0 mM KC1 and 10% ethylene glycol (all in HEPES buffer plus 50 mM
molybdate). A final elution of 6 M urea was used to remove those
receptors (and other proteins) that were strongly retained throughout
all of the elution steps. With the higher resolution resulting from
smaller fraction size, it was obvious that activated receptors were
retained longer than unactivated receptors run on butyl, pentyl and
hexyl agarose columns. On all three columns, however, both forms of the
receptor were eluted completely, though at different rates, by 600 mM
KC1. Phenyl agarose strongly retained the activated receptor, whereas
most of the unactivated form of the receptor was eluted rapidly under
the high ionic strength conditions. Octyl, decyl and dodecyl agarose
retained the receptors more than the other gels except that there were
no significant differences between the activated and unactivated forms
on these columns. Overall, the pentyl agarose exhibited the greatest
differences in retention between the activated and unactivated forms

21
while still allowing for both forms to be eluted completely under the
same buffer conditions.
Since the results of the previous experiments indicated that any
binding retained by any of the hydrophobic columns after adequately
washing with 600 mM KC1 could not be eluted with anything tested short
of denaturing solutions such as 6 M urea, hydrophobic interaction
chromatography of activated and unactivated glucocorticoid receptor
complexes on a Shaltiel series using only 600 mM KC1 was performed.
This experiment was similar to the previously described experiment
except that only one elution buffer was used instead of a series of
different buffers. The results basically replicated those of the
previous experiment in that the activated form of the receptor
consistently shows a greater degree of retardation on most of the
columns with pentyl, hexyl and phenyl agarose providing the greatest
discrimination between the two forms (Figure 2-1).
Since high ionic strength elution buffers (HEPES buffer plus 50 mM
molybdate and 600 mM KC1) were able to discriminate between the
activated and unactivated forms of the receptor on virtually all of the
columns tested while allowing all of the binding to be eluted, the
effects of using lower or higher ionic strength buffers to equilibrate
and elute the hydrophobic columns were investigated. Because of
uncertainties regarding the activation of unactivated glucocorticoid
receptors and the stability of glucocorticoid binding under high salt
conditions, hydrophobic interaction chromatography of activated and
unactivated glucocorticoid receptor complexes on a Shaltiel series
equilibrated and eluted with only HEPES buffer and 50 mM molybdate (no
KC1) was first attempted (Figure 2-2). Proply, butyl, pentyl and hexyl

Figure 2-1. Hydrophobic interaction chromatography of unactivated and activated Type II glucocorticoid
receptor complexes on a Shaltiel series equilibrated and eluted with HEPES buffer containing 50 mM
molybdate and 600 mM KC1. Brain cytosol prepared with HEPES buffer containing 20 mM molybdate and labeled
with 20 nM [3H]TA for 40 hr at 0 C, was run on Sephadex G-25 columns equilibrated and eluted with HEPES
buffer plus either 0 or 20 mM molybdate to remove the molybdate and/or free steroid. The molybdate-free
cytosol was activated by a 24 min incubation at 22 C (solid triangle), whereas the molybdate-containing,
unactivated cytosol was left at 0 C (solid circles). Both groups were subsequently run on Sephadex G-25
columns equilibrated and eluted with HEPES buffer containing 50 mM molybdate and 600 mM KC1 prior to being
run on each of the alkyl, phenyl and control agarose columns equilibrated and eluted with the same buffer.
Cytosol (0.5 ml) was applied to the columns and 10 fractions (0.5 ml) were collected. Binding is expressed
as percent of the total counts (> 15,500 cpm) applied to each column. Results presented here are
representative of 3 independent replications. 0 = unmodified agarose (control), 3 = propyl agarose, 4 =
butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose, 10 = decyl agarose and 12 =
dodecyl agarose.

Percent of Total
ro
LO
Fraction Number

Figure 2-2. Hydrophobic interaction chromatography of unactivated and activated Type II glucocorticoid
receptor complexes on a Shaltiel series equilibrated and eluted under low salt conditions (HEPES buffer
containing 50 mM molybdate and no KC1). Brain cytosol was prepared, labeled and activated (solid
triangles) or left unactivated (solid circles) as described in Figure A. Both groups were subsequently run
on Sephadex G-25 columns equilibrated and eluted with 50 mM molybdate, but no KC1, prior to being run on
each of the alkyl, phenyl and control agarose columns equilibrated and eluted with the same buffer.
Cytosol (0.5 ml) was applied to each column and 10 fractions (0.5 ml) were collected. Binding is expressed
as percent of the total counts ( > 15,000 cpm) applied to each column. Results presented here are
represetative of 3 independent replications. 0 = unmodified agarose (control), 3 = propyl agarose, 4 =
butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose, 10 = decyl agarose and 12 =
dodecyl agarose.

Percent of Totol
60
40
20
60
40
20
60
40
20
2468 10 2468 10
2 4 6 8 10
Fraction Number

26
agaroses all showed an increased affinity for the unactivated receptor,
resulting in varying degrees of increased retention when compared to
elution by higher salt. Interestingly, pentyl agarose showed the
greatest retention of the unoccupied receptor of any of the gels. In
contrast, the unactivated receptor exhibited a decrease in retention on
the phenyl agarose. There was no appreciable differences in the elution
profiles obtained from octyl, decyl or dodecyl agarose in high or low
salt. As was the case for unactivated receptors, propyl, butyl, pentyl
and hexyl agaroses all showed an increased affinity for the activated
receptor under low salt conditions, resulting in varying degrees of
increased retention when compared to elution by higher salt. Interest¬
ingly, pentyl agarose exhibited the greatest retention of any of the
gels examined for both the activated and the unactivated forms of the
receptor. There was no appreciable differences in the elution profiles
obtained from octyl, decyl, dodecyl or phenyl in high or low salt.
The finding that octyl agarose exhibited greater retention of both
forms of the receptor than decyl agarose, which exhibited greater
retention than dodecyl agarose regardless of ionic strength (one would
have expected the reverse if these interactions were truly hydrophobic),
led to a more extensive investigation of the interactions between the
glucocorticoid receptor and these highly hydrophobic gels. Although
free steroid is removed from the cytosolic preparations immediately
prior to applying these preparations on the hydrophobic columns and
binding with [3H]TA has been shown to exhibit high stability and very
slow dissociation even under high salt conditions, the possibility that
the hydrophobic alkyls might themselves be destabilizing the receptors
via strong hydrophobic interactions had not been examined. This

27
involved replicating the previously described experiment whereby
activated and unactivated receptors where applied to the complete
Shaltiel series of columns equilibrated and eluted with HEPES buffer
plus 50 mM molybdate and 600 mM KC1 except that each fraction eluted
from the column was divided into two equal halves. One half was counted
directly as before while the other half was subjected to an additional
bound/free separation on Sephadex G-25 columns. When the profiles
determined by each of these methods for each of the gels were compared,
there were no significant differences observed except in the case of the
octyl, decyl and dodecyl agaroses. None of the counts eluting from
these three columns eluted in the bound fraction during the subsequent
Sephadex G-25 bound/free separation.
Since the results from the preceding experiment indicated how
receptor instability and/or dissociation could complicate the
interpretation of the results from hydrophobic interaction chromato¬
graphy of these receptors, and since many of these experiments required
higher concentrations of molybdate and potassium chloride than are
generally encountered throughout most of the literature regarding
glucocorticoid receptors, effects of these parameters on receptor
binding properties obviously warranted further study. In this
experiment, cytosol was prepared in the usual fashion in the presence of
20 mM molybdate and 2 mM DTT and labeled with 40 nM [3H]TA plus or minus
unlabeled TA for 24 hours to achieve maximal binding. After this
initial incubation, aliquots were taken to determine total and non¬
specific binding. Next, aliquots of prelabeled cytosol were applied to
Sephadex G-25 columns preequilibrated with every possible combination of
the following concentrations of sodium molybdate (0, 20 or 50 mM) and

28
KC1 (O, 50, 150, 300, 600, 1200 or 1800 mM). Of the bound fraction
collected from each of the 42 columns (21 total and 21 nonspecific), an
aliquot was counted to represent the "zero hour" binding for each buffer
condition. Each of the samples representing total binding was then
further subdivided into two tubes, one containing 20 nM [3H]TA (for the
determination of destabilization) and one containing 4 uM unlabeled TA
(for the determination of destabilization plus dissociation). Each of
the samples representing nonspecific binding was also subdivided into
two tubes, one containing 20 nM [3H]TA and 4 uM unlabeled TA and the
other containing only 4 uM unlabeled TA. After an 8 hour incubation at
0 C, aliquots of each of these experimental groups was again subjected
to bound/free separation on Sephadex G-25 columns. Rates of destabil¬
ization and dissociation (which rarely exceeded a few percent) were very
low for all groups, to the point that neither was likely to have been a
significant factor in any of the hydrophobic interaction chromatography
studies involving high concentrations of molybdate and/or KC1. It must
be emphasized, however, that these results cannot necessarily be applied
to the binding of this receptor to other ligands, such as DEX or
corticosterone, or to the binding of this receptor under different
temperature or pH conditions. However, since the conditions of this
experiment matched very closely the conditions of the hydrophobic
studies, any significant loss in binding seen during these studies is
likely to be the result of destabilizing effects of the hydrophobic
matrix instead of the buffer itself.
Since potassium chloride concentrations of up to 1200 mM with and
without molybdate were shown to have little effect on the stability or
rate of steroid (TA) dissociation of glucocorticoid receptors maintained

29
at low temperatures, and since retention of both activated and
unactivated receptors was shown to increase dramatically on alkyl
(particularly pentyl) agarose gels when ionic strength was lowered, the
effects of ionic strength and molybdate concentration on hydrophobic
interaction chromatography of both activated and unactivated receptors
was investigated in greater detail. Unactivated receptor preparation
was applied to pentyl agarose columns equilibrated in, and then eluted
with, one of the following: HEPES only, 10 mM molybdate, 50 mM
molybdate, 150 mM KC1 and 50 mM molybdate, 300 mM KC1 and 50 mM
molybdate, 600 mM KC1 and 50 mM molybdate, 900 mM KC1 and 50 mM
molybdate or 1,200 mM KC1 and 50 mM molybdate (Figure 2-3). Despite the
finding that high ionic strength had little effect on TA dissociation
from glucocorticoid receptors, the eluted fractions were subjected to
another bound/free separation step as an extra precaution. Pentyl
agarose was chosen as the gel to study the effects of ionic strength
since it was pentyl agarose that displayed total retention of both forms
of the receptor under low salt conditions. Since the effect of
molybdate (which had been present in all prior hydrophobic interaction
chromatography experiments in this study) was unknown, the concentration
of both molybdate and KC1 were varied in this experiment. Columns
eluted with HEPES only, 10 mM molybdate or 50 mM molybdate displayed
total retention by the column. The elution profiles of all the
KCl-containing buffers were very similar except that a very slight
decrease in retention was observed going from 150 to 300 to 600 mM KC1.
The differences between these three concentrations, though possibly
significant, were not very dramatic. Elution profiles resulting from
600, 900 and 1,200 mM KC1 showed virtually no changes. In a parallel

Figure 2-3. Hydrophobic interaction chromatography of unactivated and activated Type II glucocorticoid
receptor complexes on pentyl agarose columns equilibrated and eluted with increasing ionic strength and
molybdate concentrations. Brain cytosol was prepared, labeled and activated (solid triangles) or left
unactivated (solid circles) as described in Figure 2-1. Both groups were subsequently run on Sephadex G-25
columns equilibrated and eluted with HEPES buffer plus one of the following: no molybdate or KC1, 10 mM
molybdate, 50 mM molybdate, 50 mM molybdate and 150 mM KC1, 50 mM molybdate and 300 mM KC1, 50 mM molybdate
and 600 mM KC1, 50 mM molybdate and 900 mM KC1, 50 mM molybdate and 1200 mM KC1 prior to being run on
pentyl agarose columns equilibrated and eluted with the same buffer. Cytosol (0.5 ml) was applied to each
column and 10 fractions (0.5 ml) were collected. Binding is expressed as percent of the total counts ( >
13,700 cpm) applied to each column. Results presented here are representative of 3 independent
replications. 0 = unmodified agarose (control) run only with 50 mM molybdate and 600 mM KC1, all other
columns are pentyl agarose.

60
40
20
60
40
20
60
40
20
Fraction Number

32
experiment, an activated receptor preparation was run on identical
columns under indentical conditions. Unlike the unactivated form,
however, the activated form was also very strongly (but not completely)
retained when eluted with 150 mM KC1 and again (but to a slightly lesser
degree) when eluted with 300 mM KC1. Elution profiles with 600, 900 and
1,200 mM KC1 were all rather similar.
For the purpose of comparing the hydrophobic properties of the
different forms of the glucocorticoid receptor to those of other well
studied proteins, the next two experiments examined the hydrophobicity
of two purified proteins, bovine serum albumin (BSA) and immuno-gamma-
globulin (IgG). Each of the two proteins were first radiolabeled with
[14C]formaldehyde (Rice and Means, 1971) and then run on a Shaltiel
series using 600 mM KC1 and 50 mM molybdate. All other conditions were
identical to the previously described experiments for glucocorticoid
receptors being run on a Shaltiel series under these same salt
conditions. Like the unactivated glucocorticoid receptor, BSA exhibited
some slight retention by the propyl, butyl, pentyl, hexyl and phenyl
agaroses and was totally retained by the octyl, decyl and dodecyl gels
(Figure 2-4). Like the unactivated receptor, BSA exhibited some slight
retention by the propyl, butyl, pentyl, hexyl and phenyl agaroses and
was totally retained by the octyl, decyl and dodecyl gels. The chromat¬
ographic behavior of IgG was significantly different from that of BSA on
several of the gels in the series. Of particular interest were the
differences between the two proteins on pentyl, octyl, decyl and dodecyl
agaroses. The pentyl agarose exhibited a higher affinity for the IgG
than the BSA although both proteins did elute from the column. The
biggest difference occurred on the decyl agarose where the BSA was

Figure 2-4. Hydrophobic interaction chromatography of bovine serum albumen (solid circle) and
immuno-gamma-globulin (solid triangle) on a Shaltiel series equilibrated and eluted with HEPES buffer
containing 50 mM molybdate and 600 mM KC1. Both proteins were [14C]methylated (for detection) to low
specific activity with [14C]formaldehyde and then diluted into HEPES buffer plus 50 mM molybdate and 600 mM
KC1 prior to being run on each of the alkyl, phenyl and control agarose columns equilibrated and eluted
with the same buffer. Cytosol (0.5 ml) was applied to each columns and 10 fractions (0.5 ml) were
collected. Results are expressed as percent of the total counts ( > 20,000 cpm) applied to column. The
profiles presented here are representative of 2 independent replications. 0 = unmodified agarose
(control), 3 = propyl agarose, 4 = butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose,
10 = decyl agarose and 12 = dodecyl agarose.

60
40
20
60
40
20
60
40
20
Fraction Number

35
totally retained but the IgG eluted in a pattern similar to the propyl
and butyl agarose profiles. Also of interest was the unexpected finding
that IgG exhibited a greater affinity for hexyl agarose than for pentyl
agarose and a greater affinity for decyl agarose than for octyl agarose.
The effects of changing ionic strength on these profiles was not
determined.
In an effort to improve resolution of the chromatographic profiles
of the activated and unactivated forms of the glucocorticoid receptor as
well as to increase separation between the 2 forms, these preparations
were run individually on pentyl agarose columns much longer than those
used for the experiments involving the complete Shaltiel series (7 ml as
opposed to 1.5 ml). These preparations were run on columns equilibrated
and eluted with 600 mM KC1 and 50 mM molybdate and fractions of 0.5 ml
were collected so that fraction size was smaller relative to gel volume,
thereby increasing resolution further. As the results illustrate
(Figure 2-5), the unactivated receptors were represented by the sharper,
faster eluting peak, whereas the activated receptors were represented by
a broader, more slowly eluting peak. Although a much high resolution
was attained, the two different profiles didn't change much relative to
one another when compared to their relative shapes and positions when
run under identical buffer conditions on the smaller pentyl agarose
columns. In other words, the separation between the two peaks was not
significantly increased by increasing column length and there was still
a significant amount of overlap between them. In an attempt to increase
the separation between the two peaks, the experiment was repeated under
identical conditions except that the KC1 concentration was lowered from
600 mM to 400 mM since the affinity of activated receptors for pentyl

Figure 2-5. Hydrophobic interaction chromatography of activated and unactivated Type II glucocorticoid
receptors on long (7 ml) pentyl agarose columns. Brain cytosol was prepared in HEPES buffer plus 20 mM
molybdate and 2 mM DTT and incubated with 20 nM [3H]TA at 0 C for 24 hr. Aliquots were then either run on
Sephadex G-25 columns equilibrated and eluted with 600 mM KC1 and 50 mM molybdate (unactivated, solid
circles) or run first on Sephadex G-25 columns equilibrated and eluted with only HEPES buffer, incubated at
22 C for 24 min to activate the receptor complexes and then run on a Sephadex G-25 column equilibrated and
eluted with 600 mM KC1 and 50 mM molybdate (activated, solid triangles). Aliquots (0.5 ml) of each
treatment were run on pentyl agarose columns equilibrated and eluted with 600 mM KC1 and 50 mM molybdate
and 0.5 ml fractions were collected directly into scintillation vials for counting. Binding is expressed
as percent of the total counts applied to the column eluting with each fraction. Each condition was run in
triplicate and the profiles shown are representative.


38
agarose had previously been shown to increase to a greater extent than
the affinity of pentyl agarose for unactivated receptors when lowering
the KC1 concentration from 600 mM to 300 mM. The results were that
under the 400 mM KC1 elution conditions, the activated peak was broader
than with 600 mM KC1 elution while the unactivated receptor peak did not
change significantly (data not shown). Overlap of peaks was still
substantial.
To more closely investigate the relationship between column shape
and elution profile on hydrophobic interaction chromatography columns,
the next experiment examined the profiles for both the activated and
unactivated forms of the glucocorticoid receptor on two dramatically
different shaped pentyl agarose columns with the same gel volume.
Although two different sizes of columns had been used previously, the
shape difference (ratio of gel length to width) were not great and
differences in gel volume, relative sample size and relative fraction
size made comparisons of relative chromatographic resolution difficult.
In this experiment, the gel volume for both columns was 7 ml, but the
length to width ratio for one column was 18.6 (this column was identical
in dimensions to the 7 ml column described in the previous experiment)
while the length to width ratio for the second column, which was
significantly shorter and rounder, was 0.5, but, as previously
mentioned, contained an identical volumn of gel. There was therefore a
37.2-fold difference in the length to width ratio of the two columns.
The size of the sample applied to each column as well as the size of the
fractions collected from each column were identical and the elution
buffer contained 600 mM KC1 and 50 mM molybdate. The results of this
experiment (data not shown) indicate that there were no major

39
differences in the overall chromatographic profiles (position of peak
fraction, etc.) obtained from the two columns for either the activated
or unactivated forms of the receptor although there appeared to be a
very slight decrease in resolution (sharpness of peak) on the short wide
column for the unactivated receptor. This slight decrease in resolution
may have been related to technical problems in evenly applying such a
small sample volume to such a large gel surface.
Since most of the work to date characterizing the glucocorticoid
receptor has involved steroid labeled forms, little is known about the
physicochemical properties of the unoccupied glucocorticoid receptor.
Since hydrophobic interactions are thought to account for the high
affinity binding of glucocorticoids to their receptors (Wolff et al.,
1978; Alísen, 1983; Eliard and Rousseau, 1984), the empty hydrophobic
pocket exposed on a receptor whose binding site is unoccupied by a
steroid ligand may have an effect on the overall hydrophobicity of the
receptor that could have functional implications in vivo. This
experiment examined the hydrophobicity of the unoccupied glucocorticoid
receptor using a Shaltiel series of alkyl agarose and phenyl agarose
columns under experimental conditions identical to those previously
described except post-, instead of pre-, labeling was required (Figure
2-6) for analysis of activated and unactivated receptors when 600 mM KC1
and 50 mM molybdate was used. This allowed for direct comparison of the
results between all three forms of the receptor. Basically, the
unoccupied receptor behaved almost exactly like the unactivated occupied
receptor. Only slight differences between the two were detected on
profiles from the pentyl agarose columns. To determine if the slight
difference seen on this column was meaningful, unoccupied receptor

Figure 2-6. Hydrophobic interaction chromatography of unoccupied Type II glucocorticoid receptors and free
[3H]TA on a Shaltiel series equilibrated and eluted with HEPES buffer containing 50 mM molybdate and 600 mM
KC1. For unoccupied receptor runs (open circles), brain cytosol prepared in HEPES buffer plus 20 mM
molybdate and 2 mM DTT was run on Sephadex G-25 columns equilibrated and eluted with HEPES buffer plus 50
mM molybdate and 600 mM KC1 prior to being run on each of the alkyl, phenyl and control agarose columns
equilibrated and eluted with the same buffer. Cytosol (0.5 ml) was applied to each column and 10 fractions
(0.5 ml) were collected and postlabeled with 20 nM [3H]triamcinolone acetonide for 40 hr at 0 C. For free
steroid runs (open triangles), steroid was dried in a tube and resolubilized in HEPES buffer plus 50 mM
molybdate and 600 mM KC1 before 0.5 ml aliquots were applied to each column and fractions collected as
above. Binding is expressed as percent of the total counts ( > 14,050 cpm) applied to each column.
Results presented here are representative of 3 independent replications. 0 = unmodified agarose (control),
3 = propyl agarose, 4 = butyl agarose, 5 = pentyl agarose, 6 = hexyl agarose, 8 = octyl agarose, 10 = decyl
agarose and 12 = dodecyl agarose.

60
40
20
60
40
20
60
40
20
Fraction Number

42
preparation was run on the long (7 ml) pentyl agarose column previously
described for higher resolution analysis of the activated and
unactivated forms. The chromatographic profile obtained for the
unoccupied receptor turned out to be virtually identical to that
obtained for the unactivated steroid-labeled form (data not shown).
The possibility of using hydrophobic interaction chromatography as
a means of separating bound from free steroid for glucocorticoid
receptor binding assays was investigated. The results of this
experiment may also prove useful in interpreting data from experiments
where some degree of dissociation was known to occur on the hydrophobic
columns. This experiment examined the chromatographic profiles of free
[3H]TA on a Shaltiel series of columns equilibrated and eluted with 600
mM KC1 and 50 mM molybdate under conditions identical to those used for
the hydrophobic interaction chromatography of various forms of the
glucocorticoid receptor. Results indicate (Fig. 2-6) that the chromato¬
graphic profile of the free steroid on propyl, butyl, pentyl and hexyl
agarose was identical to that for the free steroid on unmodified
agarose. There was a slight increase in the affinity of [3H]TA for
octyl and decyl agarose and an even greater retention by dodecyl and
phenyl agarose. It should be noted that all of the steroid applied to
each of the columns was eluted by the seventh or eighth fraction. When
compared to the profiles for glucocorticoid receptors, the free steroid
profile overlaped these profiles in virtually every case. When free
steroid was run on a long 7 ml column identical to that previously
described, it eluted about midway between the peaks for the unactivated
and activated receptor and overlapped both, demonstrating that
hydrophobic interaction chromatography under the conditions used in

43
these experiments is less suitable for bound/free steroid separations
than chromatography on unmodified agarose.
Discussion
The results of this study indicate the presence of hydrophobic
regions on the different forms of the glucocorticoid receptor in brain.
This is in agreement with the findings of Lamb and Bullock (1983) for
progesterone receptors in uterus and Bruchovsky et al. (1981) for
androgen receptors in prostate. Even more important is the fact that
the degree of surface hydrophobicity of the glucocorticoid receptor
complex (assumed to be directly related to the degree of retention by
the hydrophobic gels) appears to be dramatically increased upon heat
activation of the receptor to the nuclear binding form. While such a
change in the surface hydrophobicity of glucocorticoid receptor
complexes associated with activation has been suggested by other workers
reporting an increase in the partitioning of these complexes after
activation into the less polar polyethylene glycol (PEG) layer in an
aqueous dextran-PEG two phase system (Andreasen, 1978 and 1982;
Andreasen and Mainwaring, 1980) and by this lab (Luttge et al., 1984d),
which reported a large increase in the fractional adsorption of
glucocorticoid receptor complexes to glass fiber (Whatman GF/C) filters
following heat activation, hydrophobic interaction chromatography has
never been used to study this phenomenon in glucocorticoid receptors.
Furthermore, although it is possible that the dramatic increase in
surface hydrophobicity associated with heat-induced activation could
represent an artefact of the nonphysiological conditions existing during
the in vitro activation process, this increased hydrophobicity is

nevertheless consistent with the presumed function of glucocorticoid
receptors in general. It is certainly possible that the increased
hydrophobicity of the activated receptor could account, at least in
part, for the increased affinity for a number of different nuclear
components including chromatin (Sakaue and Thompson, 1977; Simons et
al., 1976), nucleosomes (Climent et al., 1977), DNA (Baxter et al.,
1972; Rousseau et al., 1975; Sluyser, 1983), RNA (Tymoczko and Phillips,
1983), the nuclear matrix (Buttyan et al., 1983; Kirsch et al., 1986)
and the nuclear membrane (Smith and von Holt, 1981). If the unactivated
receptor resides in the cytosolic/cytoplasmic compartment, as originally
thought (Szego, 1974), activation-induced increases in hydrophobicity
might facilitate the permeation of the receptor through the nuclear
membrane. If, on the other hand, the current controversial theory
stating that unoccupied and occupied unactivated receptors actually
reside in the nucleus in vivo (for review see Walters, 1985) is true,
then increased hydrophobicity could decrease the likelyhood of "leakage"
of activated receptors from nuclei by virtue of their greater affinity
for nuclear components.
A Shaltiel series of alkyl agarose, as well as phenyl agarose,
columns were used since the ideal gel(s) to study the surface hydropho¬
bicity of the various receptor forms under different buffer conditions
was unpredictable initially. Although it has been shown that the
hydrophobicity of a linear aliphatic carbon increases linearly with
carbon chain length (Tanford, 1972), it has been argued by others
(Shanbhag and Axelsson, 1975), that the effective hydrophobicity depends
also on the flexibility of the hydrocarbon chain and consequently on the
degree of interaction within such chains, particularly for long chains.

45
The mechanisms by which proteins are adsorbed to hydrophobic matrices
are no doubt complex and this problem can be exemplified in the present
study by the case of the adsorption of IGG on a series of alkyl agarose
columns, in which case a decrease in adsorption between pentyl and hexyl
and between octyl and decyl agarose. This is similar to the case of the
adsorption of erthrocytes on a series of alkyl agarose derivatives,
wherein a decrease in adsorption between hexyl and octyl agarose was
noticed to occur without an obvious explanation. The present study
found that bovine serum albumen had a higher affinity for pentyl agarose
than did IGG, whereas the reverse was true for octyl, decyl and dodecyl
agaroses.
Another problem adding to the unpredictable nature of hydrophobic
interactions stems from the fact that some hydrophobic ligands denature
proteins through a "detergent-like" action (Hofstee, 1973). This was
evidenced by the fact that what initially appeared to be a partial
elution of both activated and unactivated receptors from octyl, decyl
and dodecyl agaroses, later proved to be dissociated free steroid
resulting from the denaturation of the receptors. The degree of
denaturation increased with alkyl chain length with dodecyl agarose
producing the greatest effect. In addition, the degree of denaturation
for activated and unactivated forms appeared to be roughly equivalent,
implying the possibility that the disruptive hydrophobic interaction
leading to loss of steroid binding involved a region or regions common
to both forms of the receptor. This finding may be relevant to the
interpretation of other studies involving hydrophobic interaction
chromatography. For instance, Lamb and Bullock (1983) reported that a
large volume of buffer was required for elution of the progesterone

46
receptor from decyl agarose, which they claimed was probably due to slow
equilibrium of the column with elution buffer. Another possibility is
that the progesterone receptors tightly bound to the decyl agarose may
have been undergoing a slow denaturation resulting in broad profiles of
eluted radioactivity representing free steroid only. When dissociation
was taken into account in the present study, no steroid receptor
complexes could be eluted intact from octyl , decyl or dodecyl agarose
using any of a number of buffers including some with increased "hydro-
phobic character" such as glycerol or ethylene glycol. Thurow and
Geisen (1984) have reported that polypropylene glycol/polyethylene
glycol block polymers prevent both the adsorption of dissolved proteins
to hydrophobic interfaces and the resultant aggregation and denatur¬
ation. However, it is not known whether or not these polymers can
successfully elute the intact receptors from highly hydrophobic
interfaces if they are already attached and the presence of such
polymers prior to any hydrophobic interactions would reduce or eliminate
any such interactions, rendering the whole procedure useless.
Overall, the most promising results were obtained with the pentyl,
hexyl and phenyl agarose gels. Phenyl-agarose has reportedly been
effective in hydrophobic studies of the progesterone receptor (Logeat et
al., 1981; Lamb and Bullock, 1983) and in differentiating between the
activated and unactivated forms of the estrogen receptor (Gschwendt and
Kittstein, 1980). The activated form of the glucocorticoid receptor
bound more tightly to phenyl agarose than to alkyl agarose (excluding
octyl or longer alkyl agaroses). When calculated as the change in
standard free energy on transfer from an aqueous solution to a solution
of pure hydrocarbon, a phenyl group is less hydrophobic than a pentyl

47
group (Rosengren et al., 1975; Lamb and Bullock, 1983). A portion of
the unactivated receptor was retained by phenyl agarose under high salt
conditions, while the same receptor preparation was only slightly
retarded on the pentyl and hexyl agarose columns. While the activated
form of the receptor eluted much more slowly from the pentyl and hexyl
agaroses than did the unactivated form, the activated form was
completely retained on the phenyl agarose under low and high salt
conditions. Shaltiel (1974) has proposed that adsorption requires the
interaction of an alkyl residue of sufficient length with a hydrophobic
"pocket" within the protein. It has been suggested that the aromatic
ring on phenyl agarose may be more complementary than alkyl groups to
the contours of the hydrophobic "pocket" on the progesterone receptor
(Lamb and Bullock, 1983). Because of similarities in amino acid
sequence between the two receptor types (Conneely et al., 1986), such a
pocket may also exist in or on the glucocorticoid receptor and may be
made more accessible for hydrophobic interactions by heat activation.
Alternatively, the receptor may form a charge transfer complex with the
phenyl group, thus increasing the interaction. One might therefore
conclude that phenyl agarose is the best gel for differentiating between
the activated and unactivated forms of the glucocorticoid receptor based
on differences in surface hydrophobicity. Unfortunately, the affinity
of the activated receptor for this particular matrix prevents removal of
the receptor from the matrix in an intact form. In addition, minor
changes in hydrophobic interactions between the activated receptor and
the matrix (i.e., as caused by changes in ionic strength, temp., etc.)
cannot be readily monitored by phenyl agarose and therefore alkyl
agaroses are best suited for such studies.

48
A number of factors in addition to the hydrophobicity of the
attached groups influence the adsorption of receptors and other proteins
to hydrophobic matrices. Jennissen (1976) has presented evidence in
favor of the idea that the adsorption of proteins to alkyl agarose
derivatives takes place at a critical alkyl group density. In other
words, this hypothesis considers that the protein needs to present
multiple attachment points in order to be adsorbed by a determinate
member of the series of alkyl agarose derivatives. Another factor
affecting the chromatographic behavior of a given protein on a
particular hydrophobic matrix is the overall protein concentration
applied to the column. Small amounts of protein bind more homogeneously
on a particular column than a large amount. This should not have been a
factor, however, in the differences in hydrophobicity exhibited by
activated and unactivated receptors in this study since great care was
taken to maintain the same protein concentration in all treatment groups
and throughout all experiments. On the other hand, this could very well
have been a factor in the chromatographic differences seen between the
unpurified activated receptor and the purified activated receptor since
there were hundreds- to thousands-fold differences in protein
concentration (discussed in more detail in a later section). Another
way in which protein concentration, especially in impure, crude
preparations such as cytosol, could potentially affect the chromato¬
graphic profile of glucocorticoid receptors on these columns is through
indirect, or secondary, interactions with other proteins bound tightly
to the columns.
The influence of salt on the retention of receptors and other
proteins to hydrophobic gels is probably due to a number of factors

49
acting on the protein as well as on the hydrophobic matrix. Pahlman et
al. (1977) concluded that salting-out ions, such as potassium chloride,
cause conformational, but not structural, changes in biomolecules. The
"structure forming" properties of salting-out ions enhance intra¬
molecular, as well as intermolecular, hydrophobic bonding as reflected
by a stabilization of the hydrophobic core of the biomolecule (Hofstee,
1975). High concentrations of salting-out ions can adversely affect the
solubility of a protein by decreasing the availability of water
molecules in the bulk and increasing the surface tension of water,
resulting in an inhancement of the hydrophobic interactions (Tanford,
1968; Melander and Horvath, 1977). It has been recently shown that the
free energy of a protein is increased by addition of salting-out ions
and this unfavorable free energy is smaller for the proteins bound to
columns because of their smaller surface area exposed to solvent
(Arakawa, 1986). The present study used, for the most part, a
combination of two salts: sodium molybdate and potassium chloride. The
50 mM concentration of molybdate was required to prevent activation and
to increase stability of all forms of the receptor under conditions
requiring high concentrations of potassium chloride since lower concen¬
trations of molybdate have been shown to be inadequate in preventing
activation under high salt conditions (Weatherill and Bell, 1985). It
was therefore not feasible to run meaningful experiments in the presence
of potassium chloride only, although the possibility exists that the
presence of molybdate may have influenced the effects that potassium
chloride would have had (aside from increasing activation) on protein
conformation and therefore, possibly, hydrophobic interactions. When
the effects of salt concentration on the chromatographic profiles of

50
both activated and unactivated receptor complexes on pentyl agarose were
examined, it was clear that all of the gels tested except phenyl agarose
exhibited a greater affinity for both forms of the receptor under low
salt conditions than under high salt conditions. Even the unmodified
agarose exhibited a slight affinity for the activated form of the
receptor under these conditions which may be due to low level
electrostatic or other interactions with the gel matrix itself (Janson,
1967). Since this was opposite of what was expected, a more detailed
study was carried out using only pentyl agarose and increasing
concentrations of both molybdate and potassium chloride. Elution of
activated receptors from the pentyl agarose required a higher
concentration of potassium chloride than was required to elute the
unactivated receptors. Although electrostatic interactions with the
matrix (the alkyl groups are supposedly uncharged after attachment to an
agarose matrix (Rosengren et al., 1975)) probably contributes to the
higher affinity at lower salt concentrations, the effects observed are
likely to involve a combination of electrostatic and hydrophobic
interactions since the activated receptor elutes from ion-exchange
columns at a lower salt concentration than the unactivated form (Sakaue
and Thompson, 1977). Once the electrostatic interactions are overcome
by using 600 mM concentrations of potassium chloride or greater, the
chromatographic differences between the two forms are probably purely
hydrophobic. It was reported by Peale et al. (1985) that the assayed
levels of charged estrogen receptors, using both Sephadex gel filtration
or dextran-coated charcoal treatments, in buffers containing 150-200 mM
ionic strength (roughly physiologic) was approximately one half that of
estrogen receptor levels assayed in buffers either at 0-50 or 400-450 mM

51
ionic strength. However, molybdate was shown to eliminate this
reduction.
Although Wolf et al. (1978) have shown that the high affinity
binding of a glucocorticoid by its receptor is largely due to hydro-
phobic interactions, the present study found little evidence that the
primary site of the hydrophobic interactions with alkyl and phenyl
agarose involved the steroid binding site. In fact, the chromatographic
behavior of the unoccupied receptor differed little from that of the
occupied unactivated receptor on practically all of the gels examined.
These findings are in agreement with those of Lamb and Bullock (1983)
who found that progesterone receptors bound to, and eluted from, alkyl
agarose equally well in both the free and complexed form. This, in
fact, might be expected in light of the high degree of specificity
normally associated with steroid-receptor interactions. It has been
implied, however, that steroid binding might be involved with some
hydrophobic interactions between the rat estrogen receptor and Cibacron
Blue (Tenenbaum and Leclerq, 1980).
In an attempt to improve the chromatographic resolution of the
activated and unactivated glucocorticoid receptors eluting from alkyl
agarose columns, pentyl agarose columns with various shapes (but with
the same gel volume) were compared. In addition, elution flow rates
were varied. None of these changes, however, resulted in any
significant improvement in chromatographic resolution providing further
support to the early observation by Frank and Evans (1945) that the
hydrophobic interaction is a spontaneous process.

CHAPTER III
SULFHYDRYL REGULATION OF GLUCOCORTICOID BINDING CAPACITY
Introduction
The concentration of glucocorticoid receptors is subject to
fluctuations resulting from several factors (De Nicola et al., 1982;
Svec, 1985a), including autoregulation by the ligands themselves
(Tornello et al., 1982; Ho-Kim et al., 1983; Muramatsu et al., 1983;
Sapolsky et al., 1984; Svec, 1985b; Luttge et al., unpublished). The
glucocorticoid-induced depletion of glucocorticoid receptors is
reportedly due to an increased degradation of receptors and not due to a
decreased rate of receptor synthesis (Svec and Rudis, 1981; McIntyre and
Samuels, 1985). Androgens, on the other hand, have been associated with
an augmentation of their receptors, apparently resulting from an
increase both in receptor half-life and in rates of receptor synthesis
(Syms et al., 1985). Glucocorticoid receptors may require an
energy-dependent up-regulation or transformation before they are able to
bind the steroid. Evidence for such an up-regulation in brain was
presented by Turner and McEwen (1980), who reported a lack of depletion
of hippocampal cytosol glucocorticoid receptors after multiple
injections of [3H]corticosterone. These results could not be explained
entirely by receptor synthesis and suggested that in the hippocampus an
"excess” of glucocorticoid binding proteins exist, and that the
availability of functional cytosol receptors may be regulated to
maintain a relatively constant cellular level. Evidence of a rapid
52

53
down-regulation has also been observed. Recently, the loss of a
biological response associated with such a down-regulation of
glucocorticoid receptor levels was demonstrated in a mouse cell line
(Danielsen and Stallcup, 1984). In addition to circulating levels of
steroids, other factors may play a role in the regulation of steroid
receptor concentration. Of particular relevance to the role of
glucocorticoids in the nervous system is recent evidence that neural
input influences significantly the levels of both androgen (Bernard et
al., 1984) and glucocorticoid (DuBois and Almon, 1981) receptor levels
in striated muscle as determined from denervation studies. Similarly,
Cardinali et al. (1983) reported that pineal cytoplasmic and nuclear
estrogen and androgen receptors are modulated by norepinephrine released
from nerve endings at the pinealocyte level. These findings suggest that
target cells may utilize a variety of homeostatic mechanisms to regulate
the amount of receptor capable of interaction with the free steroid.
Little is known, however, about these regulatory mechanisms and even
less is known about the quantification, production and turnover of these
cryptic (down—regulated) receptors. In addition, factors which
stabilize or destabilize unoccupied receptors under in vitro conditions
may also provide valuable information concerning the receptor and its
regulation.
One potential mechanism of receptor up- and down-regulation for
which there is fairly substantial evidence involves the reduction and
oxidation of sulfhydryl groups on the receptor. Oxidation of receptor
sulfhydryl groups (probably located in or near the steroid binding site)
has been shown to reversibly inactivate glucocorticoid receptors in a
number of tissues (Rees and Bell, 1975; Granberg and Ballard, 1977;

54
Sando et al., 1979; McBlain and Shyamala, 1980; Housley et al., 1982;
Harrison et al., 1983; Densmore et al., 1984d; Izawa et al., 1984).
These sulfhydryl groups are probably located in or near the steroid
binding site as evidenced by the fact that mesylated derivatives of
glucocorticoids have been shown to bind covalently to the receptor,
presumably by means of an interaction between the mesylate group and a
binding-site sulfhydryl group (Simons et al., 1980). The affinity
labeling of the estrogen receptor has similarly indicated the presence
of a thiol group at the binding site that appears to be directly
involved in estradiol-receptor binding (Ikeda, 1982). Addition of
sulfhydryl reducing agents such as dithiothreitol (DTT) or
2-mercaptoethanol leads to a total recovery of lost binding under
appropriate conditions. Further evidence that the sulfhydryl-disulfide
balance may be important in the regulation of glucocorticoid binding
capacity was presented recently when it was shown that endogenous
thioredoxin was responsible for up-regulation of glucocorticoid
receptors in liver cytosol (Grippo et al., 1983). In addition to
effects on binding capacity, oxidation of yet other sulfhydryl groups on
the activated glucocorticoid-receptor complex by various sulfhydryl
reactive agents has been shown to interfere with the DNA-binding ability
of receptors from thymus (Bodwell et al., 1984) and most recently
Kaufmann et al. (1986) has presented evidence suggesting that disulfide
bonds mediate the association between the activated glucocorticoid
receptor and the nuclear matrix, representing yet another possibility
for the regulation of glucocorticoid action. Much work in
this area remains to be done, however, before it can be determined
whether these mechanisms are likely to be of significance in vivo.

55
Despite the growing body of evidence that ligand binding to the
glucocorticoid receptor as well as binding of the activated gluco¬
corticoid-receptor complex to nuclei or DNA are dependent upon the
reduction of certain sulfhydryl groups on the receptor surface, there
has been surprizingly little speculation concerning the possibility of
thiol/disulfide exchange as a control mechanism for glucocorticoid
action in vivo. The specific modulation of enzyme activity by post-
translational modification of specific amino acid side chains is well
established as a mechanism of metabolic regulation. Essentially all the
amino acids possessing chemically reactive side groups are capable of
undergoing some sort of posttranslational modification: the carboxyl
groups of glutamate and aspartate, the imidazole side chain of
histidine, the hydroxyl groups of serine, threonine and tyrosine, the
amine group of lysine and even the guanidino group of arginine.
However, the sulfhydryl group of cysteine is potentially the most
reactive nucleophile in proteins. The process of thiol/disulfide
exchange could provide a mechanism for the equilibration of the
sulfhydryl oxidation state of proteins with the thiol/disulfide status
of the surrounding environment. Although the regulation of enzyme
activity by thiol/disulfide exchange was proposed some time ago (Barron,
1951), it has received less attention than other posttranslational
modifications. According to Gilbert (1984), thiol/disulfide exchange as
a biological control mechanism must exhibit at least the following six
properties:
1. The intracellular thiol/disulfide status should vary in vivo in
response to some metabolic signal. Isaacs and Binkley (1977a) reported
that the disulfide-sulfhydryl ratios of subcellular (nuclear, microsomal

56
and cytosolic) fractions of rat hepatic tissue varied diurnally with the
ratio lowest in the early morning (after feeding) and highest in the
early evening (after fasting). The primary reaction was the reversible
formation of mixed disulfides of glutathione with proteins and the
change in the overall disulfide-sulfhydryl ratio was approximately
4-fold. These same workers (Isaacs and Binkley, 1977b) also reported
changes in the disulfide-sulfhydryl ratio associated with cyclic AMP
levels. Intaperitoneal injections of dibutyryl cyclic AMP induced an
increase in hepatic glutathione protein mixed disulfides combined with a
corresponding decrease in reduced glutathione and protein sulfhydryl.
2. Oxidation of protein sulfhydryl groups by thiol/disulfide exchange
should activate some enzymes or receptors, inactivate others and not
affect the activity of still others. Gilbert (1984) has compiled a
large number of enzyme activities that have been suggested to be
modulated by reversible thiol/disulfide exchange. Like some steroid
receptors, many enzymes are reversibly inactivated by oxidation with a
number of biologically occurring disulfides. One example is
phosphofructokinase from either rabbit muscle (Gilbert, 1982) or mouse
liver (Binkley and Richardson, 1982). Fructose-1,6-biphosphatase,
however, undergoes a 2- to 4-fold activation in the presence of such
disulfides (Gilbert, 1984).
Many enzymes have sulfydryl groups that can be chemically modified
by a variety of sulfhydryl-specific reagents such as iodoacetamide,
N-ethylmaleimide or Ellman's reagent with a resultant effect on enzyme
activity. Most of these reagents are highly reactive toward sulfhydryl
groups and somewhat less specific as a result. In addition, these
reactive disulfides are much better oxidizing agents than most

57
biologically occurring disulfides. The enzymes aldolase and thiolase,
for example, both have sulfhydryl groups that react with iodoacetamide,
N-ethylmaleimide or reactive disulfides, however, incubation with lOmM
glutathione disulfide at pH 8.0 for 24 hr (very harsh oxidizing
conditions) had only a minimal effect on their activity (Gilbert, 1984).
A similar situation has been observed with steroid receptors. Tashima
et al. (1984) reported that monoiodoacetamide, N-ethylmaleimide and
5,5'-dithiobis(2-nitrobenzoate) all inactivated the binding capacity of
unoccupied mineralocorticoid ([3H]aldosterone) receptors, while the
binding capacity of these receptors was unaffected by endogenous
oxidizing factors that lead to the temperature-dependent, reversible
inactivation of glucocorticoid ([3H]dexamethasone) receptors under
identical in vitro conditions (Emadian et al., 1985). Therefore, the
presence of a chemically reactive sulfhydryl group is not in itself
sufficient evidence to suggest regulation of any enzyme or receptor by
thiol/disulfide exchange.
3. The thiol/disulfide redox potential of a regulated protein
should be near the observed thiol/disulfide ratio in vivo. If the
thiol/disulfide status of a regulated protein is in equilibrium with the
thiol/disulfide status of the cell, the equilibrium constant for the
thiol/disulfide exchange reaction
Ered + RSSR = Eox + RSH
Keq = [Eox][RSH]/[Ered][RSSR]
must lie near the intracellular thiol/disulfide ratio (Gilbert, 1984).
According to this equilibrium model, changes in the ratio of oxidized to
reduced receptor ([Eox]/[Ered]), and hence, the binding capacity, would
result from changes in the thiol/disulfide ratio ([RSH]/[RSSR]). Such a

58
relationship has been observed, although the literature concerning the
thiol/disulfide redox potentials of individual proteins is very limited.
Two enzymes that do equilibrate with glutathione/glutathione-disulfide
(GSH/GSSG) redox buffers in vitro are rabbit muscle phosphofructokinase
(Gilbert, 1982) and chicken liver fatty acid synthetase (Walters and
Gilbert, 1984). Unfortunately, there are no reports concerning redox
potential measurement in steroid receptor systems. However,
intracellular ratios of GSH/GSSG, the most predominant low molecular
weight thiol/disulfide pair, typically decrease during in vitro
procedures if precautions are not taken to "trap” thiols and disulfides
at their original in vivo levels. This is because, at neutral pH, GSH
is oxidized in air to GSSG; and due to the large excess of GSH over
GSSG, postmortem oxidation of a small amount of the total GSH will
result in a large artifactual increase in the observed GSSG
concentration. One study reported an increase in GSSG concentration of
about 50% in 100 min in acid-soluble extracts of rat liver adjusted to
pH 7.4 (Vina et al., 1978). Such an increase would lead to changes in
the GSH/GSSG ratio that would still be within the range of change
observed to occur diurnally in rats (Isaacs and Binkley, 1977),
indicating that changes seen in receptor binding activity due to
postmortem oxidation of intracellular thiols might possibly reflect a
receptor thiol/disulfide redox potential reasonably near the
physiologically significant range. However, much work remains to be
done before such a question can be answered.
4. Thiol/disulfide exchange reactions must be kinetically competent
under physiological conditions. In order for the thiol/disulfide
exchange between protein sulfhydryl groups and biological disulfides to

59
be considered a viable regulatory mechanism, the rate of this process in
vivo must be at least as fast as the changes in the intracellular
thiol/disulfide ratio. With some exceptions, most enzymes thus far
studied would probably require catalysis of in vivo thiol/disulfide
exchange in order for the process to be considered a viable mechanism of
regulation (Gilbert, 1984). Since precise rates of thiol/disulfide
exchange for steroid receptors have not been determined under defined
conditions, it is currently unknown whether such a system would be
kinetically competent in the absence of catalysis. However, recent
reports have provided direct evidence for the existence of an endogenous
thioredoxin-mediated reducing system in rat liver cytosol that maintains
the glucocorticoid receptor in a reduced state in vitro in the presence
of NADPH (Grippo et al., 1983, 1985). As for kinetic competence of this
reaction, it should be noted that E. coli thioredoxin-(SH)2 reduces
bovine insulin at least 10,000 times more rapidly than dithiotreitol at
pH 7 (Holmgren, 1979). Endogenous mammalian thioredoxin activity has
probably been undetected in many glucocorticoid receptor studies because
of its easy inactivation by oxidation of structural SH groups (Luthman
and Holmgren, 1982). Therefore, the same in vitro conditions (oxidation
of reactive thiols) that inactivate unoccupied glucocorticoid receptors
also simultaneously inactivate the enzymes that catalyze rapid reduction
of receptor disulfides. The possible role of other enzymes that
catalyze the reverse reaction (disulfide formation) have not been
investigated with regards to steroid receptor systems. One such
possibility, protein disulfide-isomerase, has been shown to have a
rather broad substrate specificity, is widely distributed and has been
detected in most vertebrate tissues (Hillson et al., 1984).

60
5. For regulated proteins, the oxidized and reduced forms should
both be observable in vivo. The relative levels of the oxidized and
reduced forms must change in response to changes in the cellular
thiol/disulfide ratio. Although there is evidence that some enzymes,
such as phosphofructokinase, exist in vivo in both an oxidized and
reduced form, it is currently unknown whether both forms of the
glucocorticoid, or any other steroid, receptor exist in vivo. This is
for several reasons. First, in vitro preparation of cytosol for steroid
receptor binding studies is not an instantaneous process and oxidation,
as stated earlier, is most likely occurring if sulfhydryl group reducing
agents are not present. However, in the presence of such reducing
agents (such as dithiothreitol or 2-mercaptoethanol), the overall
thiol/disulfide ratio of the cytosol is being shifted in favor of thiol
formation. Even though nonenzymatic reduction of disulfides is
relatively slow, dithiothreitol and other exogenously added reductants
may reactivating or "up-regulating" enzymes such as thioredoxin that
rapidly catalyze dithiol-disulfide oxidoreductions (Holmgren, 1977,
1979). These problems are magnified by the fact that, like cytosol
preparation, the measurement of steroid binding capacity itself is very
time consuming. At saturating concentrations of DEX or TA, low
temperature incubations of 24 hours or longer are often required to
reach equilibrium (or near equilibrium) binding. Finally, ratios of
oxidized versus reduced receptors can not be assumed from receptor
binding studies alone. The possibility exists that the oxidation or
reduction of nonreceptor cytosolic components may indirectly modulate
receptor binding capacity via nonthiol/disulfide exchange mechanisms.

61
6. The response of particular enzyme or receptor activities to
oxidation by thiol/disulfide exchange should be consistent with the
metabolic function of the enzyme or receptor. If changes in the
thiol/disulfide ratio are coupled to changes in the activities of some
regulated enzymes and receptors, and if one assumes that such ratios are
based entirely on the feeding state of the animal (fasting leads to a
decrease in the ratio (Isaacs and Binkley, 1977a)), three general
consequences might follow (Gilbert, 1984). (1) Enzymes or receptors
that should be active only in the fed state should lose activity on
oxidation or gain activity on reduction. (2) Enzymes or receptors that
should be active only in the fasted state should either gain activity on
oxidation or lose activity on reduction. (3) Enzymes or receptors that
must function in both the fed and fasted state should either not undergo
thiol/disulfide exchange or the thiol/disulfide exchange should not
affect the enzyme activity. Although, as stated previously, it has been
reported that a decrease in the cellular thiol/disulfide ratio can be
induced by starvation or the administration of agents that increase the
intracellular concentration of cAMP, such as glucagon or epinephrine
(Isaacs and Binkley, 1977a,b), the effects of many other factors
(disease, stress, other hormonal systems) on cellular thiol/disulfide
ratios simply has not been investigated in great detail. Therefore,
while the relationship between some enzymes and changes in the cellular
thiol/disulfide ratio might appear to be consistent with the metabolic
function of the enzyme, the apparent absence or inconsistency of such a
relationship based merely on the feeding status of an animal should not
be taken as an absolute disqualification of redox control of a receptor
system whose functions are as vastly complex as that of the

62
glucocorticoid receptor. In addition, tissue differences in cellular
thiol/disulfide ratio, as well as levels of enzymes catalyzing
thiol/disulfide exchange, are known to exist. This may, in part,
explain the tissue differences reported in the dithiothreitol-inhibited
instability of unoccupied glucocorticoid receptors (Granberg and
Ballard, 1977), and may indicate tissue differences in the degree of
redox control.
Materials and Methods
Chemicals, Steroids and Isotopes
[6,7-3H]Triamcinolone acetonide, or 9a-fluoro-llb,16a,17a,21-tetra-
hydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide, ([3H]TA, specific
activity = 37 Ci/mmol) and [6,7-3H]dexamethasone, or 9a-fluoro-16a-
methylprednisolone ([3H]DEX, specific activity = 44.1-48.9 Ci/mmol) were
purchased from New England Nuclear (Boston, MA). Sephadex G-25 (fine)
and Dextran T70 were obtained from Pharmacia Fine Chemicals (Piscataway,
NJ). Dinitrobenzoic acid (DTNB), N-ethylmaleimide NEM), iodoacetamide,
iodoacetic acid, cystamine, glutathione disulfide (GSSG) and para-
chloro-mercurisulfonate (PCMS), sodium-m-periodate, sodium-p-periodate,
6,6'-dithionicotinic acid (DTNT), methyl methane thiosulfonate (MMTS),
mercuric chloride, sodium molybdate (Na2Mo04), calf thymus DNA-
cellulose, glycerol, sucrose, activated charcoal, PPO (2,5-diphenyl-
oxazole) and dimethyl POPOP (1,4-bis[2(4-methyl-5-phenyloxazoyl)]-
benzene) and pentyl agarose were all purchased from Sigma Chemical Co.
(St. Louis, MO). Dithiothreitol (DTT) and
4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid (HEPES) were
courtesy of Research Organics (Cleveland, OH). Scinti Verse II was

63
purchased from Fisher, Inc. (Fair Lawn, NJ). All other chemicals and
solvents were reagent grade.
Animals
All studies used female CD-I mice (Charles River Laboratories,
Wilmington, MA) that were subjected to combined ovariectomy and
adrenalectomy approximately 1 week prior to each experiment in order to
remove known sources of endogenous steroids. Both operations were
performed bilaterally via a lateral, subcostal approach under barbi¬
turate anesthesia, and mice were given 0.9% NaCl (w:v) in place of
drinking water. On the day of the experiment mice were anesthetized
with ether and perfused slowly through the heart with ice-cold
HEPES-buffered saline (20-30 ml, isotonic, pH 7.6).
Cytosol Preparation and Steroid Binding
Brains were removed from the perfused animals and homogenized (2x10
strokes at 1000 rpm) in 4 volumes of ice cold buffer containing 20 mM
HEPES and 20 mM Na2Mo04, pH 7.6 at 0 C) in a glass homogenizer with a
Teflon pestle milled to a clearance between the pestle and homogeniz¬
ation tube of 0.125 mm on the radius (to minimize rupture of the brain
cell nuclei (McEwen and Zigmond, 1972)). The crude homogenate was
centrifuged at 100,000 g for 20 min and the supernatant recentrifuged at
100,000 g for an additional 60 min to yield cytosol. During these
centrifuge runs, and during all other procedures, unless otherwise
indicated, careful attention was paid to maintaining the cytosol at 0-2
C. Final protein concentrations were typically in the 6-8 mg/ml range.
After the appropriate experimental manipulations, cytosol samples were
incubated with 20 nM [3H]TA or [3H]DEX for 24 to 40 hours at 0 C with or
without a 200-fold excess of unlabeled steroid.

64
Charcoal Pretreatment
The dextran-coated charcoal (DCC) mixture was prepared overnight by
adding 1.25% (w/v) activated charcoal and 0.625% dextran T70 to buffers
identical in composition to that in which cytosol was prepared. Just
prior to use, the desired volume of DCC suspension was centrifuged at
2,230 g for 10 min, the supernatant discarded and an equivalent volume
of cytosol added to the pellet. The DCC was resuspended by gentle
vortexing and the mixture incubated for 20 min at 0 C with 30 sec
intermittent vortexing every 5 min. At the end of the incubation
period, the dispersed DCC was pelletted by centrifugation at 2,230 g for
10 min and the resulting supernatant (i.e. the DCC-pretreated cytosol)
carefully aspirated for subsequent steps. Control groups underwent
identical procedural steps (in the absence of DCC) in parallel with
their DCC-treated counterparts. The thermal stability of unoccupied
receptors in each of these groups was tested by incubating the cytosol
in a water bath maintained at the desired temperature for the period of
time designated for each experiment. Following this "aging”, the cytosol
preparations were returned immediately to an ice bath. The protein
concentration in such preparations ranged from 3.1 to 3.9 mg/ml (Lowry
et al., 1951).
Hydrophobic Interaction Chromatography
Unlabeled cytosol was either up- or down-regulated by an
appropriate incubation in the presence or absence of DTT, respectively
(described in greater detail later). The different cytosolic
preparations were then run on Sephadex G-25 columns equilibrated and
eluted with HEPES buffer containing 600 mM KC1 and 50 mM molybdate at pH
7.6. Of the macromolecular fraction collected from these columns, 0.5 ml

65
was then run on a 7 ml pentyl agarose column equilibrated and eluted
with HEPES buffer containing 600 mM KC1 and 50 mM molybdate at pH 7.6.
Fractions (0.5 ml) were collected and incubated with 20 nM [3H]DEX +/- 4
uM [1H]DEX for 40 hr at 0 C. The fractions were then assayed for
specific binding.
Sucrose Density Gradient Sedimentation
Unlabeled cytosol was either up- or down-regulated by an
appropriate incubation in the presence or absence of DTT, respectively.
Aliquots (400 ul) were layered onto linear 5-20% sucrose density
gradients (5 ml; prepared with HEPES buffer containing 20 mM molybdate
and either with (for up-regulated receptors) or without (for down-
regulated receptors) 2 mM DTT and centrifuged at 0 C for 19-20 hr at
200-234,000 g (average) in a Beckman SW 50.1 rotor. The cellulose
nitrate tubes were punctured and 28-30 fractions (180 ul) collected and
incubated with 20 nM [3H]DEX +/- 4 uM [1H]DEX in the presence of 2 mM
DTT at 0 C for 24 hr. The individual fractions were then assayed for
specific binding. Sedimentation coefficients (S20,w) were calculated
from the linear regression of S20,w vs sedimentation distance (Martin
and Ames, 1961) for the following standard proteins run in parrallel
tubes: chicken ovalbumin (0VALB, 3.6 S), bovine serum albumin (BSA, 4.3
S), bovine gamma globulin (IgG, 7.4 S) and catalase (CAT, 11.3 S). The
standard proteins were [14C]methylated (for detection) to low specific
activity with [14C]formaldehyde by the method of Rice and Means (1971).
Steroid Binding Determination
In all experiments, bound [3H]-steroid was separated from free on
Sephadex G-25 columns (0.6 x 14 cm) pre-equilibrated in buffer identical
to that in which the cytosol was prepared. Duplicate aliquots from each

66
assay tube (BT and BNS) were layered onto separate columns, allowed to
penetrate the gel and the macromolecular (bound) fraction eluted (with
homologous buffer) directly into scintillation vials for liquid
scintillation spectrometry.
Results
The purpose of this investigation was to determine the role of
sulfhydryl oxidation-reduction in the regulation of steroid binding
capacity of the unoccupied glucocorticoid receptor. It was therefore of
importance to first characterize the time- and temperature-dependent
changes in binding capacity occurring in cytosol in the absence of
exogenously-added sulfhydryl reducing or oxidizing reagents. The first
experiment investigated the temperature-dependent reversible loss of
glucocorticoid binding capacity in the presence of molybdate, but in the
absence of sulfhydryl reducing reagents such as DTT. Unlabeled cytosol
was preincubated or "aged" at 22 C for 0, 2 and 4 hr, followed by
addition of buffer with or without DTT prior to the steroid incubation.
The experiment also examined the effects of aging of cytosol after the
addition of DTT. Results (Figure 3-1) indicate that the binding
capacity lost at 22 C in the presence of molybdate is completely
restorable upon addition of DTT. Aging of the unlabeled cytosol after
DTT addition had little effect on the binding capacity. Under these
conditions, half of the maximal binding capacity (that measured after
DTT addition) was reversibly inactivated after less than 4 hr of 22 C
aging, although approximately 25% of the maximal binding capacity
appeared to have already been lost prior to any 22 C aging whatsoever.
The next experiment was designed to more carefully investigate the
phenomenon studied in the previous experiment, except the period of

Figure 3-1. Temperature-dependent reversible loss of glucocorticoid
binding capacity of unoccupied Type II glucocorticoid receptors in the
presence of molybdate and absence of DTT. Solid circles represent brain
cytosol prepared in HEPES buffer containing 20 mM molybdate that has
been incubated at 22 C in the absence of steroid prior to a 24 hr
incubation with 20 nM [3H]DEX +/- 4 uM [1H]DEX at 0 C. Solid squares
represent the addition of DTT (2 mM final concentration) to the aged
cytosol just prior to the initiation of steroid incubations. Open
symbols represent one additional hr of 22 C aging after the addition of
HEPES buffer containing either 20 mM molybdate only (open circle) or 20
mM molybdate plus 20 mM DTT (open squares, 2 mM final DTT
concentration), but prior to steroid incubations. Bound-free steroid
separations were performed on Sephadex G-25 columns. Specific binding
is expressed as percent of the 0 hr control group (250 fmole/mg cytosol
protein).

T
T
T
T

69
preincubation aging was greatly extended to determine if the reversible
down-regulation of glucocorticoid binding capacity could reach 100%
under in vitro conditions. This experiment also sought to determine if
unlabeled glucocorticoid receptors in the sulfhydryl down-regulated
state were more or less stable for extended periods at 22 C than
up-regulated unlabeled receptors. Like the previous experiment,
unlabeled cytosol prepared with 20 mM molybdate was subjected to a
preincubation aging at 22 C, but this time for 0, 2, 5, 9, 21 and 30 hr
(Figure 3-2). In addition to the nonDTT-containing groups that received
HEPES buffer with or without DTT after the 22 C aging, another group was
included that contained DTT throughout the aging step. As in the
previous experiment, suboptimal binding was encountered even prior to 22
C aging when DTT was absent from the cytosol. Down regulation appeared
to reach virtual completion at 22 C, but preincubation on the order of
30 hr was required. Maximum binding capacity (i.e., after addition of
DTT) diminished only slightly (about 30%) during the 30 hr period.
There were clearly no significant differences in binding between the
groups provided with DTT after the preincubation and those provided with
DTT prior to the preincubation.
In an attempt to further characterize the endogenous factor(s)
involved in the reversible in vitro down-regulation of unoccupied
glucocorticoid receptors, cytosol preparations were subjected to a
dextran-coated charcoal (DCC) pretreatment with or without DTT addition
(before and/or after DCC). The effect of temperature on this
DTT-reversible binding loss was also examined under each of the possible
conditions by aging half of the cytosol from each group for an
additional 4 hr at 22 C prior to incubation with labeled steroid.

Figure 3-2. Temperature-dependent reversible loss of glucocorticoid binding capacity of unoccupied Type II
glucocorticoid receptors in the presence of molybdate and absence of DTT after extended periods of
incubation at 22 C. Open bars represent brain cytosol prepared in HEPES buffer containing 20 mM molybdate
and incubated at 22 C in the absence of steroid prior to a 24 hr incubation with 20 nM [3H]DEX +/- 4 uM
[1H]DEX at 0 C. Hatched bars represent the addition of DTT (2 mM final concentration) to the preincubated
cytosol just prior to the initiation of steroid incubations. Solid bars represent the addition of 2 mM DTT
prior to the incubation at 22 C in the absence of steroid followed by the steroid incubations. Bound-free
steroid separations were performed on Sephadex G-25 columns. Specific binding is expressed as percent of
the 0 hr control group (310 fmole/mg cytosol protein) and represents the mean +/- S.E.M. of 3 independent
replications.

100
75
50
25
5 9
Duration of 22°C Aging (hours)
0
2
21
30

72
Finally, liver and kidney cytosol preparations were treated in an
identical manner for comparative purposes since these tissues reportedly
have a higher endogenous reducing potential (Granberg and Ballard,
1977). For brain, the DCC pretreatment in the absence of DTT additions
or subsequent 22 C aging led to a significant decrease (approximately
40%) in the binding capacity for [3H]DEX (Figure 3-3). There was also
an increase in the rate of loss of binding capacity at 22 C. When DTT
was added back to cytosol after DCC pretreatment there was virtually a
100% recovery of binding capacity seen in cytosol treated with DTT but
not DCC. When DTT was added to cytosol prior to DCC treatment, there
was still a loss of binding capacity to a level similar to that seen if
no DTT was present before DCC. However, there was no significant
additional loss of binding capacity after a subsequent 22 C aging as was
the case for the DTT-, then DCC-treated group. Interestingly, when DTT
was added both before and after the DCC pretreatment, there was only a
partial restoration of the total binding capacity although there was
again very little loss of binding capacity associated with aging.
Results for liver and kidney were clearly different from those obtained
for brain and surprizingly different from what was expected based on the
findings of other workers regarding the endogenous reducing potential
for these tissues. The degree of loss of binding capacity seen in
groups not pretreated with DCC, DTT or 22 C aging was greater in liver
and much greater in kidney than that observed for brain. In addition,
the rate of loss of binding capacity for these same groups during 22 C
aging was greatly enhanced for both tissues when compared to brain.
However, similar to brain cytosol, DCC pretreatment led to a significant
decrease in binding capacity that was fully reversible upon addition of

Figure 3-3. Effects of dextran-coated charcoal pretreatment and DTT on
glucocorticoid binding capacity and thermal stability of unoccupied Type
II glucocorticoid receptors from brain, liver and kidney. Concentrated
cytosol was prepared in HEPES buffer plus 20 mM molybdate and then
diluted with either additional molybdate-containing buffer or molybdate-
containing buffer plus 20 mM DTT (2 mM final concentration) prior to a
20 min incubation in the presence or absence of dextran- coated charcoal
at 0 C. Cytosols were then further diluted with either additional
molybdate-containing buffer or molybdate-containing buffer plus 20 mM
DTT (2 mM final concentration) and then incubated at 22 C for either 0
(solid bars) or 4 (hatched bars) hr in the absence of steroid prior to
incubation with 20 nM [3H]DEX +/- 4 uM [1H]DEX at 0 C for 24 hr.
Bound-free steroid separations were performed on Sephadex G-25 columns.
Specific binding is expressed as percent of the 0 hr control groups
(300, 281, and 341 fmole/mg protein for brain, liver and kidney
cytosols, respectively) and represents the mean + S.E.M. of 5
independent replications and 3 independent replications for liver and
kidney.

120
100
80
60
40
20
100
80
60
40
20
00
80
60
40
20
DT
DC
74
BRAIN
0-HR AGING
4-HR AGING
LIVER
KIDNEY
- - + + +
+ + - + -f
+ - - +

75
DTT. Unlike brain cytosol, however, both liver and kidney cytosols
exhibited a significant degree of protection from DCC-induced binding
loss when pretreated with DTT. However, DTT pretreatment preceding DCC
pretreatment of liver and kidney cytosol appeared to be less effective
in preventing further binding loss during 22 C aging than was found to
be the case for brain cytosol treated with DTT before and after DCC
pretreatment was significantly higher for liver and kidney than it was
for brain.
Since the chemical nature of the sulfhydryl up- and down-regulation
process was unknown, an investigation was made to determine if the
process involved any major, easily measurable changes in the molecular
structure of the receptor (i.e. receptor subunit association or
dissociation or major conformational changes). The aim of this
experiment was to compare the sedimentation properties of up- and
down-regulated unoccupied glucocorticoid receptors from brain using
sucrose density gradient analysis. Cytosol was up-regulated by
incubating with DTT at 0 C prior to the gradient run on sucrose
gradients prepared and DTT. Cytosol was down-regulated by incubating
without DTT at 22 C for 4 hr prior to the gradient run on sucrose
gradients prepared without DTT. All cytosol groups and sucrose
gradients contained molybdate. After an 18 hr gradient run, gradients
of both treatment groups were subjected to fractionation followed by
postlabeling of the individual fractions with [3H]DEX. Steroid
incubation conditions were identical for both groups except that DTT had
to be added back to each fraction of the down-regulated group to allow
for up-regulation so that binding could be determined. The profiles
representing the specific binding determined for each fraction indicated

76
that both forms sediment in one major peak of approximately 9.2 S
(Figure 3-4). The results therefore provide little evidence for any
major conformational changes in the receptor associated with sulfhydryl
regulation of binding capacity under these conditions.
To determine if the changes in glucocorticoid binding capacity
associated with sulfhydryl down-regulation involve merely a decrease in
the maximal binding or, instead, a modification of the receptor binding
affinity, Scatchard analysis of DEX binding was performed for cytosol
either containing or not containing DTT (Figure 3-5). The addition of
DTT led to an apparent increase in both the affinity and maximal
binding. Equilibrium binding constants were also determined after 4 hr
of 22 C aging of the cytosol with and without DTT prior to steroid
incubation. This aging only had a significant effect on the parameters
determined for non-DTT-containing cytosol, leading to a further decrease
in both maximal binding and apparent affinity.
If the apparent reduction in affinity associated with sulfhydryl
down-regulation as determined by Scatchard analysis is real, it is
likely to be due to either a decrease in the rate of steroid
association, an increase in the rate of steroid dissociation or both.
Each of these kinetic parameters were investigated. To study the effect
of reducing reagents on the rate of steroid dissociation from the
glucocorticoid receptor, cytosol was first prelabeled with [3H]DEX in
the presence of molybdate and DTT prior to being run on a Sephadex G-25
column equilibrated in non-DTT-containing buffer to remove both the free
steroid and the DTT. DTT was then added back to half of the preparation
while the other half was appropriately diluted with non-DTT-containing
buffer. As illustrated in Figure 3-6, there was no significant

Figure 3-4. Sucrose density gradient analysis of sulfhydryl-reduced and sulfhydryl-oxidized unoccupied
Type II glucocorticoid receptors. Brain cytosol prepared in the absence of DTT (HEPES buffer plus 20 mM
molybdate) was either incubated in the absence of steroid for 4 hr at 22 C (solid squares) or supplemented
with DTT (solid triangles, 2 mM final concentration) and stored at 0 C for 4 hr. Aliquots (400 ul) from
both cytosol samples were then sedimented through 5-20% linear sucrose gradients in a SW 50.1 rotor at
200,000 x g for 18 hr. Samples containing DTT (solid triangles) were run on gradients prepared with HEPES
buffer plus 20 mM molybdate and 2 mM DTT, whereas samples not containing DTT (solid squares) were run on
gradients prepared with HEPES buffer containing only 20 mM molybdate. Fractions were collected directly
into ice-cold tubes containing either [3H]DEX in HEPES buffer plus 20 mM molybdate and 2 mM DTT (solid
triangles) or [3HJDEX plus HEPES buffer plus 20 mM molybdate and 20 mM DTT (solid squares, 2 mM DTT final
concentration). The individual fractions were then incubated for 24 hr at 0 C prior to bound-free steroid
separations on LH-20 columns. Results presented here represent the mean of 2 independent replications.

PERCENT OF THE TOTAL BSP

Figure 3-5. Scatchard analysis of [3HJDEX binding to Type II
glucocorticoid receptors. Brain cytosol was prepared in HEPES buffer
containing 20 mM molybdate plus (circles) or minus (triangles) 2 mM DTT
and with (solid symbols) or without (open symbols) a 4 hr incubation at
22 C in the absence of steroid prior to incubation with [3H]DEX +/-
200-fold [1H]DEX at 0 C for 24 hr. Bound-free steroid separations were
performed on Sephadex G-25 columns.

Bsp / F
80
0-4
02-
•
+ DTT
4 HRS
AT
22°C
0
+ DTT
0 HRS
AT
o°c
A
— DTT
4 HRS
AT
22° C
A
-DTT
0 HRS
AT
o
0
O
06 08 1-0
Bsp (nM)

Figure 3-6. Effect of DTT on the rate of dissociation at 22 C of [3H]DEX from Type II glucocorticoid
receptors. Brain cytosol prepared in HEPES buffer plus 20 mM molybdate and 2 mM DTT was incubated with 40
nM [3H]DEX for 50 hr at 0 C. Labeled cytosol was then run on a jacketed Sephadex G-25 column (1 x 50 cm)
at -4 C to remove both the free steroid and the DTT. Cytosol was then incubated in the presence of 10 uM
[1H]DEX at 22 C either with (circles) or without (squares) 2 mM DTT. Aliquots were removed periodically
for bound-free steroid separations on Sephadex LH-20 columns.

HOURS
PERCENT OF ZERO HR CONTROL
Z8
IOO

83
differences in the rate of dissociation between receptors in the
presence or absence of DTT. Although an attempt was made to study the
effect of DTT on the rate of association of [3H]DEX to glucocorticoid
receptors, the results of this experiment were confounded by the fact
that the binding capacity of cytosol lacking DTT was undergoing a
time-dependent loss that did not occur in the presence of DTT.
A series of experiments were next performed to further characterize
the sulfhydryl up and down-regulation of glucocorticoid binding capacity
by studying the reactivity of the essential sulfhydryl group(s) with a
variety of known reversible and irreversible sulfhydryl-reactive agents.
The first experiment examined the effects of a 1 hr incubation of
unlabeled cytosol with 1 mM dithionitrobenzoic acid (DTNB),
n-ethylmaleimide (NEM), iodoacetamide, cystamine, glutathione disulfide
(GSSG) and para-chloromercurisulfonate (PCMS) with or without the
»
subsequent addition of 10 mM DTT on glucocorticoid binding capacity as
measured by [3HJDEX binding (Figure 3-7). DTNB and cystamine led to a
virtual total loss in binding capacity which was completely reversible
upon DTT addition. GSSG resulted in only a partial loss in binding
capacity, but this loss was also completely reversible upon DTT
addition. NEM, iodoacetamide and PCMS led to varying degrees of loss
which were not completely reversible with DTT. In a follow-up
experiment, conditions identical to those described in the previous
experiment were used to investigate the effects on glucocorticoid
binding capacity of several additional sulfhydryl reactive reagents
including iodoacetic acid, sodium-m-periodate, sodium-p-periodate,
6,6'-dithionicotinic acid (DTNT), methyl methane thiosulfonate (MMTS)
and mercuric chloride (Figure 3-8). Iodoacetic acid was relatively

Figure 3-7. Effects of O C incubation of cytosol with various known reversible and irreversible
sulfhydryl-reactive reagents on Type II glucocorticoid receptor binding capacity with and without
subsequent addition of excess DTT. Brain cytosol prepared in HEPES buffer plus 20 mM molybdate was
incubated at 0 C for 1 hr with either additional HEPES buffer dilution (BUFF) or a 1 mM final concentration
of dithionitro- benzoic acid (DTNB), n-ethyl maleimide (NEM), iodoacetamide (IAM), cystamine (CYS),
glutathione dissulfide (GSSG) and para-chloro- mercurisulfonate (PCMS). Cytosol samples were then
supplemented with (solid bars) or without (open bars) and excess of DTT (10 mM final concentration) prior
to incubation with 20 nM [3H]DEX +/- 4 um [1H]DEX at 0 C for 40 hr. Bound-free steroid separations were
performed on Sephadex G-25 columns. Specific binding is expressed as percent of the BUFF control group
after addition of DTT (317 fmol/mg protein) and represents the mean +/- S.E.M. of 3 independent
replications.

100
75
50
25
BUFF DTNB CYS GSSG I AM NEM PCMS
00
i_n

Figure 3-8. Effects of O C incubation of cytosol with additional known reversible and irreversible
sulfhydryl-reactive reagents on Type II glucocorticoid receptor binding capacity with and without
subsequent addition of excess DTT. Brain cytosol prepared in HEPES buffer plus 20 mM molybdate was
incubated at 0 C for 1 hr with either additional HEPES buffer (BUFF) or a 1 mM final concentration of
6,6'-dithionicotinic acid (DTDN), methyl methane thiosulfonate (MMTS), sodium-m-periodate (SMP),
sodium-p-periodate (SPP), iodoacetic acid (IAA) or mercuric chloride (HgC12). Cytosol samples were then
supplemented with (solid bars) or without (open bars) an excess of DTT (10 mM final concentration) prior to
incubation with 20 nM [3H]DEX +/- 4 um [1H]DEX at 0 C for 40 hr. Bound-free steroid separations were
performed on Sephadex G-25 minicolumns. Specfic binding is expressed as percent of the BUFF control group
after addition of DTT ( 347 fmol/mg protein) and represents the mean +/- S.E.M. of 3 independent
replications.

Percent of Control

88
ineffective in reducing the binding capacity, possibly due to the pH of
the system or the low concentration used, but it did produce some
irreversible losses. There was no difference between the two isomers of
sodium periodate which were intermediate in reducing binding capacity
which was not completely DTT-reversible. DTNT resulted in nearly a
total loss of binding capacity which was completely reversible while
MMTS was much less effective in reducing binding capacity. However,
total reversibility with DTT in the case of MMTS was less certain than
for DTNT. Finally, exposure to mercuric chloride resulted in a complete
loss of binding capacity which was partially reversible.
The next step was to investigate the ability of DTT-reversible
sulfhydryl reactive reagents to protect unoccupied receptors from
irreversible inactivation due to non-DTT-reversible sulfhydryl reactive
reagents. This experiment was designed to determine if unoccupied
receptors, down-regulated by endogenous sulfhydryl oxidizing factors,
were more or less protected from irreversible sulfhydryl reactive
reagents than were receptors down-regulated by the DTT-reversible
reagents used in the previous experiments. This was an effort to
possibly shed light on the nature of the down-regulated form in vivo as
well as to provide information on a possible means of "trapping" up-
and/or down-regulated forms of the receptor so that the true status of
up- and down-regulated receptors in vivo may someday be determined.
This experiment involved first, a 1 hr incubation of unlabeled cytosol
at 0 C with DTNB, cystamine, GSSG or no reagent for the DTT-reversible
down-regulation of the unoccupied receptors. This was followed by an
additional 1 hr incubation of each of the above groups with either NEM,
iodoacetamide, mercuric chloride or no reagent. This second incubation

89
with DTT-irreversible sulfhydryl reactive reagents was followed by
further dilution with or without DTT prior to [3H]DEX binding. Binding
data obtained for all 32 possible groups are illustrated in Figure 3-9.
DTNB and cystamine provided almost complete protection against
irreversible losses caused by either NEM or iodoacetamide. GSSG, on the
other hand, provided only a slight protection against the NEM- and
iodoacetamide-induced losses in binding capacity. Interestingly, none
of the reversible reagents provided any significant degree of protection
against the irreversible effects of mercuric chloride when compared to
the control that subsequently received mercuric chloride. These later
findings imply that either mercuric chloride is ineffective against the
endogenous down-regulated form, while being effective against the forms
down-regulated via reaction with DTNB, cystamine or GSSG, or that
mercuric chloride's effects are relatively nonspecific against both up-
and down-regulated receptors.
If one makes the assumption that mercuric chloride is acting
specifically on the sulfhydryl up-regulated form of the receptor, a
possibility implied by the results of the previous experiment, then one
would expect the incubation of cytosol containing quantitatively known
populations of both up- and down-regulated glucocorticoid receptors with
mercuric chloride, followed by addition of excess DTT (to neutralize the
reaction) at various time intervals, to result in a decreasing binding
capacity that approaches asymptotically the level of down-regulated
receptors. The results, shown in Figure 3-10, basically confirm this.
In the presence of 2 mM mercuric chloride, the binding capacity was
reduced by around 35% of maximum, a level approximating that of the
down-regulated receptors determined under similar conditions.

Figure 3-9 . The ability of DTT-reversible sulfhydryl reagents to
protect unoccupied Type II glucocorticoid receptors from irreversible
inactivation due to non DTT-reversible sulfhydryl-reactive reagents.
Brain cytosol prepared with HEPES buffer plus 25 mM molybdate was
incubated at 0 C with either additional HEPES buffer (BUFF) or a 1 mM
final concentration of dithionitrobenzoic acid (DTNB), cystamine (CYS)
or glutathione dissulfide (GSSG). Cytosol samples were then incubated
at 0 C for a second hr with either additional HEPES buffer (BUFF) or a 2
mM final concentration of n-ethyl maleimide (NEM), iodoacetamide (IAM)
or mercuric chloride (HgC12). Each subset of cytosol was then incubated
at 0 C for an additional 1 hr prior to the addition of either HEPES
buffer (open bars) or HEPES buffer containing excess DTT (20 mM final
concentration, solid bars) followed by incubation at 0 C for 40 hr with
20 nM [3HJDEX +/- 4 um [1H]DEX. Bound-free steroid separations were
performed on Sephadex G-25 columns. Specific binding is expressed as
percent of the BUFF-BUFF plus DTT control group (302 fmol/mg protein)
and represents the mean +/- S.E.M. of 3 independent replications.

Percent of Control Percent of Control
91
BUFF BUFF BUFF BUFF DTNB DTNB DTNB DTNB
BUFF NEM 1AM HgCI2 BUFF NEM 1AM HgCI2
CYS CYS CYS CYS GSSG GSSG GSSG GSSG
BUFF NEM I AM HgCI2 BUFF NEM I AM HgCI2

Figure 3-10. Time course of the effects of 1 mM mercuric chloride on Type II glucocorticoid receptor
binding capacity in the absence of steroid and with the subsequent addition of DTT. Brain cytosol prepared
with HEPES buffer plus 20 mM molybdate was either brought to a final concentration of 1 mM mercuric
chloride (triangles) or was diluted with an appropriate volume of HEPES buffer (circles). Cytosols were
then incubated at 0 C for the times indicated prior to addition of excess DTT (10 mM final concentration)
followed by incubation at 0 C for 40 hr with 20 nM [3H]DEX +/- 4 urn [1H]DEX. Bound-free steroid
separations were performed on Sephadex G-25 columns. Specific binding is expressed as the percent of the 0
hr control (no mercuric chloride) (297 fmol/mg protein) and represents the mean +/- S.E.M. of 3 independent
replications.

Percent of Control
Duration of 0°C Aging (Hours)

94
The next experiment was designed to determine if DTT-reversible
sulfhydryl reactive reagents were capable of interacting with prelabeled
glucocorticoid receptors and affecting the rate of steroid dissociation,
inactivation, etc. for the steroid receptor complex. Cytosol was
prepared in the absence of any sulfhydryl reactive or reducing reagents
and prelabeled with 20 nM [3H]DEX for 40 hr at 0 C. After a bound/free
separation on Sephadex G-25 columns, cytosol was incubated with 1 mM
concentration of DTNB, cystamine, GSSG or an appropriate dilution of
HEPES buffer devoid of any sulfhydryl reagents for 4 hr at 0 C in the
presence or absence of 4 uM [1H]DEX. The results from this experiment
(data not shown) confirmed expectations that these DTT-reversible
reagents could only affect the steroid binding properties of unoccupied
glucocorticoid receptors since no enhancement of the rate of
dissociation of [3H]DEX was evident. It was evident, however, that
interactions between these reagents and receptors unoccupied by virtue
of normal steroid dissociation did reversibly prevent steroid rebinding.
When the previous experiment was replicated using mercuric
chloride, instead of known DTT-reversible reagents, very different
results were obtained. When cytosol containing both [3H]DEX-labeled
(up-regulated) receptors and down-regulated unoccupied receptors was
incubated in the presence of 1 mM mercuric chloride for 4 hr, there was
a near total loss of receptor binding. Upon addition of DTT, and after
subsequent reincubation with [3HJDEX, binding equivalent to the
population of down-regulated unoccupied receptors reappeared (data not
shown), indicating the possibility that bound steroid was not protective
of the inactivating effects of the mercury, but that the down-regulated
form was somehow protected from such inactivation.

95
The fact that the in vivo down-regulated receptor is protected from
inactivation by mercurial reagents, while steroid binding or reversible
interactions with DTNB, cystamine and GSSG are ineffective in protecting
the receptor from inactivation could have potentially important
functional implications for the role and/or the mechanism of
down-regulation in vivo if proven to be true. The aim of the next
experiment was to further investigate this phenomenon by looking at the
effects of mercuric chloride on receptors that had been completely
up-regulated and labeled in the presence of DTT as well as a mixed
population as was studied before. If the original theory is correct,
one would expect to see a total irreversible loss of the DTT-incubated
samples while incubation of the mixed population would result in a total
binding loss that was partially restorable (representing the
down-regulated unoccupied receptors) upon addition of excess DTT. The
experiment was also different from the previous experiment in that both
plus and minus DTT cytosol samples were run through Sephadex G-25
columns equilibrated with buffer devoid of DTT prior to incubation with
mercuric chloride. In addition to removing DTT, this column run also
removed endogenous small thiols that compete or otherwise interfere with
the mercury-thiol reaction. After these Sephadex G-25 column runs,
cytosol was incubated with mercuric chloride, followed by another G-25
column run and subsequent reincubation with [3H]DEX in the presence of
high concentrations of DTT. The results provided evidence that the
original theory regarding the selective action of mercury against
up-regulated receptors only was flawed (Figure 3-11). To begin with,
the incubation with mercuric chloride did not lead to a complete loss of
previously bound receptors as previously thought. Although this

Figure 3-11. Effects of mercuric chloride on occupied type II
glucocorticoid receptors labeled in the presence or absence of DTT.
Brain cytosol prepared in HEPES buffer plus 25 mM molybdate was divided
into half and supplemented with either HEPES buffer (B) or HEPES buffer
plus DTT (D, 10 mM final concentration). Both groups were then
incubated at 0 C for 4 hours to allow for reduction of all reversibly
oxidized sulfhydryls in the DTT group. Both groups were then run
through Sephadex G-25 columns to remove DTT and any small endogenous
thiols present. Each group was then treated with either HEPES buffer
plus mercuric chloride (DHg, BHg; 2 mM final concentration) or HEPES
alone (DB, BB) and then incubated for 4 hours at 0 C. Aliquots of the
different cytosolic groups were run directly on Sephadex G-25 columns
for bound-free separations (hatched bars). HEPES buffer plus DTT (10 mM
final concentration) and 20 nM [3H]TA + 4 uM [1H]TA was added to the
remainder of each of the cytosol groups and another aliquot was
immeadiately taken from each for bound-free separation (screened bars).
The remaining cytosol was incubated for an additional 40 hr at 0 C prior
to running the final bound-free separation (solid bars). The binding is
expressed as percent of the initial control binding of the DTT only
group (DB open bar, 340 fmole/mg protein) and represents the mean of 2
replications. All binding is corrected for sequencial dilutions.

PERCENT of CONTROL
ro d) CD o
o.o o o o

98
appeared to be the case when the first bound/free separations were
performed after the incubation with mercuric chloride, but prior to the
readdition of DTT, it was discovered that addition of excess DTT caused
an immeadiate "restoration" of part of the binding, too fast to be
accounted for by rebinding of dissociated receptors or up-regulation and
subsequent binding of down-regulated forms. It was obvious from changes
in the physical appearance of the cytosol after the addition of mercuric
chloride and again after the subsequent addition of excess DTT that the
mercury was causing an aggregation of cytosolic protein leading to a
faintly cloudy suspension. Dissolution of the suspension occurred
instantly upon addition of DTT indicating that a portion of the "lost”
binding was simply complexed or trapped in the suspension, unable to
pass through Sephadex G-25 gel. Perhaps even more notable was that
there was no significant increase in binding in either the DTT- or
non-DTT-pretreated cytosols from the time that DTT was readded after
mercury treatment until 40 hours later. In other words, the mercury
seemed to affect the receptor binding capacity the same regardless of
whether or not the cytosol contained only up-regulated (bound) receptors
or a mixed population of bound and down-regulated unbound receptors.
There was still a possibility that there may be some difference in the
degree to which mercury will inactivate unoccupied up-regulated as
opposed to unoccupied down-regulated receptors and this was examined in
the following experiment. However, it is clear from the results of this
experiment that the down-regulated receptor is not immune from the
inactivating effects of mercuric chloride as was previously thought and
is therefore less likely to be a useful tool in the determination of
relative populations of up- and down-regulated receptors in situ.

99
Although previous experiments investigated the effects of the
potentially irreversible sulfhydryl reactive reagents mercuric chloride,
NEM and iodoacetamide on a standard cytosol preparation (containing a
mixed population of up- and down-regulated DEX receptors) or, in the
case of mercuric chloride, prelabeled receptors, the next experiment
studied the effects of these reagents on receptor preparations
containing either entirely up-regulated or down-regulated unoccupied
receptors. The preparations were made by first preparing cytosol in the
absence of DTT, then either adding DTT and incubating in ice for 6 hours
(for up-regulated receptors) or not adding DTT and incubating at 22 C
for 6 hours (for down-regulated receptors). Admitedly, this procedure
may not have resulted in 100% down-regulation, but it appears to have
been better than 90% effective. Both treatment groups were then
subjected to Sephadex G-25 chromatography to remove DTT (where present)
as well as endogneous small thiols that would interfere with the
reaction prior to incubation with the reagent. Addition of excess DTT
halted the reaction prior to steroid incubation. Consistent with the
findings of the previous experiment, mercuric chloride incubation
resulted in the same degree of inactivation of binding capacity for both
up- and down-regulated groups (Figure 3-12). Both NEM and iodoacetamide
significantly inactivated the binding capacity of the up-regulated group
(about 90% binding loss), whereas they were differentially effective
against the down-regulated group. Most of the down-regulated binding
capacity was restorable after incubation with NEM (about 80% or better),
while only about half was restorable after incubation with
iodoacetamide. Since the ideal reagent would rapidly inactivate
up-regulated receptors while remaining ineffective against

Figure 3-12. Effects of mercuric chloride, NEM and iodoacetamide on sulfhydryl up-regulated and
down-regulated unoccupied Type II glucocorticoid receptors. Brain cytosol was prepared in HEPES buffer
plus 25 mM molybdate and then either brought to 10 mM DTT and incubated at 0 C for 6 hr (up-regulated
receptors, solid bars) or diluted with an appropriate volume of HEPES buffer only and then incubated at 22
C for 6 hr (down-regulated receptors, open bars). Each group was then run on Sephadex G-25 columns to
remove DTT and/or other small thiol-containing molecules, followed by incubation with a 2 mM final
concentration of mercuric chloride (Hg), n-ethyl maleimide (NEM) or iodoacetamine (IAM) for 1 hr at 0 C.
Each group was then incubated with 20 nM [3H]DEX +/- 4 um [1HJDEX in the presence of 50 mM DTT for 40 hr at
0 C followed by bound-free steroid separations on Sephadex G-25 columns (open bars for B and DTT groups
were incubated with steroid in the absence of additional DTT). Specific binding is expressed as percent of
the DTT control group (open bar, 325 fmole/mg protein) and represents the mean +/- S.E.M. for 3 indepedent
replications.

DTT H q N E M IA M
no
o
i
PERCENT of CONTROL
o
o
1
& CD CD
O O O
J I L
101

102
down-regulated forms, NEM appears to be the most appropriate reagent
based upon these results. However, dose response and time course
studies were required to more accurately characterize these reactions.
Time course and dose response studies of the effects of NEM and
iodoacetamide on down-regulated and up-regulated unoccupied receptors
(mixed population) were next performed in an effort to more clearly
understand the effects of these two sulfhydryl reagents. Cytosol
containing a mixed population of up- and down-regulated receptors was
incubated at 0 C with 4 different concentrations (0, 2, 10 and 50 mM) of
each of the two reagents for .5, 2 or 4 hours prior to the addition of
excess DTT and incubation with [3H]DEX. Dose studies were necessary
since it had not been determined how much of each of these reagents was
required to saturate the endogenous thiols and other potentially
reactive groups in crude cytosol (unchromatographed cytosol was used in
this experiment for this reason). A time course was required to
determine how rapidly the sulfhydryl oxidizing reaction occurs as well
as if there are two or more reactions occurring at different rates (this
might occur if the up-regulated form undergoes a rapid oxidation with a
half-life of seconds or minutes and the down-regulated form undergoes a
slow up-regulation and is subsequently oxidized quickly). Three
replications of the experiment were performed with a high degree of
replicability. As might have been expected from the previous
experiment, NEM provided the most promising results (Figure 3-13) in
that there appeared to be a "ceiling" effect with both time and dose
(except for the highest dose). It is possible that a ceiling wasn't
observed for the high dose because of a potentially inadequate
concentration of DTT added to neutralize the reaction. With

Figure 3-13. Time course and dose response studies of the effects of NEM and iodoacetamide on a mixed
population of sulfhydryl down-regulated and up-regulated unoccupied Type II glucocorticoid receptors.
Brain cytosol was prepared in HEPES buffer plus 25 mM molybdate and incubated with a 0 (BD, BB), 2, 10 or
50 mM final concentration of either iodoacetamide (I) or n-ethyl maleimide (M) for 0.5 (open bars), 2
(screened bars) or 4 (solid bars) hr prior to the addition of 100 mM DTT (except for BB, which received an
equivalent dilution with HEPES buffer only) and incubation with 20 nM [3H]DEX +/- 4 uM [1H]DEX at 0 C for
40 hr. Bound-free steroid separations were performed on Sephadex G-25 columns. Specific binding is
expressed as percent of the BD, 0.5 hr, control group (360 fmole/mg protein) and represents the mean + /-
S.E.M. of 3 independent replications.

100
80
60
40
20
I I 5-Hr Aging
SÜ 2-Hr Aging
H 4-Hr Aging
BD BB 12 110 N2 NIO N50
104

105
iodoacetamide, loss of binding capacity was time dependent regardless of
concentration and higher concentrations resulted in total loss of all
binding (including both up- and down-regulated forms). Perhaps
iodoacetamide is less specific than NEM and may be inactivating
down-regulated receptors by acting on nonbinding-site sulfhydryl or
other reactive groups. Mercuric chloride was not examined in this
experiment since the previous 2 experiments provided strong evidence
that mercury does not distinguish between up- and down-regulated
receptors.
Further attempts to characterize the differences in hydrodynamic
properties that exist between the up- and down-regulated unoccupied
glucocorticoid receptors included the hydrophobic interaction
chromatography of both forms of the receptor on long pentyl agarose
columns. Cytosol preparations that were either up- or down-regulated
were equilibrated with HEPES buffer containing 600 mM KC1 and 50 mM
molybdate, but not DTT, prior to running on a pentyl agarose column
equilibrated and eluted with the same buffer. Fractions were then
incubated with 20 nM [3H]DEX +/- 4 uM [1H]DEX in the presence of 2 mM
DTT. As shown in Figure 3-14, the down-regulated form appeared to have
a slightly higher affinity for the hydrophobic matrix than the
up-regulated form although the differences were not large.
The next experiment investigated the possibility that free thiol
groups, other than those alledged to be in the steroid binding site,
exist on the accessible surface of the glucocorticoid receptor. Cytosol
labeled with [3H]TA for 40 hr at 0 C was chromatographed on each of
several sulfhydryl affinity gels including activated thiol sepharose 4B,
thiopropyl-sepharose 6B, glutathione disulfide agarose and sulfhydryl

Figure 3-14. Hydrophobic interaction chromatography of the sulfhydryl
up-regulated and down-regulated Type II glucocorticoid receptors. Brain
cytosol was prepared in HEPES buffer plus 25 mM molybdate and either
brought to a concentration of 10 mM DTT and then incubated at 0 C for 8
hr (up-regulated receptor, open circles) or provided with an equivalent
dilution of HEPES buffer only and Incubated at 22 C for 8 hr (down-
regulated receptor, solid circles). Each group was then run on a
Sephadex G-25 column equilibrated and eluted with HEPES buffer plus 600
mM KC1 and 50 mM molybdate. Aliquots (0.5 ml) from the resulting
macromolecular fractions were run on long (7 ml) pentyl agarose columns,
equilibrated and eluted with the same HEPES, KC1, molybdate, buffer and
collected (0.5 ml/fraction) into tubes and placed on ice. Half of the
contents of each tube was transfered to tubes containing 20 nM [3H]DEX
and a sufficient concentration of DTT in HEPES buffer to result in a
final concentration of 2 mM. The other half of each fraction was added
to tubes containing the same concentration of labeled steroid and DTT
plus 4 uM [1H]DEX for nonspecific binding profile determination (solid
triangles, same profile results for both groups). Each condition was
run in triplicate and the profiles shown are representative.

107

108
cellulose. The sulfhydryl reagents covalently linked to the gel
matrices react to varying degrees with the accessible free thiols of
cytosolic proteins resulting in mixed disulfide formation and the
retention of the thiol-containing protein by the gel. Since the thiol
group(s) whose status determines the receptor's ability to bind steroid
ligand is supossedly protected by the presence of a bound ligand (as
indicated by the results of the previous experiment), the retention of
bound glucocorticoid receptors by sulfhydryl affinity gels would
indicate the likely presence of additional surface thiol groups. The
existence of such extra-binding site thiols could affect the
interpretation of quantitative sulfhydryl studies as well as allow for
partial purification of the bound receptor. Varying degrees of
retention of the [3H]TA-labeled receptors were displayed by the gels
with thiopropyl-sepharose 6B having the highest affinity for receptors
and thiol sepharose 4B exhibiting the lowest affinity (data not shown).
This step was followed by an elution with DTT-containing buffer which
reduces the mixed disulfide bonds, thereby freeing the receptor from the
gel matrix and allowing for its removal from the column. The receptor
elution profiles after DTT elution again varied depending upon the gel,
but nevertheless providing unquestionable evidence for additional
surface free thiols.
Discussion
Since sulfhydryl group oxidation was implicated in the inactivation
of estrogen receptor binding (Jensen et al., 1967), the phenomenon has
been reported for practically all other steroid receptor types including
glucocorticoid receptors. Unfortunately, sulfhydryl oxidation and
reduction of steroid receptors has been viewed by most workers in the

109
field as merely an in vitro inconvenience to be overcome by including
sulfhydryl reducing reagents, such as DTT or mercaptoethanol, in their
buffers. Few viewed the process as a potential mechanism by which
preexisting receptor binding sites were up- or down- regulated in vivo
and, as a result, many aspects of glucocorticoid receptor sulfhydryl
oxidation/reduction have not been thoroughly investigated. This study
sought to examine the process in much greater detail than had been
previously reported in an attempt to determine if it was even feasible
to suggest such an in vivo role for oxidation/reduction in the regula¬
tion of glucocorticoid binding capacity. The initial experiments in
this study confirmed in brain, liver and kidney cytosol what had
previously been reported regarding the requirement for sulfhydryl
reducing reagents in order to optimize glucocorticoid receptor binding
in thymus cytosol and that there is a temperature-dependent decline in
this binding in the absence of such reagents (Rees and Bell, 1975). The
results obtained here for brain and other tissues also confirmed the
findings of Sando et al. (1979) for thymic lymphocyte cytosol, who
reported a complete restoration of this lost binding capacity upon
readdition of DTT if molybdate had been present in the buffers. A
restoration of binding upon DTT addition had been reported earlier by
Granberg and Ballard (1977) for cytosol from several tissues, including
brain, but this restoration was only partial because of the lack of
molybdate in their buffers.
The fact that nearly 30 hr were required in order to completely
down-regulate glucocorticoid binding capacity in brain cytosol may not
be very relevant, in a physiological sense, to the understanding of
down-regulation in vivo, if, in fact, it does occur there. However, the

110
fact that binding capacity could eventually be reversibly reduced to
nothing has important implications for the understanding of how
sulfhydryl oxidation interferes with measured glucocorticoid binding
capacity. Another potentially important finding during these initial
experiments was the fact that unoccupied receptors in the down-regulated
or oxidized state do not appear to be any more or less susceptible to
irreversible inactivation than those receptors in the up-regulated or
reduced state. It is known that a number of biochemical processes
involving proteins, such as phosphorylation (Chen and Kim, 1985), are
dependent on the sulfhydryl-disulfide state of the protein. It is
possible, however, that the presence of molybdate may have prevented any
increase in the rate of irreversible inactivation induced by either
sulfhydryl oxidation or reduction.
In order to gain a better understanding of the nature of the
»
factors in cytosol responsible for maintaining the receptor in an up-
regulated state, cytosol was pretreated with dextran-coated charcoal and
the rate of reversible binding loss was evaluated at elevated
temperatures as it was in previous experiments. It is clear that an
endogenous factor responsible for the up-regulation of unoccupied
receptors is removed by the charcoal treatment, since the rate of
sulfhydryl down-regulation is dramatically increased. One must,
however, consider the possibility that charcoal is removing a factor
which indirectly affects the up-regulation of receptors. According to
Gilbert (1984), for thiol/disulfide exchange to be considered as a
feasible biological control mechanism for protein action (enzyme
activity, receptor binding, etc.), the thiol/disulfide redox potential
of a regulated protein should be near the observed thiol/disulfide ratio

Ill
in vivo. Since reduced thiols are far more reactive than disulfides,
the charcoal treatment is likely to have removed a proportionately
larger amount of the free thiol-containing molecules than the
disulfide-containing molecules, leading to a potentially significant
shift in the overall thiol/disulfide ratio. One piece of evidence that
charcoal is removing small molecular weight thiols comes from the
finding in this study that charcoal treatment of cytosol already
containing DTT still results in a loss of binding capacity that is
reversible upon readdition of DTT after charcoal treatment.
Interestingly, the binding loss due to charcoal treatment of
pre-DTT-treated cytosol was not fully reversible in brain cytosol,
whereas it was fully reversible in liver and kidney cytosol when DTT was
readded. One possible explanation is that pretreatment with DTT, by
reducing not only the sulfhydryl(s) involved with steroid binding but
also other sulfhydryls, probably on the receptor's surface (Bodwell et
al., 1984b), increased the likelihood that some of the receptors would
themselves be adsorbed by the activated charcoal. The probability of
this occurring in liver or kidney cytosol was probably minimized by the
fact that these cytosols had a higher protein concentration.
It was discovered from these same experiments that when the
charcoal treatment was omitted, cytosol from both liver and kidney
exhibited a much greater loss of binding due to sulfhydryl oxidation
(resulting from omission of DTT) than was observed for brain cytosol.
This is in direct opposition to the findings of others, particularly
Granberg and Ballard (1977), who reported that binding activity in both
liver and kidney cytosol in the absence of DTT was 100% of the level
measured in the presence of DTT, whereas binding activity for brain

112
cytosol in the absence of DTT was approximately 61% of that measured in
the presence of DTT, a level similar to our own findings for brain.
These workers suggested that the lack of a DTT requirement in liver may
have been, in part, due to the high level of "endogenous reducing
agents" and indicated that the levels of non-protein sulfhydryl groups
were approximately 6-fold greater than they were in brain cytosol. On
the other hand, the levels for non-protein sulfhydryl groups in kidney
cytosol were reportedly identical to those in brain cytosol, leaving the
impression that knowledge of sulfhydryl group levels alone, without
knowledge of disulfide levels, may be insufficient to predict the
relative binding levels in the absence of exogenously added sulfhydryl
reagents. The fact that the two studies differed so widely in their
findings regarding the DTT requirement for maximal glucocorticoid
binding in liver and kidney cytosols may be an indication of the
sensitivity of this process to variation in experimental procedure.
Since binding capacity cannot be determined instantaneously, the
duration and temperature of steroid incubation as well as buffer
composition, pH, steroid concentration, etc. will almost certainly
affect the outcome of this type of experiment.
The inactivating effects of charcoal treatment on glucocorticoid
receptor binding capacity and the ability of various factors to reverse
these effects have been reported on by Grippo et al. (1983), who also
found that lost binding capacity was restored by adding DTT. These
workers also reported that incubating charcoal-treated cytosol with
boiled liver cytosol partially restored lost binding capacity. Two
components of the boiled cytosol were found to be required for restor¬
ation of charcoal-induced losses in receptor binding capacity: NADPH and

113
an endogenous heat-stable protein later "proven" to be thioredoxin
(Grippo et al., 1985). Though they claimed to have presented proof that
the endogenous heat-stable factor was, in fact, thioredoxin, their
research does not completely rule out the possibility that other
charcoal-extractable, heat-labile factors may also play an important
role in direct or indirect glucocorticoid receptor sulfhydryl reduction.
One enzyme, designated thiol:protein-disulfide oxidoreductase, catalyzes
the reductive cleavage of insulin and is also heat-stable, but requires
reduced glutathione, or possibly other small thiols that are also
extracted by charcoal, as a cosubstrate (Bjelland et al., 1984).
The fact that antibodies against thioredoxin reductase inhibit up
regulation of glucocorticoid receptor binding capacity in
charcoal-treated cytosol by boiled liver cytosol has been used by these
workers as the ’’proof” that the endogenous heat stable glucocorticoid
receptor up-regulating factor is thioredoxin. However, one of the
coauthors of the report had previously shown thioredoxin reductase from
mammalian tissues to have a fairly wide substrate specificity (Holmgren,
1977). Although Holmgren (1977) claimed that in a purified system,
disulfide bond reduction in insulin or L-cystine by NADPH and
thioredoxin reductase was dependent upon thioredoxin as an intermediate
electron carrier, the same study indicated that the enzyme could reduce
other potentially relevant endogenous compounds, such as the oxidized
forms of glutathione and lipoic acid as well as the artificial substrate
DTNB "directly without thioredoxin". Lipoamide dehydrogenase and
glutathione reductase are both capable of catalyzing net reduction of
disulfides. Glutathione reductase catalyzes the reduction of oxidized
glutathione and, like thioredoxin reductase, also requires NADPH.

114
Reduced glutathione, in turn, can reduce a wide variety of disulfides by
transhydrogenation. The later reactions are catalyzed by thiol-
transferases (Mannervik and Axelsson, 1975) and glutaredoxin (Luthman
and Holmgren, 1982), both of which are cytosolic and are similar in
molecular weight to each other (about 11,000) as well as to thioredoxin
(about 12,000). Although the substrate specificities of these enzymes,
like thioredoxin reductase, are rather broad, thiol transferase
catalyzes transhydrogenation of a larger variety of different thiols and
disulfides as well as reversible formation of protein-mixed disulfides
(Axelsson and Mannervik, 1980). One point in favor of thioredoxin being
of significant importance in the maintenence of the up-regulated form of
the unoccupied glucocorticoid receptor comes from a comparison of the
reductive capacity of the thioredoxin system in rat liver with that of
the glutathione-thiol transferase system (Mannervik et al., 1983),
wherein it was shown that while the thiol transferase system is more
efficient in catalyzing reduction of small disulfides, thioredoxin was
slightly more effective in reducing exposed disulfides in peptides.
Since the discussion thus far has focussed on enzymes and small
molecules potentially involved in the reduction or up-regulation of
glucocorticoid receptors, it is important to consider, momentarily,
those endogenous factors that might be involved with thiol oxidation.
This cannot be overlooked if sulfhydryl down-regulation of gluco¬
corticoid receptor binding capacity is to be considered an active
physiological process. Although nonenzymatic oxidation of thiols within
intact cells is probably insignificant (Ziegler, 1985), enzyme-catalyzed
reactions capable of net generation of disulfides within cells are quite
limited and only a few have been well characterized. The oxidation of

115
glutathione by H202, catalyzed by glutathione peroxidase, is usually
considered the major route for enzymatic generation of cellular
disulfide. However, additional intracellular disulfides are contributed
as the result of the desulfuration of 3-mercaptopyruvate, catalyzed by
the mercaptopyruvate transsulfurase, and to a lesser extent by the
oxidation of cysteamine to cystamine, catalyzed by the membrane-bound
flavin-containing monooxygenase (Ziegler, 1985). A fact relevant to the
interpretation of experiments regarding sulfhydryl down-regulation of
binding capacity over time in cytosolic preparations is that much of the
enzyme activity associated with sulfhydryl oxidation/reduction is either
membrane associated or extracellular. Most, if not all, of the
membrane-associated activity will be absent from cytosol, and therefore
not contribute to sulfhydryl oxidation after cytosol preparation is
complete. However, some of the membrane associated enzymes, such as
thiol oxidase (Ziegler, 1985), apparently have their active sites
directed extracellularly, where the thiol:disulfide ratio is
dramatically (50- to 100-fold) lower. These enzymes would normally not
influence the glucocorticoid receptor binding capacity in vivo since
these receptors are intracellular. However, tissue homogenization could
potentially lead to an increase in enzyme-induced receptor oxidation
during cytosol preparation in the absence of excess sulfhydryl reducing
agents. In addition, the overall thiol/disulfide ratio of the
homogenate and, subsequently, the resulting cytosol will be much lower
and the potential for sulfhydryl oxidation much higher as a consequence
of the contribution of high disulfide concentration and soluble
extracellular nonspecific thiol oxidases characteristic of extracellular
fluids.

116
Finally, Grippo et al. (1985) have provided no evidence as to
whether thioredoxin simply reduces a disulfide bond to yield a reduced,
steroid binding form of the glucocorticoid receptor or whether, as in
the case of T7 DNA polymerase (Mark and Richardson, 1976), reduced
thioredoxin must be bound to the receptor for it to be in a steroid
binding conformation. The possibility that charcoal treatment may have
an affect directly on the glucocorticoid receptor itself has been
proposed by Bell et al. (1984) who reported that charcoal pretreatment
of unlabeled receptors led to a significant reduction in steroid
dissociation rate after steroid labeling and suggested that this change
was a consequence of the removal of a lipid component from the complex.
Although these authors don’t implicate sulfhydryl oxidation/reduction in
the reduced steroid dissociation rates, it has been reported by
Takabayashi et al. (1983) that the binding of fatty acids to bovine
serum albumen increases the rate of oxidation of a free sulfhydryl
group, while the oxidation of the sulfhydryl enhances the binding of
fatty acids to the protein. Fishman (1983) reported similar effects of
charcoal treatment on estrogen receptors when he showed that treating
uterine cytosol with dextran-coated charcoal in the absence of ligand
causes the subsequently formed receptor-estradiol complex to be stable
at 37 C, although again, the results were too inconclusive to implicate
sulfhydryl oxidation/reduction.
The effect of sulfhydryl up- and down-regulation on the structure
of the unoccupied glucocorticoid receptor has never before been
investigated. Although the characterization of nonsteroid-labeled
receptors entails a number of obvious difficulties, such an
investigation might provide valuable clues as to whether or not

117
sulfhydryl up- and down-regulation involves merely a thiol/disulfide
exchange reaction within the receptor or mixed disulfide formation with
another small molecule, peptide, protein, etc. as was suggested by
Grippo et al. (1985) as a possible means of thioredoxin interaction with
the glucocorticoid receptor. In addition, even if the oxidation
reaction is completely internal (inter- or intra-subunit disulfide
formation), significant conformational changes are possible which could
lead to measurable changes in sedimentation coefficient, hydrophobicity,
etc. One experiment in this study provided evidence that whatever
changes that occur during sulfhydryl oxidation/reduction do not affect
the receptor conformation or overall molecular mass to an extent
detectable by changes in sedimentation coefficient. Because of the
relative insensitivity of such a measurement, one cannot rule out the
possibility of mixed disulfide formation with a small molecule such as
glutathione, and it is conceivable that simultaneous changes in
molecular mass and conformation could result in a new form with
sedimentation characteristics very similar to the original form.
However, the sedimentation results indicate that at least no major
changes are likely. Although Wilson et al. (1986) recently reported
changes in sedimentation properties of the androgen receptor associated
with sulfhydryl oxidation and reduction, these changes occurred in the
absence of molybdate and presumably were linked to the process of
receptor activation.
Possible changes in hydrophobicity associated with sulfhydryl up-
and down-regulation were also investigated using pentyl agarose
hydrophobic interaction chromatography. Again, no significant changes
were detected. This is somewhat of a surprize since it is well known

118
that reduced proteins are generally less soluble and have a strong
tendency to aggregate (Gilbert, 1984). In addition, the oxidation of a
thiol that presumably resides in or near the hydrophobic steroid binding
site of the receptor might be expected to interfere to some extent with
hydrophobic interactions. It must again be emphasized, however, that
the nature of these experiments and the lability of the unoccupied
glucocorticoid receptor under such conditions necessitated the use of
molybdate in most buffers. Although the molecular mechanism by which
molybdate stabilizes steroid receptors is not clearly known, it is
possible that molybdate may be interacting to form bridge structures
between adjacent sulfhydryl groups (Wilson, 1986). Molybdate, therefore,
might influence otherwise detectable sulfhydryl oxidation/reduction-
mediated changes in receptor properties.
Scatchard analysis of [3H]DEX binding to cytosol in the presence or
absence of DTT led to the impression that not only did DTI increase the
maximum binding capacity, but also the apparent affinity of the receptor
for DEX. Because of the potential implications of such a finding for
the role of sulfhydryl oxidation/reduction in steroid binding and,
consequently, steroid action, this phenomenon was further investigated.
Kinetic studies showed that neither the presence or absence of DTT had
any effect on the rate of dissociation of [3H]DEX from the prelabeled
receptor. These results differ from those reported in a similar study
on the effects of DTT on DEX dissociation kinetics, in which case DTT
was found to increase the rate of dissociation under certain conditions,
while it was reportedly Ineffective under other conditions (Buell et
al., 1986). Unfortunately, this study was flawed In that 12 mM
monothioglycerol was present in all buffers, even in the nonDTT

119
experimental groups. It is puzzleing why these workers included
monothioglycerol in their study since it is a sulfhydryl reducing agent
and, by their own admission, has been shown to have "an effect on
binding similar to DTT". One might argue then, that the appropriate
controls were absent from this study. In addition, these workers found
that dissociation rate was not affected by DTT if removal of free DEX
was initiated by a dextran-coated charcoal treatment (without a
subsequent readdition of DTT). Our own work and that of others (Grippo
et al., 1983, 1985) has shown that charcoal treatment removes much, if
not all, of the reducing capacity of cytosol, including exogenously
added DTT as well as endogenous reducing factors. Buell et al. (1986)
did observe an increased dissociation rate in the presence of DTT if
dissociation was initiated by addition of a 1000-fold concentration of
either unlabeled DEX or unlabeled RU5020, a synthetic progestin shown to
facilitate glucocorticoid dissociation. A potentially important
difference between our study and that of Buell et al. (1986) was that
our own work used a DTT concentration of only 2 mM, shown in a number of
experiments in this study to be more than sufficient to up-regulate all
glucocorticoid binding capacity, whereas the other workers used 20 mM
DTT. It is possible that if the DTT effect reported by these workers is
real, it may be concentration dependent and may involve effects on
glucocorticoid receptors very different from the reduction of the
binding site sulhydryl group(s). These researchers reported earlier in
abstract form on the effect of DTT on the rate of association of [3H]DEX
to glucocorticoid receptors (Buell et al., 1986). They claimed that DTT
led to a slight decrease in the association rate constant. Results from
our own efforts to determine effects of DTT on association rate were

120
deemed uniterpretaable because of the fact that unoccupied receptors in
cytosol not containing DTT are undergoing down regulation for the
duration of time that the association rate is being determined, thereby
significantly complicating the determination of rate constants.
In light of the findings of Buell et al. (1986), wherein DTT
increased dissociation rate and decreased association rate, one would
assume that Scatchard analysis of DEX binding in the presence and
absence of DTT would result in an apparent reduction in affinity for
cytosol containing DTT. However, as previously stated, our own work
showed the opposite to be the case in that DTT led instead to an
apparent increase in affinity for DEX binding. It is now thought that
this apparent change in affinity can be explained, for the most part, by
the selective down regulation of the unoccupied, but not the occupied,
form of the receptor. Factors that can differentially affect the
stability of the bound and unbound forms of the glucocorticoid receptor
(or any other type of receptor for that matter) can potentially result
in artefactual changes in various binding parameters as determined by
Scatchard analysis as has been shown by computer simulation (Beck and
Goren, 1983; Ketelslegers et al., 1984). Since Scatchard analysis
involves the incubation of identical cytosols with varying concentra¬
tions of free ligand, receptors incubated with relatively small
concentrations of steroid will, on average, be in an unbound form for a
much greater percentage of the incubation period, and, thus, be more
susceptible to the types of inactivation that are inhibited by the
binding of a steroid ligand, including sulfhydryl down-regulation.
Therefore binding in these samples will appear artefactually small.
However, receptors in samples incubated with steroid concentrations in

121
ess of saturation levels will remain unbound for a relatively short
iod of time, resulting in little appreciable loss of binding capacity
ing the incubation period. If such a situation is true, changes in
ubation time and temperature will have additional influence on the
ree of artefactual change observed in these types of determinations,
course, these artefactual changes would not occur in the presence of
since both occupied and unoccupied forms of the receptor appear to
equally stable under the experimental conditions since sulfhydryl
i-regulation is no longer a factor.
The next phase of this study, that dealing with the effects of
.ous reversible and irreversible sulfhydryl reactive reagents on
ocorticoid receptor down-regulation was carried out with several
s in mind. The first of these was to determine how effective these
erent reagents were in down-regulating receptors under the buffer
itions used. Most sulfhydryl reactions are very pH dependent,
gh they are much less affected by temperature. In addition,
bility, reactivity with other buffer components and the ability of
effects of the reagent to be neutralized by DTT all had to be
stigated early on. Of major concern was whether the loss of
jcorticoid binding capacity attributed to a particular reagent was
rsible upon addition of excess DTT and to what extent. The
Lficity of the reaction against up-regulated or down-regulated
‘.upied receptors or even occupied forms of the receptor was also
.cally important. The effectiveness of different reagents might
de some indication of the nature of the sulfhydryl group(s) being
zed during down-regulation. An excellent example of this was
ted by Formstecher et al. (1984) who determined the

122
pseudo-first—order kinetics for the irreversible inactivation of
glucocorticoid receptor binding capacity in liver using a series of
n-alkylmaleimides. These workers demonstrated a striking increase of
receptor inactivation with increasing chain length of the maleimide
derivative while steroid binding continued to afford full protection
against inactivation. It was suggested that the chain length effect
observed in the inactivation process was related to nonpolar
interactions in the binding of maleimides to the receptor prior to the
irreversible alkylation of sulfhydryl groups. Because of the
hydrophobic nature of the environment where these sulfhydryl groups were
located, the authors were able to conclude that these groups were
probably in the binding site itself. Because one of the goals of the
present study was to determine, if possible, the in vivo ratios of up-
and down-regulated forms of the glucocorticoid receptor, most of the
experiments were performed using either crude cytosol or crude
homogenate or, in some cases, a partially purified preparation. The
kind of conclusions drawn by Formstecher et al. (1984) regarding the
molecular localization of the sulfhydryl group(s) critical to steroid
binding required that at least a partially purified system be used.
This was in large part because the pseudo-first-order kinetics of
receptor inactivation would be difficult to obtain in the presence of
high concentrations of free thiol groups, particularly in liver. This
brings up several problems relevant to not only sulfhydryl studies of
steroid receptor binding capacity, but to sulfhydryl studies in impure
systems in general. First, as stated previously, numerous enzymes (both
intra- and extracellular in origin) with broad substrate specificities
can be found in cytosol preparations. Secondly, small thiol and

123
disulfide "contaminants", again introduced by mixing of intra- and
extracellular fluids during homogenization, can dramatically alter the
thiol/disulfide status relative to normal intracellular levels.
Thirdly, air oxidation of free thiols, though generally a slow process,
can be a greater problem for a particular protein thiol in an impure
system (because of indirect oxidation) than in a pure system. Finally,
most sulfhydryl reactive reagents are relatively nonspecific and
modification of one particular sulfhydryl group in a system is generally
accompanied by a similar modification of many, if not most, of the other
sulfhydryls in the system. This, of course, often perturbs the entire
thiol/disulfide balance for the system which, in turn, may have
consequences for those protein sulfhydryls or disulfides that aren't
directly impacted by the reagent in question, further complicating the
interpretation of results.
Although many of the experiments in the present study indicated a
relatively slow rate of down-regulation in cytosol at low temperatures,
the situation for receptors in homogenate, for the reasons already
discussed, appears to be more complex. For this reason the determina¬
tion of in vivo levels of up- and down-regulated receptors would require
that the two forms of the receptor be rapidly trapped in their
particular sulfhydryl configuration either during or immediately after
tissue homogenization. Initial experiments in this series, performed on
cytosolic preparations, demonstrated that some reagents, such as DTNB,
cystamine, glutathione disulfide, DTNT and MMTS were capable of down¬
regulating glucocorticoid receptors in a fashion that was completely
reversible with DTT. A potentially more valuable finding, however, was
that receptors down-regulated with some of these reversible reagents

124
appeared to be almost completely protected from irreversible
inactivation by some of the irreversible sulftiydryl reactive reagents.
If the endogenously down-regulated form of the receptor possesed the
same resistance to irreversible inactivation by NEM or iodoacetamide,
then it could later be up-regulated to an assayable steroid binding form
by addition of [3H]DEX and an excess of DTT, which would simultaneously
neutralize the inactivating reagent. The original population of
up-regulated receptors could be determined indirectly by measuring total
binding capacity in the presence of DTT, but with no inactivation
treatment, and then subtracting the down-regulated level. This, in
fact, was the goal for the experiments that followed. However, the
matter proved to be far more complicated than originally thought, and
the chemical reactions involved with this "trapping" procedure had to be
further investigated and characterized in detail.
The potential up regulation of down-regulated receptors during the
incubation of homogenate or cytosol with sulfhydryl inactivating
reagents was another problem that had to be considered since the
inactivation reaction was not instantaneous. This is likely to be a
major problem only if the inactivation reaction is slow. Otherwise, the
small thiols (such as glutathione) necessary as electron acceptors for
reduction of down-regulated receptors would themselves be consumed by
the reaction, thereby shifting the reaction in favor of receptor
oxidation. The rate of the inactivation reaction was therefore of
serious importance. Since the rate of such reactions depends most
heavily on the concentrations of the reactants and since the binding
site sulfhydryl group(s) of the up-regulated receptor is competing with
a many thousand-fold greater concentration of free sulfhydryl groups in

125
cytosol or homogenate, a relatively high concentration of either NEM or
iodoacetamide was required to achieve acceptably fast inactivation.
Unfortunately, raising the concentration of these reagents presented yet
another problem in that high concentrations of iodoacetamide has been
shown to produce modifications of other protein groups. Although it
isn't known for sure how such non-sulfhydryl modifications of proteins
attributed to iodoacetamide could lead to inactivation of the
normally-protected down-regulated glucocorticoid receptor, but results
from several experiments in the present study indicated that higher
levels of iodoacetamide eliminated all of the binding capacity in
cytosol known to contain down-regulated receptors. Like iodoacetamide,
reaction of NEM with proteins is not completely specific for sulfhydryl
groups, but also occurs with imidazole and alpha-amino groups via a
similar mechanism. However, reaction with sulfhydryl groups occurs much
faster, and the rate of the reaction with sulfhydryls is markedly
enhanced with increasing pH. At pH 7, the reaction rate of NEM with
simple thiols is approximately 1000-fold greater than with corresponding
simple amino compounds. Therefore, at the pH of 7.6 used in the present
study, NEM reaction with amino groups does not appear to have been a
problem.
Another related problem that had to be dealt with simultaneously
involves the effects of high concentrations of DTT on glucocorticoid
receptor binding capacity in tissue homogenate. When high
concentrations of either iodoacetamide or NEM are used, even higher
concentrations of DTT are required to neutralize the reaction and
up-regulate the down-regulated forms. It was found in the present study
that concentrations of 50 mM and higher DTT, when added to homogenate,

126
inactivated glucocorticoid receptor binding, even in the absence of the
other inactivating reagents. Even though DTT has a similar effect on
binding when added to cytosol, much higher concentrations are required.
The mechanism of this DTT-induced inactivation is unknown, although it
is possible that protein aggregation was enhanced by these high
concentrations of reductant. If this is the case, aggregation would
expect to be even more of a problem in a homogenate suspension than in
cytosol. This unexpected problem placed even further restrictions on
inactivating agent concentration.

CHAPTER IV
THE PURIFICATION AND SUBSEQUENT ACTIVATION OF THE GLUCOCORTICOID
RECEPTOR
Introduction
Purification
A major problem in interpreting much of the published work
concerning the physicochemical properties of glucocorticoid (and other
steroid) receptors is that all but the most recent studies were
conducted on either crude or only partially purified receptors. In
light of the numerous demonstrations that various endogenous "factors”
and exogenously added substances can dramatically affect receptor
stability, binding and activation kinetics, specificity and affinity of
nucleic acid binding, the purification of the receptor is clearly an
essential next step in many of these studies. A number of approaches
have been taken to achieve varying degrees of purification of gluco¬
corticoid and other steroid receptors. The strategy of choice depends
on the form of receptor desired (unoccupied, occupied-unactivated or
occupied-activated), the degree of purification required for a given
procedure and various other limitations or requirements. A combination
of different purification steps will likely be required to produce
optimal results in most cases.
Any attempt at glucocorticoid receptor purification, however, must
take into account the following difficulties: (1) limited sources of a
tissue abundant in free receptor, (2) the presence of proteins other
127

128
than the receptor which bind to glucocorticoids, (3) the high degree of
purification necessary to obtain homogeneous preparations and (4) an
apparent instability of all forms of the receptor (Santi et al., 1979).
The last difficulty, receptor instability, was a major factor limiting
many early attempts at receptor purification, particularly in light of
the fact that the apparent instability of the receptor from some sources
increases as the receptor is purified (perhaps due to the removal of
endogenous stabilizing factors). However, the findings of Leach et al.
(1979) demonstrating the stabilization of each of the glucocorticoid
receptor forms by molybdate greatly diminished the limitations placed on
purification by receptor instability. Of course, the fact that the
precise nature of the mechanism by which molybdate (and other transition
state elements) stabilizes the glucocorticoid receptor remains unknown
should not be overlooked when interpreting the results of experiments
performed in the presence of molybdate.
Earlier attempts to purify the glucocorticoid receptor generally
involved a series of chromatographic steps typically including ion-
exchange, gel exclusion and DNA-cellulose (Santi et al., 1979; Govindan
and Manz, 1980; Romanov and Gorshkova, 1984). The degree of purifi¬
cation, as well as the yield, varied considerably from study to study,
although impressive results were recently obtained by Govindan and
Gronemeyer (1984) and Moudgil et al. (1985) using a refined DNA-
cellulose procedure and by Wrange et al. (1984) using a sequential
chromatography on DNA-cellulose and DEAE-Sepharose. The use of steroid
affinity resins has increased markedly the degree of purification
attainable in recent studies. Deoxycorticosterone hemisuccinate coupled
covalently to BSA-Sepharose 4B was used to achieve a relatively low

129
level of purification of glucocorticoid receptors from rat brain (De
Kloet and Burbach, 1978). More recently, deoxycorticosterone-agarose
has been used for the purification of both progesterone (Grandics et
al., 1982) and glucocorticoid (Grandics and Litwack, 1982) receptors.
Basically, cytosol is applied to the affinity gel and binding to the
deoxycorticosterone is allowed to reach equilibrium. The gel is then
washed free of nonbinding components and is incubated with high
concentrations of the free tritiated steroid of choice. The highly
specific synthetic [3H]triamcinolone acetonide was used for the exchange
assay for glucocorticoid receptors since it binds neither progesterone
nor mineralocorticoid receptors nor corticosteroid binding globulin
(CBG), which are all likely to bind to some extent to the deoxycortico¬
sterone resin. After the exchange reaction has gone to (or near)
completion, the radiolabeled receptors can simply be washed from the
resin and further purification steps can be applied as necessary. Very
recently a 4000-fold purification of rat liver glucocorticoid receptor
was achieved using this procedure (Grandics et al., 1984b). Govindan
and Manz (1980) and Lustenberger et al. (1981) achieved similar results
using an affinity column containing either dexamethasone-17B-carboxylic
acid or dexamethasone-21-methanesulfonate coupled to an aminocystamido-
succinylamidohexyl-Cl-Sepharose 4B column. Govindan and Gronemeyer
(1984), using this same ligand affinity procedure, have recently
increased the yield of purified receptor significantly. Other
procedures using affinity resins for the purpose of purification have
included the binding of glucocorticoid-biotin analogs to the receptor
prior to incubation with an avidin-Sepharose affinity gel (Manz et al.,
1983). These workers reported a 19,000-fold purification of the

130
glucocorticoid receptor from human spleen tumor cytosol. Other steroid
receptor systems have been purified by various types of affinity
chromatography. Progesterone receptor from chick, oviduct has been
purified to a high degree (1500-2700-fold) using a N-(12-amino-dodecyl)-
3-oxo-4-androsten-17B-carboxamide-substituted Sepharose gel (Renoir et
al., 1982). As with glucocorticoid receptors, combining this affinity
chromatography step with an ion-exchange chromatography step
significantly increased the degree of purification (to >6700-fold).
Smith et al. (1981) reported a 67,000-fold purification of human uterine
progesterone receptor using a combination of ammonium sulfate
fractionation and affinity chromatography (deoxycorticosterone-BSA-
Sepharose 4B). More recently, Van Oosbree et al. (1984) used
diethylstilbestrol coupled to epoxy-activated agarose to yield highly
purified rabbit uterine estrogen receptors upon elution with
p-sec-amylphenol and NaSCN (a chaotropic salt). More recently,
amino-aryl controlled pore glass beads coupled to a variety of steroids
(including several glucocorticoids) using a bifunctional cross-linker
with amino reacting and photogenerated nitrene functions (Lingwood,
1984) have become commercially available. However, no reports have yet
appeared concerning the use of these steroids immobilized to glass beads
for steroid receptor purification purposes. Krajcsi and Aranyi (1986)
most recently reported the use of cortexalone-Sepharose for the
purification of thymus glucocorticoid receptors. This new affinity
matrix only resulted in a 75-150-fold purification with a yield of
20-30%, but the relatively fast dissociation of the glucocorticoid
receptor-cortexalone complex allows for a much more rapid purification
step.

131
Another form of affinity chromatography that has been reportedly
used for the purification of estrogen and glucocorticoid (Weisz et al.,
1984) receptors involves the use of immobilized heparin. However,
heparin has been shown to cause inactivation of unoccupied progesterone
(Thorsen, 1981) and glucocorticoid (Hubbard and Kalimi, 1983; McBlain
and Shyamala, 1984; Densmore et al., unpublished results) receptors as
well as activation of bound receptors (Thorsen, 1981; Yang et al., 1982;
Hubbard and Kalimi, 1983c; McBlain and Shyamala, 1984). In addition,
the degree of purification obtained with heparin chromatography (only
about 10-fold (Weisz et al., 1984)) is exceptionally low and would best
be used for nonquantitative characterization or receptor activation
studies (Blanchardie et al., 1984). More recently Weisz et al. (1986)
used Sepharose-heparin to partially purify Type I, or mineralocorticoid,
binding activity of kidney cytosol. Although a yield of around 90% was
reported and all CBG was removed by the process, the degree of
purification was once again in the range of only 10-fold.
Finally, immunopurification was recently described as a means of
purifying [3H]dexamethasone mesylate-labeled receptors (Smith and
Harmon, 1985). Labeled cytosol was first incubated with anti¬
glucocorticoid receptor serum followed by adsorption of the antibody-
receptor complexes to protein A immobilized to Sepharose CL-4B. These
workers did not report the degree of purification or the yield from this
procedure. Although this study used prelabeled receptors, the technique
offers a means of purifying nonsteroid-labeled glucocorticoid receptors.

132
Activation
Probably the most accepted definition of "activation” is simply the
process whereby the steroid-receptor complex is converted to a form able
to bind to its nuclear acceptor sites. Precisely what physicochemical
changes occur in the receptor molecule during this process, what
endogenous factors, if any, regulate this process and what, exactly,
constitutes an "acceptor" site remains controversial. However, despite
the lack of total agreement on these matters, certain changes in the
basic properties of the glucocorticoid-receptor complex during
activation are now generally accepted: 1) an increase in the affinity of
the receptor complex for natural and synthetic polyanions (apparently by
virtue of an increase in surface positivity), 2) a decrease in the
overall size of the receptor complex and 3) a decrease in the dissocia¬
tion rate constant for the receptor complex (although this now appears
to depend upon the ligand bound). Thus, the most obvious means by which
the process of activation could be qualitatively and quantitatively
studied would involve the separation of the activated and unactivated
forms of the receptor complex based on their size and/or surface charge
differences. A variety of chromatographic and other techniques have
been used to study some of the physicochemical changes in the gluco¬
corticoid-receptor complex which accompany activation. However, much of
the results remains controversial due to a failure to control for
receptor lability and activation during the long procedures required for
determination of these physiochemical parameters. As mentioned
previously, the discovery that molybdate prevents both the loss of
unoccupied receptors and the activation of occupied receptors (Leach et
al., 1979) has removed many of these difficulties and has allowed a much

133
greater resolution of unactivated from activated glucocorticoid-receptor
complexes (Luttge and Densmore, 1984; Luttge et al., 1984a,b,d). The
following section will briefly review some of the more promising
techniques of activation analysis which now demand renewed attention in
light of recent developments in receptor stabilization and purification.
Historically, it was the observation that glucocorticoid-receptor
complexes translocated and bound to the nuclei of glucocorticoid target
cells that provided the most convincing evidence of their genomic
mechanism of action. Early studies by several laboratories demonstrated
that when nuclei isolated from hepatoma cells in tissue culture or liver
cells were incubated with the synthetic glucocorticoid [3H]dexa-
methasone, very low levels of nuclear binding were detected. However,
if the steroid was first equilibrated with cytosol at 0 C and this
mixture was then incubated with nuclei at 20 C, time-dependent specific
nuclear binding of the tritiated glucocorticoid was observed (Baxter et
al., 1972; Kalimi et al., 1973; Litwack et al., 1973). It is not
surprizing then that the binding of glucocorticoid-receptor complexes to
isolated nuclei was developed as one of the first cell-free assays for
activation for a number of glucocorticoid target tissues including,
among others, thymus (Munck et al., 1972), lymphoma cells (Rosenau et
al., 1972) and embryonic chick retina (Sarkar and Moscona, 1974), and is
still used frequently. There are some potential drawbacks to the
nuclear-binding assay, however. The tissue specificity of the binding
of activated complexes to nuclei in cell-free systems is not high
(Kalimi et al., 1973; Lippman and Thompson, 1974; Feldman et al., 1975;
Romanova et al., 1983) and apparently the nuclear binding sites in
cell-free systems are non-saturable (Simons et al., 1976; Milgrom and

134
Atger, 1975), suggesting the possibility that the nuclear sites to which
glucocorticoid-receptor complexes are bound in cell-free systems may
differ from those to which they are bound in intact cells which may be
saturable (Higgins et al., 1973). This and the fact that the activated
complex has an increased affinity for a number of different nuclear
components including chromatin (Sakaue and Thompson, 1977; Simons et
al., 1976), nucleosomes (Climent et al., 1977), DNA (Baxter et al.,
1972; Rousseau et al., 1975; Sluyser, 1983), RNA (Tymoczko and Phillips,
1983), the nuclear matrix (Buttyan et al., 1983; Kirsch et al., 1986)
and the nuclear membrane (Smith and von Holt, 1981; Kaufman and Shaper,
1984), has prevented a precise analysis of the physicochemical changes
associated with activation based on the nuclear binding assay alone. In
addition, the question of what exactly comprises the specific nuclear
acceptor sites has not been resolved satisfactorily. Finally, much more
time and care are required to prepare clean isolated nuclei than are
required for various other activation assay systems.
Another, more defined system for quantifying and characterizing
activation involves the binding of activated glucocorticoid-receptor
complexes to DNA-cellulose (Alberts and Herrick, 1971; Kalimi et al.,
1975; Luttge and Densmore, 1984). Advantages over nuclear binding
assays include the ease of preparation (DNA-cellulose is available
commercially) and the high degree of replicability obtained. An
interesting observation was made by LeFevre et al. (1979) who reported
that when the same hepatic cytosol containing heat-activated
[3H]triamcinolone acetonide-receptor complexes was titrated by high
concentrations of nuclei or DNA-cellulose, the former bound 75% of the
complexes while the latter bound only 40%. These workers suggested that

135
this decreased binding was due on the one hand to a lower initial
interaction between DNA-cellulose and activated complexes than between
nuclei and these complexes and on the other hand to increased losses
during washing when DNA-cellulose was used. However, these reasons can
be discounted partially based on the results of Luttge and Densmore
(1984) and Luttge et al. (1984 a,b,d) who used much longer incubation
times to allow for increased interaction between DNA-cellulose and
activated complexes, yet still observed a maximum binding of only
30-40%. In addition, these workers found losses during washes to be
insignificant when washing pellets as many as 5 times. More likely
explanations for the roughly two-fold difference between nucleus and
DNA-cellulose binding include the possibility that free steroids may be
binding directly to nuclei (Kaufman and Shaper, 1984) or that two forms
of activated glucocorticoid-receptor complexes exist, both of which can
bind to nuclei with relatively high affinity, but only one of which can
bind to DNA-cellulose. Hirose and co-workers (1983) reported two such
activated forms for rat mammary glucocorticoid-receptor complexes which
they claim have slightly different sedimentation properties. These
workers compared their findings to those of others who have studied
extensively the progesterone receptor from avian oviduct. This
progesterone receptor was shown to consist of two subunits, A and B, and
that as nuclear acceptor sites, A and B subunits recognize DNA and
chromosomal proteins, respectively. If such a situation does exist for
glucocorticoid receptors, the separation and subsequent detailed
characterization of each of the two or more forms of the activated
glucocorticoid-receptor complexes would surely help to clarify a number
of nuclear-receptor interactions and would probably provide much greater

136
resolution in the physicochemical characteristics determined for the
glucocorticoid receptor. Further evidence for existence of multiple
populations of activated glucocorticoid receptors was presented by Munck
and Foley (1980) who showed that the "activated” peak eluted from a
DEAE-cellulose column could be separated into two populations based on
their binding to DNA-cellulose. Cidlowski and Munck (1980) used
differential salt extraction of nuclei from cells incubated with
glucocorticoids under various conditions to illustrate "that
glucocorticoids form physiologically distinct classes of nuclear
acceptor-receptor complexes."
Ion-exchange chromatography has proved to be a particularly useful
tool for simultaneously separating activated from unactivated
glucocorticoid-receptor complexes and providing information about
surface charge changes during activation. A number of studies have
employed the popular anion-exchangers diethylaminoethyl cellulose
(DEAE-cellulose) and DEAE-Sephadex. Investigators in two labs reported
simultaneously the successful separation of the unactivated and
activated forms by rapid ion-exchange chromatography. Parchman and
Litwack (1977) reported that the more acidic unactivated rat liver
glucocorticoid-receptor complexes, which do not bind to carboxymethyl-
Sephadex (CM-Sephadex, a cationic exchanger) or DNA-cellulose, are
eluted from minicolumns of DEAE-Sephadex in phosphate buffer containing
0.4 M KC1. The more basic activated glucocorticoid-receptor complexes
that bind to both CM-Sephadex and DNA-cellulose are eluted from DEAE-
Sephadex at a lower salt concentration (0.2 M KC1). The fact that this
form of the receptor bound to both DEAE- and CM-Sephadex indicates the
presence of both positively and negatively charged regions on its

137
surface. Since it was more easily eluted from CM-Sephadex, it is
probably an acidic protein with a localized basic region. Sakaue and
Thompson (1977) resolved activated and unactivated glucocorticoid-
receptor complexes by chromatography on DEAE-cellulose with potassium
phosphate as the eluting salt. With this anion-exchange resin the peak
of radioactivity corresponding to the unactivated complexes, which do
not bind to either DNA or chromatin, was eluted by 0.24 M potassium
phosphate, whereas the peak corresponding to the activated complexes,
which bind to nuclei, chromatin and DNA, is eluted by 0.06 M potassium
phosphate. These investigators reported that virtually identical
chromatographic results were obtained with extracts from rat HTC and
human LA9 cells, rat thymus and rat brain, indicating that the procedure
can apparently be used to study glucocorticoid-receptor complexes in a
variety of responsive tissues. Recently, DEAE-cellulose chromatography
has been used as a step in the purification of unactivated gluco¬
corticoid receptors (Manz et al., 1983), and DEAE-Sepharose has been
used in the purification of activated receptors (Wrange et al., 1984).
Another chromatographic procedure with potential for studying the
process of glucocorticoid receptor activation is dye-ligand affinity
chromatography. Perhaps the most commonly used dye-ligand for protein
purification has been Cibacron Blue F3GA, originally a textile dye and
now refered to as a "universal pseudoaffinity ligand" (for review see
Subramanian, 1984). Cibacron Blue was initially thought to possess a
discriminating ability to bind to selected proteins containing a
specific structure called a "dinucleotide fold", often associated with
the ability of a protein to bind nucleotides. Although the dye
eventually proved to be less specific for dinucleotide fold-containing

138
proteins than originally thought, it was shown to inhibit the binding of
activated mouse uterine estradiol receptors to oligodeoxynucleotide
cellulose (Kumar et al., 1979), and the activated and unactivated forms
of the glucocorticoid receptor have been shown to bind differentially to
Cibacron Blue affinity columns. Mouse brain glucocorticoid receptors
have also been observed to bind to Cibacron Blue (Blue A)-agarose
columns with apparent high affinity (Luttge and Densmore, unpublished)
and affinity immobilization on Cibacron Blue-Sepharose 6B has been used
most recently as a microassay for the determination of various binding
parameters of estrogen and androgen receptors (Iqbal et al., 1985).
Until the precise nature of the interaction between the receptor and
Cibacron Blue F3GA is known, however, results from such studies can be
of only limited value in the interpretation of the qualitative aspects
of steroid receptor activation.
»
Although a change in the sedimentation coefficient (typically 9-10
S to approximately 3-4 S) of the glucocorticoid-receptor complex is
considered by many to be associated with the process of activation,
published reports of sedimentation data have often been confusing and
inconsistent. Excluding known proteolytic degradation products,
sedimentation coefficient values of 3.2-3.5S (Wrange et al., 1979; Raaka
and Samuels, 1983; O'Brien and Cidlowski, 1981), 3.8-4S (Luttge et al.,
1984a,b,d; Eisen and Glinsman, 1978; Beato and Feigelson, 1972;
Carlstedt-Duke et al., 1977; Norris and Kohler, 1983; Middlebrooks and
Aronow, 1977), 4-6S (Sherman, 1984; Holbrook et al., 1983a; Weatherill
and Bell, 1985; Alexis et al., 1983; Vedeckis, 1983; Raaka and Samuels,
1983), 6-8S (Raaka and Samuels, 1983; Beato and Feigelson, 1972; Baxter
and Tomkins, 1971; Kalimi et al., 1975), 8-9S (Failla et al., 1975;

139
Vedeckis, 1983; Middlebrooks and Aronow, 1977) and 9-10S (Alexis et al.,
1983; Sherman, 1984; Holbrook et al., 1983a; Raaka and Samuels, 1983;
Norris and Kohler, 1983; Grandics et al., 1984b; Luttge et al.,
1984a,b,d) have been reported. While tissue and species differences can
not be ruled out as being at least partially responsible for these
inconsistencies, widely variable experimental conditions are certainly a
factor. Variation in the ionic strength, which is well known to effect
the hydrodynamic properties of proteins (Sherman, 1984), is common from
one study to the next. Perhaps even more important is the fact that
most earlier studies ran gradients that provided no protection for the
receptor against either activation or inactivation. Therefore receptor
complexes that had initially been identified as unactivated prior to
going on a gradient might have undergone activation during the long
centrifugation (typically 16-24 hours). Since the rate of activation
would increase as the salt concentration of the gradient increased, it
is easy to see why there were so many difficulties associated with the
interpretation of these studies. One method of dramatically shortening
the long centrifugation runs, but which has seen very limited use,
involves the use of a vertical tube rotor. Run times on vertical rotors
(typically less than 2 hours) can be reduced to between one-fifth and
one- thirteenth the time required for swinging bucket rotors. One
possible reason why vertical tube rotor sucrose gradient
ultracentrifugation has been utilized much less frequently since its
first reported use in the study of steroid hormone receptors (Hofman et
al., 1978) is that the technique has been claimed to result in poor
resolution of various receptor forms (Traish et al., 1981). However,
recent reports have described conditions which yield very good

140
resolution of the multiple forms of the mouse glucocorticoid receptor
(with peaks at 3.8 S, 5.2 S and 9.1 S) which occur before, during and
after the activation process (Eastman-Reks et al., 1984; Reker et al.,
1985). The technique has also been reported to produce good results
with progestin receptors (Schneider et al., 1984).
In agreement with the decrease in sedimentation coefficient of the
receptor complex associated with activation, are reports of decreases in
both the apparent molecular mass and Stoke's radius during activation.
The Stoke's radius of the unactivated form from mouse brain decreased
from 7.7 to 5.8 nm during activation, while the apparent molecular mass
decreased from 297,000 to 92,000 daltons as determined by Sephacryl
S-300 gel exclusion chromatography (Luttge et al., 1984d). This agrees
closely with the recent findings of Gehring and Arndt (1985), who
reported molecular weights of 325,000 and 94,000 daltons for the
unactivated and activated forms of the S49.1 lymphoma cell
glucocorticoid receptor as also determined by Sephacryl S-300
chromatography.
Most recently, gel exclusion high performance liquid chromatography
(HPLC) has been employed to characterize steroid receptors and the
physicochemical changes they undergo during activation. The advantages
of HPLC over conventional (open-column) gel exclusion chromatography
include a decrease in the time required to separate the various forms of
the receptor and an apparent increase in the resolution. Wiehle et al.
(1984) used gel exclusion HPLC to separate estrogen, progestin and
androgen receptors from several target tissues within 50 min on the
basis of size and shape (Stoke's radius). These workers reported that
the system provided for the detection of heterogeneity of receptor forms

141
in a manner superior to that observed with sucrose density gradient
centrifugation. Barbey et al. (1983) achieved similar results with
estrogen, progesterone and glucocorticoid receptors from a large number
of breast cancer cytosols, detecting five forms each for progesterone
and glucocorticoid receptors, and six forms for estrogen receptors.
Again, these researchers found the HPLC results favourable to those
obtained with either conventional gel exclusion chromatography or
density gradient ultracentrifugation. However, recently it has been
reported that certain forms of the glucocorticoid-receptor complex
extracted from mouse AtT-20 cells appeared to be less stable during a
15-20 min HPLC run than during a 65 min vertical tube rotor sucrose
density gradient ultracentrifugation run (LaPointe and Vedeckis, 1984;
LaPointe et al., 1986). The instability of a 5.2 S intermediate form of
the receptor during the short HPLC run was unaffected by the presence or
absence of sodium molybdate. Hutchens et al. (1984) reported that gel
exclusion HPLC of estrogen binding proteins from human uterine cytosol
demonstrated a predominant 8-8.5 nm species, whereas conventional size
exclusion chromatography revealed two to three distinct regions of
estrogen binding proteins with Stokes radii of about 8.5, 6 and 3 nm
(major species). Unlike the previously cited reports concerning HPLC,
however, this study did not include molybdate in the elution buffers,
and therefore an increased lability (or susceptibility to activation) of
the larger forms of the receptor would be much more apparent during the
very long open-column chromatography runs than during the very short
HPLC runs.
Other means by which activation-induced changes in steroid receptor
structure have been studied include polyacrylamide gel electrophoresis

142
and isoelectric focusing. Like vertical rotor sedimentation analysis
and HPLC, these techniques have been used rather infrequently for the
characterization of steroid receptors. Ben-Or and Chrambach (1981) were
able to resolve three molecular species of glucocorticoid receptors from
chick neural retina by gel electrophoresis (see also Ben-Or, 1983).
These same workers later used isoelectric focusing techniques along with
molybdate containing buffers to determine the apparent isoelectric
points of three major forms of the receptor (ranging from 5.4 to 7.6)
and the relationship between each of these forms and the process of
receptor activation (Ben-Or and Chrambach, 1984). Cidlowski and Richon
(1984) more recently combined these two techniques for a two-dimensional
gel analysis of affinity labeled (dexamethasone mesylate) human
glucocorticoid receptors by first subjecting the receptors to
isoelectric focusing and then to SDS-polyacrylamide gel electrophoresis.
Their data suggested that the human glucocorticoid receptor consists of
a family of at least five proteins with molecular masses of approxi¬
mately 88,000 which have discrete isoelectric points ranging from 6.5 to
7.5, possibly representing post transcriptional modification of a single
protein. However, when a nonaffinity label (such as dexamethasone) was
used, such microheterogeneity was not seen and only a single isoelectric
peak of about 6.2 was detected for hippocampal or renal glucocorticoid
receptors from rat (Wrange and Yu, 1983). However, this same lab
(Wrange et al., 1984) has more recently used sodium dodecyl sulfate-gel
electrophoresis to characterized purified activated glucocorticoid
receptor from rat liver cytosol. They reported finding 94,000, 79,000,
and 72,000 molecular mass species of the activated receptor. Another
investigator has used polyacrylamide gel-electrophoresis to measure

143
various kinetic binding parameters of glucocorticoid receptors from
hepatoma cells (Lenger, 1983).
Only recently, however, has there been any attempt to activate
highly purified unactivated glucocorticoid receptors. The investigation
of activation under purified conditions should provide important new
clues regarding the potential role of endogenous factors, enzymes, etc.
in the activation process(es). Although the reported results vary
somewhat, perhaps dependent upon the degree of purification, one finding
seems to be relatively consistent. It has been shown in at least three
different studies that activation of purified receptors is not followed
by the same increase in binding to DNA as is the case with unpurified
receptor preparations (Grandics et al., 1984b; Webb et al., 1985;
Schmidt et al., 1985). It has been shown by each of these studies that
unpurified cytosol will dramatically increase the binding of prepurified
activated glucocorticoid receptors when added to the preparation,
although the nature of the cytosolic factor(s) responsible for
increasing DNA binding are currently unknown.
Materials and Methods
Chemicals, Steroids and Isotopes
[6,7-3H]Triamcinolone acetonide, or 9a-fluoro-llb,16a,17a,21-
tetrahydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide, ([3H]TA,
specific activity = 37 Ci/mmol) and [6,7-3H]dexamethasone, or
9a-fluoro-16a-methylprednisolone ([3H]DEX, specific activity = 44.1-48.9
Ci/mmol) were purchased from New England Nuclear (Boston, MA). Sterogel
A affinity resin (deoxycorticosterone agarose) was purchased from
Sterogene Biochemicals. Sephadex G-25 (fine) was obtained from
Pharmacia Fine Chemicals (Piscataway, NJ). Dithiothreitol (DTT), and

144
4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid (HEPES) was courtesy
of Research Organics (Cleveland, OH). Sodium molybdate (Na2Mo04), calf
thymus DNA-cellulose, glycerol, sucrose, PPO (2,5-diphenyloxazole) and
dimethyl POPOP (1,4-bis[2(4-methyl-5-phenyloxazoyl)jbenzene) and pentyl
agarose were all purchased from Sigma Chemical Co. (St. Louis, MO).
Scinti Verse II was purchased from Fisher, Inc. (Fair Lawn, NJ). All
other chemicals and solvents were reagent grade.
Animals
All studies used female CD-I mice (Charles River Laboratories,
Wilmington, MA) that were subjected to combined ovariectomy and
adrenalectomy approximately 1 week prior to each experiment in order to
remove known sources of endogenous steroids. Both operations were
performed bilaterally via a lateral, subcostal approach under
barbiturate anesthesia, and mice were given 0.9% NaCl (w:v) in place of
drinking water. On the day of the experiment mice were anesthetized
with ether and perfused slowly through the heart with ice-cold
HEPES-buffered saline (20-30 ml, isotonic, pH 7.6).
Cytosol Preparation and Steroid Binding
Brains were removed from the perfused animals and homogenized (2x10
strokes at 1000 rpm) in 4 volumes of ice cold Buffer A (20 mM HEPES, 2
mM DTT and 20 mM Na2Mo04, pH 7.6 at 0 C) in a glass homogenizer with a
Teflon pestle milled to a clearance between the pestle and
homogenization tube of 0.125 mm on the radius (to minimize rupture of
the brain cell nuclei (McEwen and Zigmond, 1972). The crude homogenate
was centrifuged at 100,000 g for 20 min and the supernatant
recentrifuged at 100,000 g for an additional 60 min to yield cytosol.
During these centrifuge runs, and during all other procedures, unless

145
otherwise indicated, careful attention was paid to maintaining the
cytosol at 0-2 C. Final protein concentrations were typically in the
6-8 mg/ml range. For experiments involving unpurified occupied
(activated or unactivated) receptors, cytosol samples were incubated
with 20 nM [3H]TA or [3H]DEX for 24 to 40 hours at 0 C with or without a
200-fold excess of unlabeled steroid.
Affinity Chromatography
Unlabeled cytosol was applied to small columns containing 1-2 ml of
Sterogel affinity resin and incubated at 0 C for 2 to 8 hours prior to
elution of the column with a minimum of 10 volumes of HEPES buffer
containing 2 mM DTT and 50 mM molybdate. Direct steroid exchange was
then carried out by incubating the washed resin with either [3H]DEX or
[3H]TA (concentrations varied from 100 to 500 nM) for periods varying
from 20 to 72 hr. Radiolabeled receptors were then eluted from the
column using the previously described buffer and then subjected to
Sephadex G-25 chromatography to remove free steroid. Samples were then
either used for subsequent experimental analysis, counted to determine
specific binding or assayed for protein content. Indirect steroid
exchange involved first incubating the washed resin with 5 uM [1H]DEX
for 4 hr, followed by elution from the affinity column and separation of
free [1H]DEX on a Sephadex G-25 column prior to a second steroid
exchange reaction involving incubation of the [1H]DEX-labeled receptors
with 50 nM [3H]DEX or [3H]TA for periods varying from 20 to 72 hr.
Activation and Removal of Unbound Steroid
[3H]TA-labeled purified receptor preparations or [3H]TA-labeled
cytosol samples (for experiments involving unpurified receptor
preparations) to be activated were first run on Sephadex G-25 columns

146
(0.6 x 14 mm) equilibrated with buffer containing 20 mM HEPES and 2 mM
DTT only. This column run resulted in the removal of both free steroid
and molybdate, allowing for uninhibited activation (Luttge et al.,
1984a,b,d; Luttge and Densmore, 1984). The bound fraction was then
incubated at 22 C for variable periods of time to determine the minimum
period of time required to achieve total activation. The degree of
activation was determined by DNA-cellulose binding assay. For the
purpose of hydrophobic interaction chromatography of activated
receptors, the maximally activated receptor preparation was again run on
a second Sephadex G-25 column equilibrated with the appropriate buffer
(the same buffer used to equilibrate and elute the subsequent alkyl
agarose columns). For unactivated receptors, the cytosol was treated in
an identical fashion except that the first Sephadex G-25 column was
equilibrated and eluted with HEPES buffer containing 20mM molybdate and
2 mM DTT and the bound fraction was subjected to a 0 C (rather than a 22
C) incubation prior to running on the second Sephadex G-25 column
(identical to the second column run for the activated preparation).
DNA-cellulose Binding Assay
Calf thymus DNA-cellulose, prepared originally by the method of
Alberts and Herrick (1971), was added to HEPES buffer containing 2 mM
DTT to yield a final concentration of 10 mg/ml (4.1 mg DNA/g
DNA-cellulose). In a typical assay (run as duplicates) a 100 ul aliquot
of [3H]TA-labeled cytosol or purified receptor preparation was added to
300 ul of the DNA-cellulose slurry. The mixture was then vortexed
gently and incubated at 0 C for 60 min. Assay tubes were oscillated at
150 rpm throughout the incubation and vortexed gently every 10 min. The
DNA-cellulose was collected by centrifugation (2000 x g for 5 min), the

147
supernatant discarded and the DNA-cellulose pellet washed 3 times with 1
ml of HEPES buffer plus 2 mM DTT. The entire pellet was resuspended in
deionized water and transferred to scintillation vials for determination
of bound radioactivity. DNA-cellulose was prepared in buffer not
containing molybdate in order to reduce the concentration of molybdate
during the binding assay, since high concentrations of molybdate were
found to reduce the efficacy of receptor binding to DNA-cellulose
(Luttge et al., 1984b).
Hydrophobic Interaction Chromatography
[3H]TA-labeled purified receptor preparations were run on Sephadex
G-25 columns equilibrated and eluted with HEPES buffer containing 600 mM
KC1 and 50 mM molybdate at pH 7.6. Of the bound fraction collected from
these columns, 0.5 ml was then run on a 7 ml pentyl agarose column
equilibrated and eluted with HEPES buffer containing 600 mM KC1 and 50
mM molybdate at pH 7.6. Fractions (0.5 ml) were collected and assayed
for specific binding.
Sucrose Density Gradient Sedimentation
Purified receptor samples (400 ul) were layered onto linear 5-20%
sucrose density gradients (4.6 ml; prepared with HEPES buffer containing
2 mM DTT and 20 mM molybdate) and centrifuged at 0 C for 1.5-2 hr at
370,000 g (average) in a Sorvall TV-865 vertical tube rotor. The
cellulose nitrate tubes were punctured and 26-28 fractions (180 ul)
collected and assayed for radioactivity by liquid scintillation
spectrometry. Sedimentation coefficients (S20,w) were calculated from
the linear regression of S20,w vs sedimentation distance (Martin and
Ames, 1961) for the following standard proteins run in parrallel tubes:
chicken ovalbumin (0VALB, 3.6 S), bovine serum albumin (BSA, 4.3 S),

148
bovine gamma globulin (IgG, 7.4 S) and catalase (CAT, 11.3 S). The
standard proteins were [14C]methylated (for detection) to low specific
activity with [14C]formaldehyde by the method of Rice and Means (1971).
Results
Initial attempts to purify the glucocorticoid receptor from brain
using deoxycorticosterone agarose followed very closely the "batch"
procedure originally described by Grandics et al. (1984b) for the
purification of glucocorticoid receptors from liver except that HEPES
buffer was used instead of potassium phosphate for cytosol preparation
and for ecomomic reasons, a 1 uM concentration instead of a 2 uM
concentration of [3H]TA was used for the steroid exchange reaction. For
reasons that are not entirely clear, this procedure resulted in
relatively low yields of purified receptors in the range of 5%, much
lower than the 50-70% recoveries reported by Grandics et al. (1984b).
While the 50% lower concentration of [3H]steroid used in the exchange
probably contributed to the lower yields, it is not likely to be
entirely responsible for a 10- to 14-fold lower receptor recovery rate.
Some modifications to the procedure involved running cytosol through a
column of deoxycorticosterone agarose as opposed to using the batch
approach. In addition, it was assumed that due to the excellent
stability of the [3H]TA-receptor complex in cytosol under these
particular buffer conditions and because of the relatively very slow
dissociation of [3H]TA (the free ligand) as compared to deoxycorti¬
costerone (the matrix-associated ligand), a much higher yield should be
obtained for a given concentration of [3H]TA if a much longer exchange
incubation is used.

149
The next series of experiments involved applying a volume of
cytosol to the affinity resin column which was roughly equivalent to the
void volume of the column, thereby exposing all of the gel to receptor-
containing cytosol. The columns were allowed to incubate for
approximately 8 hr at 0 C prior to washing the affinity resin with at
least 10 times the bed volume of the column. The column was washed with
a buffer identical to that used in the preparation of cytosol containing
both molybdate (to stabilize and prevent activation) and DTT (to prevent
oxidation of any dissociated receptors which would preclude
reassociation to either the affinity resin or the tritiated exchange
ligand). Aliquots of each were checked for glucocorticoid binding
activity by incubating with 20 nM [3H]TA +/- 4 uM [1H]TA for an
additional 24 hr. No binding activity whatsoever was detected in any of
these column washes indicating that the concentration of, glucocorticoid
receptors in cytosol as prepared for these experiments was far from
saturating the affinity resin and that the incubation time of 8 hr at 0
C was more than sufficient to achieve virtually 100% binding of all
receptors. After extensive washing of the column to remove any
non-binding or low affinity-binding components, the steroid affinity gel
was incubated with a lower concentration of [3H]TA (100 nM) than was
used before, but the steroid exchange incubation period was extended
considerably from 16 hr to 48 hr. The recovery of glucocorticoid
receptors from brain cytosol was up from approximately 5% to 15%, a
3-fold increase over the previous procedure. When liver cytosol was
used in these experiments, a similar yield of 15-20% was obtained but
much more binding was obtained since the concentration of glucocorticoid
receptors per gram wet weight of tissue is several times greater for
liver than brain.

150
It was observed in subsequent studies using the purified receptor
preparation obtained by the previously described procedure that these
[3H]TA-labeled receptors appeared to be far more labile than unpurified
[3H]TA-labeled receptors in cytosol. The dissociation rate for [3H]TA
is extremely slow in cytosol with a half time of days at 0 C (Gray,
1982). Likewise, the rate of receptor degradation leading to an
irreversible loss in binding is very slow at low temperatures,
especially in the presence of molybdate and is generally not a factor in
most experiments. However, for reasons that are not yet clear, affinity
resin-purified [3H]TA-labeled glucocorticoid receptors undergo an
approximately 50-60% loss at 0 C, even when excess free [3H]TA is
present at concentrations greater than 100 nM, indicating that the loss
probably represents an irreversible loss of binding capacity independent
of steroid dissociation. Similar losses were seen when [3H]DEX was used
as the exchange steroid. This finding had important implications for
both the purification of glucocorticoid receptors and the subsequent
studies regarding activation of the glucocorticoid receptor.
Due to the extended time and expense required for receptor
purification and the unexpected lability under supposedly ideal
conditions, there was an obvious need for reducing the losses in
receptor binding until subsequent experimental manipulations could be
carried out. One possibility was to freeze the receptor preparation
immediately after purification, store at a very low temperature and thaw
immediately prior to experimental use. High enough concentrations of
purified bound receptor would have to be used in the subsequent
experimental procedures to allow for detection of chromatographic peaks,

151
DNA-cellulose binding, etc. after substantial losses during some of the
procedures. A series of experiments were therefore carried out to
determine the lability of glucocorticoid binding in non-steroid-labeled
cytosol, steroid-labeled cytosol and an affinity resin-purified
preparation of unactivated receptors during freezing/thawing and during
long-term storage in a deep-frozen state (-75 to -85 C). In addition,
the effect of glycerol (0, 10 and 30% w:v) on glucocorticoid receptor
lability under these conditions was simultaneously investigated. The
initial part of the study involved unpurified receptor preparations
(standard cytosol preparations) to provide, first, a comparison for
purified preparations and second, potentially practical information for
preparatory purposes when dealing with unpurified preparations. Cytosol
prepared in the usual manner was split into two equal fractions for the
two parts of this experiment, that being radiolabeled with [3H]TA prior
to freezing and thawing (for purposes of studying the occupied form of
the receptor), and that being radiolabeled with [3H]TA after freezing
and thawing (for purposes of studying the unoccupied receptor).
Appropriate volumes of glycerol and/or deionized water were added to
subfractions of each of these groups to achieve the desired
concentrations of glycerol while maintaining the concentration of
cytosolic protein as well as other endogenous and exogenous compounds
equal in all of these subfractions. The effects of freezing and thawing
on the unoccupied receptor were determined by placing the unlabeled
cytosolic samples in a -85 C freezer and subsequently thawing at various
time intervals prior to incubation with 20 nM [3H]TA +/- 200-fold
concentration of unlabeled TA (for determination of nonspecific
binding). Since the melting point for all the groups, including the

152
nonglycerol group, was below 0 C, thawing was accomplished by simply
placing the frozen sample tubes in an ice water bath at 0 C. Thawing
took somewhat longer for the nonglycerol group, but in every case took
no more than 15 min. The thawed samples were incubated with the
indicated steroids for 24 hours prior to bound/free steroid separations
performed on Sephadex G-25 columns. The effects of freezing and thawing
on the occupied receptor were studied by first radiolabeling the cytosol
(again with 20 nM [3H]TA +/- 4 uM [1H]TA) and then subjecting the
labeled cytosol to freezing at -85 C followed by thawing after various
time intervals. Bound/free separations were performed immeadiately
after the freeze/thaw step. Later, purified [3H]TA-labeled receptor
preparations were treated identically to the prelabeled cytosolic
fraction. It is clear from the results (Table 4-1) that the process of
freezing and thawing led to only a slight loss (10-15%) in cytosolic
binding which was almost completely preventable by the addition of 10%
glycerol. Increasing the glycerol concentration to 30% further
decreased the freeze/thaw-induced binding loss to virtually zero.
Interestingly there was very little difference between the stability of
the bound versus the unbound receptors in cytosol during freezing and
thawing. Perhaps even more important was the fact that virtually no
further losses in binding capacity were encountered once the cytosol was
frozen for periods of up to 5 to 11 days. Purified [3H]TA-labeled
receptors, on the other hand, appeared more susceptible to
freeze/thaw-induced losses, even in the presence of glycerol, though
glycerol did decrease the degree of loss slightly. However, despite the
more dramatic loss of binding capacity associated with the freezing and
thawing of purified preparations, the degree of loss was nevertheless

153
acceptable when compared to the rapid rate of loss in an unfrozen state
and further losses during long term storage at -85 C were almost
negligible.
The discovery of the relative instability of the bound purified
glucocorticoid receptor led to yet another change in the affinity
purification procedure in an attempt to increase the yield of purified
receptor. This involved reducing the steroid exchange incubation period
to an intermediate level from 48 to 30 hr and again increasing the
concentration of [3H]TA from 100 nM to 500 nM. These changes were based
on the assumption that the increase in receptor lability occurs
immediately after washing the affinity column free of non-binding
components and that extended steroid incubations under these conditions
would result in receptor degradation that would eventually outpace the
rate of exchange between deoxycorticosterone and [3H]TA binding to the
glucocorticoid receptor. The rate of exchange could be increased during
a shorter incubation period by increasing the concentration of free
[3H]TA relative to the concentration of resin-associated
deoxycorticosterone. Although increasing the temperature would increase
the rate of steroid exchange, it would simultaneously increase the rate
of receptor degradation (the temperature dependence of purified receptor
lability will be discussed later in this section). An additional step
to enhance purified receptor stability and thereby increase recoveries
during purification was to increase the concentration of molybdate from
20 to 50 mM in the buffers used in washing affinity columns and present
during steroid exchange reactions and many subsequent steps. Although
20 mM molybdate is generally sufficient to almost completely inhibit
both glucocorticoid receptor degradation that leads to a loss in binding

154
and activation of the steroid receptor complex to the nuclear binding
form (Dahmer, 1983; Luttge et al., I984a-d), a higher concentration of
molybdate was found to further decrease the lability of the purified
receptor, though only slightly (data not shown). The net result of
these changes in the purified procedure was that a 20-45% recovery was
now obtainable using both brain and liver. These recoveries, though
still less than those reported by some labs (Grandics et al., 1984b)
were finally in a range where ample quantities of purified receptor
could be obtained for at least some physicochemical studies.
Since binding of the cytosolic glucocorticoid receptor to the
affinity resin should occur very rapidly, because of a very high
concentration of deoxycorticosterone attached to the resin, it was
thought that even higher yields of purified receptor could be obtained
even more economically by performing a two-step steroid exchange
reaction. This involved an initial steroid exchange reaction, after
washing the affinity resin free of non-binding components, with a very
high concentration of [1HJDEX (4 uM) for approximately 8-12 hr. The
[1H]DEX-Iabeled receptors, which after the incubation period should have
represented a very large proportion of the total population of
glucocorticoid receptors, were then eluted free of the affinity resin
column prior to being subjected to Sephadex G-25 chromatography in order
to remove free [1H]DEX from the bound purified receptors. The bound
fraction was then incubated with 40 nM [3H]TA at 0 C for the second
steroid exchange reaction. Aliquots of this preparation were
periodically assayed for [3H]TA binding by again running bound/free
steroid separations on Sephadex G-25 columns. Using [1H]DEX for the
first exchange ligand served two purposes. First, DEX is roughly equal

155
to TA in its specificity for the type II glucocorticoid receptor (it
should be noted that progesterone and type I adrenocorticoid
(mineralocorticoid) receptors have some affinity for
deoxycorticosterone). Second is the fact that DEX dissociates from the
receptor much faster than TA (Gray, 1982). Since the [3H]TA had only to
compete with the very low, almost negligible, levels of free [1H]DEX
that had dissociated from glucocorticoid receptors in the preparation,
much lower concentrations (40 nM versus 500 nM) could be used for this
exchange and, theoretically, result in a high yield of [3H]TA-labeled
receptor. Unfortunately, this two-step steroid exchange procedure did
not result in any greater recovery than was obtained with a single-step
steroid exchange, and, in fact, the maximal recoveries (12-20%) were
typically about half of what had been achieved with the single-step
process. One advantage of the procedure, however, is the lower cost per
quantity of receptor recovered.
Another two-step exchange procedure for purifying glucocorticoid
receptors attempted involved an intermediate sulfhydryl down-regulation
step in order to remove the bound receptors from the affinity column in
an unoccupied state. The procedure offered the potential for yet
another economical method of glucocorticoid receptor purification, while
also allowing for the possibility of obtaining fairly pure
glucocorticoid receptors in an unoccupied form, another goal of this
research. This procedure involved first incubating brain or liver
cytosol with the deoxycorticosterone agarose as previously described
followed by extensive washing of the column to remove non-binding
components. A change in the washing procedure from previously described
experiments involved using a buffer not containing DTT during the final

156
3-4 washes. This non-DTT washing step was then followed by incubation
of the affinity column with 10 mM DTNB. DTNB had previously been shown
to reversibly down-regulate the binding capacity of unoccupied
glucocorticoid receptors by apparently interacting with a binding site
sulfhydryl group(s) while having no effect on prebound receptors (see
Chapter III). It was assumed that the free DTNB would compete with the
matrix-associated deoxycorticosterone for the steroid binding site of
dissociated receptors, down-regulating them and preventing any further
rebinding to the steroid affinity matrix. The down-regulated unoccupied
receptors were then washed free of the affinity matrix and the resulting
preparation was run on a Sephadex G-25 column in order to remove any
free DTNB. The macromolecular fraction was then incubated with 20 nM
[3H]TA in the presence of 10 mM DTT, which had previously been shown to
up-regulate virtually 100% of unoccupied receptors down-regulated in
cytosol by DTNB. For reasons that are not entirely clear, absolutely no
[3H]TA binding was ever detected, even after several modifications of
the procedure involving incubation times, DTNB concentrations, etc.
Another modification of the procedure which greatly increased the
quantity of receptor purified per ml of affinity resin was based on the
high concentration of deoxycorticosterone attached to the gel matrix.
It was found that when the affinity column was continually "washed" with
yet another application of the original cytosol, allowing for 2 to 4 hr
of incubation at 0 C between each application of cytosol. Up to 10
volumes of cytosol were applied to the affinity resin before the wash
procedure (using buffer only) was initiated. All washes eluting from
the column were checked for glucocorticoid binding activity by
incubating aliquots with 20 nM [3H]TA +/- 4 uM [1H]TA for 24 hr at 0 C.

157
No [3H]TA binding was ever detected, indicating that the affinity column
had not been saturated with receptors even after 10 applications of
cytosol. Another positive point regarding this procedure is that
receptor lability is not a problem during the entire multiple cytosol
application step since the receptors bound to the affinity matrix are
continuously stabilized by the cytosolic factors later removed by
washing with buffer only. After the steroid exchange reaction with
[3H]TA was complete, total recoveries were still in the 20-45% range
meaning that multiple applications of cytosol onto the steroid affinity
resin resulted in up to 10 times the total amount of receptor normally
obtainable per ml of resin with a single application of cytosol. It is
likely that even more cytosol could be applied to the gel, resulting in
even greater quantities of purified receptor per ml of affinity resin
per experiment, but this was not further investigated in the present
study.
Activation of the steroid affinity resin-purified glucocorticoid
receptor was carried out by using procedures nearly identical to those
described previously by this lab for the activation of cytosolic
[3H]TA-labeled glucocorticoid receptors (Luttge et al., 1984a-d; see
also Chapter II). Previously frozen samples of purified receptor were
thawed by incubation in a 0 C water bath, then run on Sephadex G-25
columns equilibrated and eluted with HEPES buffer containing 2 mM DTT
and either containing or not containing 50 mM molybdate. The bound
fraction collected from the Sephadex G-25 column equilibrated with
non-molybdate-containing buffer was subjected to a 24 min incubation at
22 C after which the sample was again cooled quickly to 0 C and
sufficient molybdate was added to achieve a final concentration of 50

158
mM. Both activated and unactivated preparations were then again run on
Sephadex G-25 columns equilibrated and eluted with a buffer of variable
composition depending upon the particular experiment that followed.
This second column run acted to reequilibrate both activated and
unactivated preparations in the same buffer and also acted to remove any
free steroid occurring as the result of receptor degradation resulting
in steroid dissociation. One finding that was immediately apparent was
that heat-induced activation led to a significant loss (approximately
50-75%) in receptor binding, far more than generally had been observed
during the activation of cytosolic glucocorticoid receptors (Luttge et
al., 1984a-d; Luttge and Densmore, 1984). This finding resulted in the
use of higher concentrations of purified receptor to allow for expected
losses in binding during heat activation and subsequent activation assay
procedures.
Initial attempts to activate the purified receptor investigated
changes in binding of the receptor to DNA-cellulose associated with
activation. The first experiment looked at DNA-cellulose binding before
and after the single activation incubation previously described (24 min
at 22 C). Unpurified cytosolic receptor preparations, run
simultaneously as controls, displayed the expected changes in DNA
affinity upon activation (less than 1% of unactivated unpurified
receptors bound to DNA-cellulose whereas 45-50% of the activated
unpurified receptors bound to DNA-cellulose, a greater than 50-fold
increase). Interestingly, the purified unoccupied receptor sometimes
appeared to have a higher affinity for DNA-cellulose (2-10% binding)
than did the cytosolic unoccupied receptor ( <1% binding), but the
purified activated receptor always displayed precisely the same affinity

159
for DNA-cellulose as did the purified unactivated receptors within a
given experiment. This slight and variable binding of purified receptor
to DNA-cellulose is not completely understood, but it was clear after
several replications that no further increases in binding were
associated with heat activation under the conditions tested.
The possibility that activation of purified receptors may require
conditions different from those needed for activation of unpurified
receptors was investigated in part. While the temperature of activation
(22 C) was left constant, the duration of activation was varied to
include periods that were both shorter (6 and 12 min) and longer (48
min) than the original incubation period of 24 min. In addition,
purified receptor groups were included at each of those time points
which were activated in the presence of 150 mM KC1 to determine if ionic
strength was a more important factor in the activation of purified
receptors. The KC1, where added during activation, was removed from the
samples during the run on the second Sephadex G-25 column which was
always equilibrated and eluted with HEPES buffer containing 2 mM DTT and
20 mM molybdate. It should be noted that all of the samples were
equilibrated to 20 mM molybdate instead of 50 mM molybdate, the
concentration which had been used during the purification procedure,
storage, etc., because this lab had previously shown higher
concentrations of molybdate to inhibit the binding of activated
glucocorticoid receptors to DNA-cellulose (Luttge et al., 1984b). The
results of this limited study (Table 4-2) showed that neither duration
of activation nor ionic strength were important factors in the
activation of the purified glucocorticoid receptor as measured by
DNA-cellulose binding. The increased heat lability of the purified

160
receptor made interpreting the DNA-binding data for the 48 min
activation a problem since the total level of [3H]TA binding was reduced
to the point that a small change in the already small percentage of
receptor bound to DNA-cellulose was hard to determine accurately.
Again, however, any major increases in binding would have been evident,
but they were not. The effect of including 150 mM KC1 during the 22 C
activation incubation on DNA-cellulose binding seemed to also be
negligible. The change in ionic strength also had virtually no effect
on the temperature-dependent lability of the purified glucocorticoid
receptor.
In order to determine if the inability of activated purified
glucocorticoid receptors to bind to DNA represented a
purification-induced modification in only the DNA binding properties of
the receptor or an overall inability of the receptor to undergo the
major physical changes generally associated with activation, other means
of monitoring changes in the properties of the receptor associated with
heat activation were employed. One of the analytical tools used to
study this phenomenon (or lack thereof) was hydrophobic interaction
chromatography. Measurements of surface hydrophobicity had previously
been used to differentiate between activated and unactivated
glucocorticoid in unpurified preparations of brain cytosol (see Chapter
II). The current investigation of purified receptors used a 7 ml pentyl
agarose column of the precise demensions previously used in the
hydrophobic analysis of unpurified receptors. Again, the purified
receptor preparation was activated in the absence of molybdate at 22 C
for 24 min prior to being run on a Sephadex G-25 column equilibrated and
eluted with HEPES buffer containing 600 mM KC1 and 50 mM molybdate which

161
was also used to equilibrate and elute the pentyl agarose column. The
profiles of both the purified activated and unactivated receptors
(Figure 4-1) were strikingly different from the profiles obtained with
their unpurified counterparts. Perhaps even more interesting is the
fact that the purified activated and unactivated profiles differed
significantly from one another. The purified unactivated receptor
resulted in a profile containing one major and one minor peak. The
major peak eluted first and at the identical position that the
unpurified unactivated receptor eluted at under identical conditions
(Figure 2-5). The minor peak eluted only 3 fractions later and
represented a fraction of the total binding that varied slightly from
one replication to the next but averaged approximately 25%. Both peaks
were relatively sharp and well defined and their positions were very
replicable. The profile of purified activated receptor was a single
»
sharp peak eluting at the same position as the second minor peak of the
purified unactivated receptor profile. While all of the peaks obtained
with the purified preparations were sharper than those obtained with the
unpurified preparations, the greatest contrast was between the purified
and unpurified activated profiles. The unpurified activated peak is
dramatically broader and elutes slower than its purified counterpart.
Discussion
This section of the dissertation sought primarily to investigate
the process of receptor activation using a receptor preparation as free
as possible of non-receptor cytosolic components that could interfere
with, or contribute to, the conversion of the receptor from a nuclear
non-binding to a nuclear binding form. The first step required for such
an investigation was the purification of the unactivated form of the

Figure 4-1. Hydrophobic interaction chromatography on purified samples
of unactivated and activated Type II glucocorticoid receptors. Brain
cytosol prepared in HEPES buffer containing 50 mM molybdate and 2 mM DTT
was applied to a deoxycorticosterone agarose affinity column and
incubated for 4 hr at 0 C. The column was then washed free of
nonbinding components using 10 washes of the same buffer and then
incubated for 40 hr at 0 C with 250 nM [3H]TA to facilitate a steroid
exchange. The purified receptors, labeled with [3H]TA, were then eluted
with the original buffer and run on Sephadex G-25 columns equilibrated
and eluted with HEPES buffer plus 600 mM KC1 and 50 mM molybdate.
Aliquots (0.5 ml) from the macromolecular fractions collected from these
columns were then run on 7 ml pentyl agarose columns equilibrated and
eluted (0.5 ml fractions) with the same HEPES, KC1, molybdate buffer.
Binding is expressed as percent of the total counts (12,000 cpm) applied
to the column. The profile shown here is representative of two
independent replications.

163

164
glucocorticoid-receptor complex. It was also important that the process
of purification did not in itself lead to activation of the receptor.
Although several labs have recently undertaken the task of purifying the
glucocorticoid receptor (see introduction of this chapter for details),
numerous experimental factors, which can vary from one lab to another
have been shown to dramatically affect receptor stability, receptor
activation, steroid association and dissociation. The effects of these
variable factors on receptor parameters may actually be magnified by the
process of purification since many inhibitors of receptor degradation
and activation are removed from the preparation. For this reason, the
methodology for unactivated glucocorticoid receptor purification had to
be perfected in order to work effectively under the experimental
conditions previously and currently used by this lab for a number of
extensive studies regarding glucocorticoid receptor regulation and
activation. Although receptor recoveries of 45% may have been somewhat
lower than those reported by Grandics et al. (1984b) and Govidan and
Gonemeyer (1984) which ranged from 50 to 70%, they were sufficiently
high enough to carry out the subsequent investigations of
activation-induced changes in purified glucocorticoid receptor
properties. This is partly true because of the relatively high
concentrations of glucocorticoid receptors in the tissues used for
purification (brain and liver). In addition, only relatively low levels
of receptor were needed to perform many of the qualitative and
quantitative determinations.
Unfortunately, purificaton of unoccupied receptors using
deoxycorticosterone agarose affinity chromatography proved entirely
unsuccessful. Although the reasons for this are not entirely known,

165
these negative findings may, nevertheless, provide additional clues as
to the nature of the purified receptor. All indications from other work
in this dissertation regarding sulfhydryl down-regulation of
glucocorticoid receptor binding capacity (see Chapter III) were that
DTNB treatment of either liganded or unliganded receptors led to no
irreversible loss of binding capacity, beyond what was normally
encountered in the absence of DTNB, when all other conditions were
constant. It therefore came as a surprise that DTNB appeared to
irreversibly inactivate the binding capacity of purified glucocorticoid
receptors on the affinty column. Even if the high concentration of DTNB
had only been a poor competitor for the lower concentration of affinity
resin-linked deoxycorticosterone, one would have expected some recovery
of receptor binding capacity since the reaction between the receptor and
DTNB is irreversible until DTT is added after the prepartion is washed
from the column. Because the total amount of receptor binding activity
associated with the affinity column is so high and because nonspecific
binding activity removed from the column as a result of the DTNB
incubation is, for all practical purposes, zero, specific binding
recoveries as low as 0.02 to 0.05% could have been easily and accurately
detected. It can therefore be safely assumed that the inability to
recover any binding activity by this method was not merely due to a
concentration-related problem regarding the DTNB. It should also be
noted that even if the method had been successful in purifying the
unoccupied glucocorticoid receptor, the final preparation probably would
not have been as pure or homogeneous as that obtained by the
steroid-exchange purification of steroid-labeled receptors using the
same steroid affinity resin. This is because the exchange with DEX or

166
TA is very specific for glucocorticoid type II receptors, whereas DTNB
or other sulfhydryl reagents have been shown to reversibly inactivate
the binding capacity of progesterone (Kalimi and Banerji, 1981) as well
as glucocorticoid type I (Emadian and Luttge, unpublished) receptors,
both of which also bind to deoxycorticosterone, but have very low
affiinities for DEX or TA.
Since this work was done, a report has very recently appeared which
described the partial purification of the ligand-free glucocorticoid
receptor (Krajcsi and Aranyi, 1986). These workers used a
cortexolone-substituted affinity matrix and eluted receptors from the
column via an exchange with either free TA or cortexolone. Owing to the
relatively fast dissociation of the glucocorticoid receptor-cortexolone
complex, a partially purified free glucocorticoid receptor capable of
steroid binding, was obtained by merely allowing for the rapid
dissociation of the purified complex. It should be noted, however, that
like deoxycorticosterone, cortexolone is not specific for only the
glucocorticoid type II receptor (Krajcsi and Aranyi, 1986).
Using a dissociation approach to purification of unoccupied
receptors probably would have resulted in some degree of success in the
present study. However, the yields would have been lower than those for
purified bound receptors because of binding losses encountered during
the relatively long periods required for dissociation of DEX. Although
purification recoveries as high as 20% were achieved with a two-step
steroid exchange using DEX as the intermediate steroid, it should be
remembered that receptors dissociated from DEX rapidly rebound TA.
Since it was never determined in the present study what impact, if any,
steroid binding has on the stability of the purified unoccupied

167
receptor, it would not be possible to predict what yields could have
been achieved by such a procedure.
The successful long-term storage of both purified and unpurified
(cytosol), steroid-bound and free glucocorticoid receptors at -75 to -85
C with virtually no losses in binding capacity provided an additional
degree of convenience when running long, complex, multi-step
experiments. The losses that did occur during the freezing and thawing
were not considered a serioius drawback to cold storage and these losses
could generally be eliminated, or nearly so, by the inclusion of the
cryoprotective agent glycerol. The greater losses encountered during
the freezing and thawing of purified receptor preparations, even in the
presence of glycerol, were probably related to the absence of some
endogenous component(s) acting in a cryoprotective manner. These
missing factors may have some relation to the increased lability of the
purified glucocorticoid receptor, even in the presence of molybdate.
The losses are still acceptable, however, considering the importance of
storage of a form that is so costly (in time and money) to prepare each
time. McLusky et al. (1986) recently examined the effects of freezing
and thawing on steroid receptor concentrations in the brain and
pituitary of the rat. These workers found that freezing and thawing
resulted in measurable losses of cytoplasmic androgen, progestin and
glucocorticoid receptors, while estrogen receptors were relatively
stable. They also found that in all cases except, interestingly, that
of cytoplasmic glucocorticoid receptors, these losses could be prevented
by freezing the tissue in 10% aqueous dimethylsulfoxide, another reagent
with known cryoprotective properties. With regards to the effects of
freezing on other properties of steroid receptors, Janes et al. (1982)

168
earlier compared the sucrose density gradient sedimentation profiles of
uterine estrogen and progesterone receptors that were either frozen or
lyophilized and found little difference between the two. Unfortunately,
these workers did not compare their findings directly with those for
fresh cytosolic preparations, but the implication was that freezing and
thawing of either cytosol or whole tissue had little effect on
subsequent receptor stability, activation, etc..
One of the primary reasons for purifying the glucocorticoid
receptor was to allow for a more complete understanding of the process
of activation of the receptor to its nuclear binding form. The relative
importance of examining glucocorticoid receptor activation using a
purified preparation is exemplified by a number of recent reports that
appeared during the planning and progress of the present study (Grandics
et al., 1984b; Wrange et al., 1984; Schmidt et al., 1985; Webb et al.,
1985; Krajcsi and Aranyi, 1986; Schmidt et al., 1986). The finding of
the present study that purified activated receptors have a dramatically
reduced affinity for DNA is in general agreement with the findings of
Grandics et al. (1984b), Schmidt et al. (1985 and 1986) and Webb et al.
(1985). In contrast, Krajcsi and Aranyi (1986) were able to achieve an
increase in DNA binding that was close to the levels attained when crude
cytosol preparations were used. This is probably because of the fact
that the cortexolone-substituted affinity procedure used by these
workers was reported to result in a lower degree of purification (only
75 to 100-fold) than was achievable by the deoxycorticosterone affinity
procedure used in the present study and by the other workers (several
hundred to thousand-fold). It is likely that a very high degree of
purification is required in order to remove whatever factor(s) are

169
involved in the transformation of the receptor to a form able to bind
DNA.
Despite the lack of activation-induced increases in DNA binding,
the finding in this study that heat activation did lead to changes in
the purified receptor normally associated with activation of unpurified
receptors, such as an increase in hydrophobicity, leads to the notion
that activation may be a multi-step process and that some changes, such
as hydrophobicity, are not dependent upon cytosolic factors removed by
purification, whereas other changes, such as DNA binding, are. Such a
possibility has also been suggested by Schmidt et al. (1985), who found
that whereas purified glucocorticoid receptors underwent a temperature-
dependent, molybdate-sensitive change in DEAE-cellulose binding, a
heat-stable cytoplasmic macromolecule was required to subsequently
achieve an increase in DNA binding. This second step involving
cytoplasmic factor(s) was reportedly molybdate-insensitive and
temperature-independent. More recently, a preliminary report by Schmidt
et al. (1986) showed that bovine pancreatic ribonuclease (RNase) A and
S-protein (an enzymatically inactive proteolytic fragment of RNase A
which contains the RNA binding site) stimulated the activation, as
evidenced by increased DNA-cellulose binding, of highly purified rat
hepatic glucocorticoid receptors. These authors concluded that their
findings were consistent with numerous observations which suggest that a
small RNA molecule(s) may be an integral component of the glucocorticoid
receptor and may influence activation. A multistep process for
activation was actually proposed earlier when it was demonstrated that
the rate of activation of glucocorticoid receptors in mouse brain
cytosol varied according to which assay for activation was used (Luttge

170
et al., 1984a, b & d; Luttge and Densmore, 1984). Changes in binding to
both DEAE-cellulose filters (ion exchange) and glass fiber filters
(hydrophobic interaction) occurred at about the same rate which was
twice the rate at which DNA-cellulose binding increased. Changes in
sedimentation rate (indicating subunit dissociation) occurred at yet a
different rate, indicating the possibility that even more than two steps
may be involved in the overall activation process. It is clearly
obvious that purification of the glucocorticoid receptor has greatly
enhanced the understanding of the process of steroid receptor
activation, but much work remains to be done in this area before final
resolution of this important process has been reached.

CHAPTER V
CHARACTERIZATION OF THE UNOCCUPIED GLUCOCORTICOID RECEPTOR
Introduction
Numerous studies have revealed that glucocorticoid (as well as most
other steroid) receptors are very unstable molecules in vitro,
particularly when they are not bound to a steroid ligand (Kirkpatrick et
al.,1972; Koblinsky et al., 1972; Bell and Munck, 1973). These
receptors are stabilized considerably when they are bound by
glucocorticoids (Kirkpatrick et al., 1972; Pratt et al., 1975;
Rafestin-Oblin et al., 1977), and the degree of stabilization appears to
be roughly proportional to the binding affinity of the steroid (Nielsen
et al., 1977a,b). In light of recent reports that unbound
glucocorticoid (Raaka and Samuels, 1983) and estrogen (Eckert et al.,
1984) receptors appear to be relatively stable in situ (with half-lives
of up to 9.5 Hr at 37 C), an understanding of those stabilizing factors
that are effectively removed or diluted out during cytosol preparation
would not only allow for a more accurate quantification of receptor
binding data, but is virtually essential before in vitro studies of
receptor up- and down-regulation can be interpreted with any degree of
confidence. A number of substances added exogenously have been shown to
influence the stability of unbound steroid receptors. Varying degrees
of stabiliza- tion have been achieved with glycerol (Schaumburg, 1972;
Korge and Timpmann,1983; Ogle, 1983), molybdate (Leach et al., 1979),
sulfhydryl protecting agents (Rees and Bell, 1975; Granberg and Ballard,
171

172
1977; Sando et al., 1979; McBlain and Shyamala, 1980; Densmore et al.,
1984 ), nucleotides (Sando et al., 1979; Barnett et al., 1983; Densmore
and Luttge, 1985), and protease inhibitors (Sherman et al., 1978;
Ratajczak et al., 1981; Kalimi et al., 1983). Conflicting results have
been obtained with the addition of alkali salts (Schaumburg, 1972;
Schmid et al., 1976; Krieger et al., 1976; Young et al., 1977; Densmore
et al, 1984c), divalent cations (Schmid et al., 1976; Nakai et al.,
1978; Aranyi and Naray, 1980; Ratajczak et al., 1981; Rousseau et al.,
1982; Hubbard and Kalimi, 1983a,b; Kalimi et al., 1983; Densmore et al.,
1984a) and various chelating agents (Bell and Munck, 1973; Rees and
Bell, 1975; Schmid et al.,1976; Rafestin-Oblin et al., 1977; Ratajczak
et al., 1981; Hubbard and Kalimi, 1983 a,b; Densmore et al., 1984a).
Glycerol, other polyhydric alcohols and sugars are known to
increase the thermal stability of many proteins (Gerlsma and Stuur,
1972; Donovan, 1977; Back et al., 1979), including steroid receptors
(Sherman, 1975). Early studies of glucocorticoid receptors reported a
requirement for high concentrations (40%) of glycerol for maximum
stabilization of the receptor in rat thymocyte cytosol (Schaumburg,
1972) and a similar requirement was reported for progesterone receptors
from rat and mouse uteri (Feil et al., 1972), although the mechanism of
glycerol stabilization was unknown. More recently, Korge and Timpmann
(1983) reported that not only did glycerol stabilize dexamethasone
binding in rat heart cytosol, but it also reduced markedly both the
association and dissociation rate constants. It is likely that
glycerol's ability to enter the layer formed in aqueous solution at the
receptor surface accounts for its stabilizing effect (Ogle, 1983). It
has been suggested that glycerol molecules distribute themselves

173
throughout the solvation sheath of proteins in accordance with the
balance of forces between repulsion from nonpolar regions and attraction
to the polar regions of the protein surface, as well as attraction
between water and glycerol molecules (Gekko and Timasheff, 1981a).
These influences tend to stabilize the more folded, or native, state of
the protein and increase its chemical potential due to the preferential
exclusion of glycerol from the domain of the protein. It follows then
that the more hydrophobic a protein's surface is, the greater the
concentration of glycerol required to enter the hydration layer and,
hence, stabilize the native conformation, will be. Denaturation, or
unfolding, involves an increase in the surface of contact between
receptor protein and solvent, and in particular exposes additional
hydrophobic residues to contact with solvent. The presence of glycerol
would lead to a thermodynamically less favorable situation and require
the use of more free energy for unfolding than in water. As a result,
the presence of glycerol should tend to favor the folded native state of
the receptor (Gekko and Timasheff, 1981a,b), possibly accounting for its
increased stability at elevated temperatures as well as the additional
effect of holding the ligand-binding site more rigidly in its native
conformation, thus protecting high affinity binding (Ogle, 1983). In
addition, the presence of high concentrations of glycerol has been shown
to reduce the loss of proteins by adsorption to glass or plastic
surfaces (Suelter and DeLuca, 1983) and acts as a cryoprotective agent
to reduce receptor losses encountered during freezing and thawing
(Densmore, see Chapter IV). One must also consider the possibility that
some of glycerol's effects on receptor stability, association rates,
etc., may be related to concentration-dependent changes in viscosity.

174
Recent work with the glucocorticoid type I receptor has indicated that
glycerol has little or no effect on the stability of this receptor
(Emadian et al., unpublished).
Molybdate, vanadate and tungstate are group 6A transition metal
oxyanions that inhibit both alkaline (Lopez et al., 1976) and acid
(VanEtten et al., 1974) phosphatases as well as some phosphohydrolases
like (Na+ and K+)ATPases (Cantley et al., 1978; Karlish et al.,1979;
Simons, 1979) in micromolar and submicromolar concentrations. It was
for this reason that Nielsen et al. (1977a,b) added molybdate to
cytosols prepared from several tissues and cultured cells where it was
found to produce a profound inhibition of temperature-mediated
inactivation of glucocorticoid receptors. Although molybdate's
stabilizing effects were thought originally to be via a direct effect on
a phosphatase enzyme (Nielsen et al., 1977a,b), several later
observations provided evidence that the transition metal's inhibition of
phosphatase activity and receptor activation may not be related. To
begin with, the concentrations of molybdate, vanadate and tungstate
required to stabilize unoccupied glucocorticoid receptors and to prevent
the activation of the glucocorticoid receptor complex are generally
greater (millimolar rather than micromolar) than those required for
effective phosphatase inhibition (Leach et al., 1979; Wheeler et al.,
1981; Gray, 1982; Densmore et al., unpublished). In addition, 10 mM
molybdate reportedly did not inhibit calf intestine alkaline-phosphatase
activity assayed with para-nitrophenyl phosphate as substrate, nor did
it prevent inactivation of glucocorticoid-binding capacity when this
enzyme was added to rat liver cytosol, although lost binding could be
reactivated upon addition of a sulfhydryl protecting (reducing) reagent

175
such as dithiothreitol (Dahmer, 1983; Dahmer et al., 1984). In
contrast, receptor inactivation occurring in the presence of reducing
agents, but in the absence of transition metal oxyanions, is
irreversible. These and many other observations strongly suggest that
molybdate interacts directly with the receptor. Such a direct
interaction has also recently been proposed to account for molybdate's
similar actions on estrogen receptors (Braunsberg, 1984). This
interaction is apparently a weak one because of the relatively high
(millimolar) concentrations required for receptor stabilization. The
interaction between the transition metals and the receptor is not likely
to be the same as the interaction between vanadate and Na+, K+ ATPases
since these enzymes are inhibited by several orders of magnitude lower
vanadate concentration (Cantley et al., 1978). In the case of acid
phosphatases, there is evidence that molybdate complexes with a histidyl
residue in the enzyme active site, functioning as an analog of the
transition state (VanEtten et al., 1974). Again, the concentration of
the oxyanion required for inhibition of these enzymes is very low
(10-1000 mM) and unlikely to represent the type of interaction occurring
between molybdate and the receptor. Although the type of functional
groups required for this interaction are not known for sure, it has been
reported that molybdate, vanadate and tungstate can form chelates with a
variety of functional groups, including oxygen, nitrogen and sulfur
groups (VanEtten et al., 1974). Kay and Mitchell (1968) reported the
formation of molybdenum-cysteine complexes and suggested that such
interactions may be involved in molybdenum-containing enzymes. Weathers
et al. (1979) have also reported that molybdenum ions have a high
affinity for sulfur groups and, in fact, appear to bind proteins via

176
thiol group interactions, which in itself may be sufficient to stabilize
proteins (Brandt and Andersson, 1976). It has been suggested that
binding of the molybdate ion to thiol groups may form a molybdenum
bridge between receptor subunits (Ogle, 1983; Renoir and Mester, 1984;
Wilson et al., 1986). A more detailed understanding of this interaction
would likely provide valuable clues as to the actual molecular
mechanisms involved in receptor activation, in vivo stabilization and,
possibly, mechanisms of receptor up and down regulation.
Although molybdate has clearly been shown to inhibit activation of
bound receptors and irreversible loss of binding capacity in unoccupied
receptors, Mendel et al. (1985), by analyzing parallel cytosol samples
under denaturing conditions, have been able to demonstrate that the
stability of the nonactivated glucocorticoid receptor complex in the
presence of molybdate is only apparent. These workers reported that
while gel filtration chromatography showed that molybdate does
"stabilize" the nonactivated complex, so that after 2 hr at 3 C it
remained as a multimer with Stokes radius of about 8 nm; SDS-PAGE, by
contrast, indicated that this complex is made up primarily of 50,000
dalton fragments instead of intact 90,000 dalton monomers. Degradation
of the receptor to this size did not, according to these workers, affect
the ability of the complex to undergo cell-free activation. To
determine if the effects of molybdate on steroid receptor stability and
activation in vitro reflect the nature of molybdate-receptor
interactions in vivo, Raaka et al. (1985) examined the effects of
treating intact GH1 cells with molybdate and reported an inhibition of
the subsequent rate of nuclear accumulation of hormone-occupied
glucocorticoid and estrogen receptors. These workers also reported that

177
although molybdate did not affect the rate of receptor occupancy with
either steroid, cells treated with molybdate had more occupied cytosolic
and fewer occupied nuclear receptors than control cells. Svec (1985b),
however, reported that neither activation nor nuclear translocation of
glucocorticoid receptors was affected in vivo when intact AtT-20 mouse
pituitary tumor cells were incubated with molybdate.
Okret et al. (1985) reported evidence that the molybdate-stabilized
glucocorticoid receptor is actually a heteromeric complex of
approximately 302,000 daltons consisting of a single 94,000 dalton
steroid-binding subunit and other non-steroid-binding subunit(s) which
apparently dissociate during activation. More recently, Mendel et al.
(1986) reported that a 90,000 dalton non-steroid-binding phosphoprotein,
which is lost upon activation, is associated with the molybdate-
stabilized glucocorticoid receptor from WEHI-7 mouse thyoma cells.
Since the activation-associated dissociation of these heterologous
subunits is greatly facilitated by, and probably dependent upon, ligand
binding, a more appropriate way of studying the native form of the
receptor in cytosol would be to use the non-liganded form, both in the
presence and in the absence of molybdate.
Materials and Methods
Chemicals, Steroids and Isotopes
[6,7-3H]Triamcinolone acetonide, or 9a-fluoro-llb,16a,17a,21-tetra-
hydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide, ([3H]TA, specific
activity = 37 Ci/mmol) and [6,7-3H]dexamethasone, or 9a-fluoro-16a-
methylprednisolone ([3H]DEX, specific activity = 44.1-48.9 Ci/mmol) were
purchased from New England Nuclear (Boston, MA). Sephadex G-25 (fine)
was obtained from Pharmacia Fine Chemicals (Piscataway, NJ).

178
Dithiothreitol (DTT) and 4-(2-hydroxyethyl)-l-piperazineethane-sulfonic
acid (HEPES) was courtesy of Research Organics (Cleveland, OH). Sodium
molybdate (Na2Mo04), calf thymus DNA-cellulose, glycerol, sucrose, PPO
(2,5-diphenyloxazole) and dimethyl POPOP (1,4-bis[2(4-methyl-5-phenyl-
oxazoyl)]benzene) and pentyl agarose were all purchased from Sigma
Chemical Co. (St. Louis, MO). Scinti Verse II was purchased from
Fisher, Inc. (Fair Lawn, NJ). All other chemicals and solvents were
reagent grade.
Animals
All studies used female CD-I mice (Charles River Laboratories,
Wilmington, MA) that were subjected to combined ovariectomy and
adrenalectomy approximately 1 week prior to each experiment in order to
remove known sources of endogenous steroids. Both operations were
performed bilaterally via a lateral, subcostal approach under
barbiturate anesthesia, and mice were given 0.9% NaCl (w:v) in place of
drinking water. On the day of the experiment mice were anesthetized
with ether and perfused slowly through the heart with ice-cold
HEPES-buffered saline (20-30 ml, isotonic, pH 7.6).
Cytosol Preparation and Steroid Binding
Brains and other tissues were removed from the perfused animals and
homogenized (2x10 strokes at 1000 rpm) in 4 volumes of ice cold buffer
containing 20 mM HEPES and 2 mM DTT, pH 7.6 at 0 C in a glass
homogenizer with a Teflon pestle milled to a clearance between the
pestle and homogenization tube of 0.125 mm on the radius (to minimize
rupture of the brain cell nuclei (McEwen and Zigmond, 1972). The crude
homogenate was centrifuged at 100,000 g for 20 min and the supernatant
recentrifuged at 100,000 g for an additional 60 min to yield cytosol.

179
During these centrifuge runs, and during all other procedures, unless
otherwise indicated, careful attention was paid to maintaining the
cytosol at 0-2 C. Final protein concentrations were typically in the
6-8 mg/ml range. For prelabeled receptor preparations, cytosol was
incubated in the presence of 20 mM molybdate with 20 nM [3H]TA or
[3H]DEX for 24 to 40 hours at 0 C with or without a 200-fold excess of
unlabeled steroid.
Preparation of Heat-stable Cytosolic Factors
Brain or liver cytosol (prepared as above) was incubated in a
boiling water bath for 10 min and precipitated proteins were removed by
centrifugation at 100,000 g for 20 min.
Sucrose Density Gradient Sedimentation
Aliquots (400 ul) of either unlabeled or prelabeled cytosol were
layered onto linear 5-20% sucrose density gradients (4.6 ml; prepared
with HEPES buffer containing 2 mM DTT and either with (for
molybdate-stabilized receptors) or without (for nonmolybdate-stabilized
receptors) 20 mM molybdate and centrifuged at 0 C for 1.5-2 hr at
370,000 g (average) in a Sorvall TV-865 vertical tube rotor. The
cellulose nitrate tubes were punctured and 26-28 fractions (180 ul)
collected and incubated with 20 nM [3H]DEX +/- 4 uM [1H]DEX in the
presence of 20 mM molybdate at 0 C for 24 hr. The individual fractions
were then assayed for specific binding. Sedimentation coefficients
(S20,w) were calculated from the linear regression of S20,w vs
sedimentation distance (Martin and Ames, 1961) for the following
standard proteins run in parallel tubes: chicken ovalbumin (OVALB, 3.6
S), bovine serum albumin (BSA, 4.3 S), bovine gamma globulin (IgG, 7.4
S) and catalase (CAT, 11.3 S). The standard proteins were

180
[14C]methylated (for detection) to low specific activity with
[14C]formaldehyde by the method of Rice and Means (1971).
Steroid Binding Determination
In all experiments, bound 3H-steroid was separated from free on
Sephadex G-25 columns (0.6 x 14 cm) pre-equilibrated in buffer identical
to that in which the cytosol was prepared. Duplicate aliquots from each
assay tube (BT and BNS) were layered onto separate columns, allowed to
penetrate the gel and the macromolecular (bound) fraction eluted (with
homologous buffer) directly into scintillation vials for liquid
scintillation spectrometry.
Results
A major problem in the early studies of glucocorticoid, as well as
most other steroid, receptors before the discovery of molybdate's
effects on receptor complex activation was that activation was often
occurring throughout many, if not all, of the experimental steps,
frequently rendering the results uninterpretable. Never-the-less,
attempts were often made to interpret these results with little or no
concern for the impact of uncontrolled receptor activation. The
principle effect of molybdate on unoccupied glucocorticoid receptors,
perhaps via a mechanism similar to its inhibition of steroid-receptor
complex activation, is to inhibit the irreversible inactivation of
glucocorticoid binding capacity. This receptor lability is greatly
enhanced when the receptor is unoccupied by steroid ligand and even more
so when cytosolic stabilizing factors are separated from the receptor
(either intentionally or not) by any number of analytical procedures. As
a consequence, uncontrolled instability generally results in no data at
all, instead of confounded data that is likely to be misinterpreted.

181
For this reason and because of the fact that many physicochemical
studies of unoccupied receptors require a complicated postlabeling
procedure that would not be required for studies of prelabeled
receptors, few studies have been conducted on the physicochemical
properties of glucocorticoid receptors and virtually none of these have
been carried out in the absence of molybdate. The initial experiments
in this series sought to determine if the overall size and shape of the
unoccupied receptor, as determined by sedimentation analysis, was the
same or different from that of the occupied unactivated receptor. The
first experiment used the traditional swinging bucket rotor method which
typically requires a 16 to 24 hr ultracentrifugation. Because of the
long centrifuge times, this experiment could only be carried out using
sucrose gradients containing 20 mM molybdate. The results indicated
that the molybdate-stabilized unoccupied receptor was indeed of the same
sedimentation coefficient as the molybdate-stabilized unactivated
glucocorticoid receptor (see Chapter III, Figure 3-4). These same
results also revealed that sulfhydryl modification of the molybdate-
stabilized unoccupied receptor in or around the steroid-binding site, an
action of potential physiological relevance, had no effect on sedimenta¬
tion characteristics. Hydrophobic interaction chromatography was also
used to compare the properties of the occupied and unoccupied gluco¬
corticoid receptors (see Chapter II, Figure 2-6), but again the
procedure (because of high ionic strength requirements) required the use
of molybdate-stabilized forms and again the binding of steroid ligand
was found to be ineffectual in altering surface hydrophobicity
properties.

182
A relatively recent modification of sedimentation analysis that has
markedly reduced the centrifugation time required involves the use of a
vertical tube rotor (for a discussion of vertical tube sedimentation
analysis of steroid receptors, see Chapter IV). Since the use of a
vertical tube rotor can decrease the centrifugation time by an order of
magnitude or better, the sedimentation properties of unoccupied
receptors in both molybdate- and non-molybdate-containing sucrose
gradients (5-20%) was investigated using such a rotor. In the same run
were included unactivated [3H]TA-labeled glucocorticoid receptor
complexes also run on molybdate-containing gradients for direct
comparative purposes. The 8 position rotor allowed each of these groups
to be run in duplicate along with protein standards run on both
molybdate-containing and non-molybdate-containing gradients. After a
two hour centrifuge run at 0 C, the non-molybdate-containing gradients,
because of their high lability, were removed first and rapidly
fractionated into test tubes containing 20 nM [3H]TA and then incubated
at 0 C for 24 hr. The unoccupied receptors sedimented on molybdate-
containing gradients were next fractionated in an identical fashion and
incubated with [3H]TA as well. The gradients containing prelabeled
receptors and [14C]-labeled protein standards were fractionated directly
into scintillation vials for counting.
As expected, 20 mM molybdate had no effect whatsoever on the
elution profiles of the standard proteins. The prelabeled unactivated
receptor sedimented at the rate predicted (9 S) based on previous
findings by this lab using the more traditional swinging bucket rotors
(Luttge et al., 1984a-d; Luttge and Densmore, 1984). After bound/free
steroid separations had been performed using Sephadex G-25

183
chromatography on the individual postlabeled fractions from the unoc¬
cupied receptor gradients, it was evident that the molybdate-stabilized
form of the unoccupied glucocorticoid receptor sedimented at precisely
the same rate that the molybdate-stabilized prelabeled unactivated
glucocorticoid receptor complex did. Unfortunately, the sucrose
gradients containing the non-molybdate-stabilized unoccupied receptors
exhibited no binding activity whatsoever. This finding was unexpected
since although unoccupied glucocorticoid receptors are much less stable
than their steroid-bound counterparts, they are generally not this
labile, even in the absence of molybdate, when low temperature is
maintained. Obviously the process of centrifugation must have been
removing some cytosolic factor(s) responsible for stabilizing the
unoccupied receptor from irreversible degradation that is prevented by
the presence of molybdate.
The next attempt at characterizing the unoccupied glucocorticoid
receptor in the absence of molybdate required a number of modifications
of the vertical rotor sedimentation procedure and the subsequent steroid
incubation step. One change involved the precooling of the vertical
rotor to approximately -4 C prior to the centrifuge run. Previous runs
had been made with a rotor prechilled to approximately 4 C and although
the centrifuge run was made at 0 C, the temperature of the rather
massive vertical rotor was likely to have remained well above 0 C for
the 2 hr under the high vacume required for a 65,000 rpm spin. The low
temperature of -4 C was considered warm enough to prevent the possibil¬
ity of freezing of the sucrose gradients during the centrifuge run which
would seriously disrupt, if not destroy, the resolution of the gradient.
Another corrective measure taken was the inclusion of 20 mM molybdate in

184
each of the postlabeling incubation tubes. This addition of molybdate
after the fractionation of the non-molybdate-containing gradients would
not alter the sedimentation profile of the non-molybdate-stabilized
form, but would act to prevent much of the receptor degradation
occurring after gradient fractionation and during the steroid incubation
period. A final modification to the procedure involved the inclusion of
KC1 in the sucrose gradients. It had been shown previously that the
addition of a number of monovalent cations (i.e. Na, K, Li, Cs and Rb)
to cytosol dramatically increased the thermal stability of
non-molybdate-stabilized unoccupied receptors (Densmore et al., 1984a).
Since small molecules including these cations are quickly separated from
the relatively fast-sedimenting receptor early in the centrifugation
run, the receptor is suddenly forced into an environment of lower ionic
strength than exists in cytosol. To counteract the possibility of low
ionic strength-induced destabilization of non-molybdate-stabilized
receptors, 2 different concentrations of KC1 were introduced into the
gradients including 50 and 150 mM. Protein standards were run on
gradients containing each of these concentrations of KC1 and unoccupied
receptors were also run on gradients containing 20 mM molybdate and 150
mM KC1. The postlabeling of unoccupied receptors was carried out as
previously described except for the addition of molybdate to the
incubation tubes of the non-molybdate-stabilized form.
As was the case with the presence or absence of molybdate, the
sedimentation profiles of the protein standards were not affected by the
addition of either 50 or 150 mM KC1. Likewise, the unoccupied receptors
run on molybdate-containing gradients displayed the same sedimentation
properties when run in the presence or absence of 150 mM KC1. Finally,

185
the precautionary measures taken to reduce the degree of degradation of
unoccupied receptors when run on gradients lacking molybdate appeared to
have been successful. A major peak was evident at 9 S, or precisely the
same position as the single peak exhibited by the molybdate-stabilized
unoccupied and steroid-bound forms (Fig. 5-1). A minor peak sedimenting
at 4-5 S was also apparent in all of the profiles (50 mM and 150 mM KC1,
both run in duplicate). Although the positions of both peaks were
highly replicable across all 4 gradients, the total quantity of viable
receptors measured from these gradients was only a small fraction of
what was measured from molybdate-containing gradients on which equal
aliquots from the same pool of cytosol were run.
In order to further stabilize both peaks sedimenting on the
non-molybdate gradients, an effort was made to isolate whatever
factor(s) present in cytosol were responsible for stabilizing the
unoccupied receptor. The first experiment sought to determine whether
or not these factors were macromolecular or micromolecular. Cytosol
prepared in the absence of molybdate was simply run on a small Sephadex
G-25 column equilibrated and eluted with non-molybdate containing
buffer. The macromolecular fraction was then incubated with 20 nM
[3H]DEX +/- 4 uM [1H]DEX for 24 hr at 0 C. A similar aliquot of
non-gel-filtered cytosol from the original pool was incubated likewise.
After bound/free separations were performed on Sephadex G-25 columns and
dilutions occurring as the result of the initial gel-filtering of
unlabeled cytosol were taken into account, it was determined that
removal of small molecules led to a 90% reduction in binding capacity
(data not shown).

Figure 5-1. Vertical tube rotor sucrose density gradient sedimentation
analysis of nonmolybdate-stabilized unoccupied Type II glucocorticoid
receptors on nonmolybdate-containing sucrose gradients. Brain cytosol
was prepared in HEPES buffer containing only 2 mM DTT. Aliquots (400
ul) were applied to 4.6 ml, prechilled, 5-20% linear sucrose gradients
(made in the absence of molybdate) which were then placed in a TV-865
vertical tube rotor prechilled to approximately -2 C and run at 65,000
rpm (370,000 x g (average)) for 2 hr. Tubes were then quickly
fractionated into steroid incubation tubes containing HEPES buffer plus
molybdate (20 mM final concentration) and DTT (2 mM final concentration)
and 20 nM [3H]TA. After a 40 hr incubation, bound-free steroid
separations were performed on Sephadex G-25 columns. Binding is
expressed as percent of the total counts (mean > 1600 cpm) collected
from each gradient and represents the mean value of the same fraction
from three gradients. The profile shown here is representative of 4
independent replications.

187

Figure 5-2. Vertical tube rotor density gradient sedimentation analysis
of the molybdate-stabilized unoccupied Type II glucocorticoid receptor.
Brain cytosol was prepared in HEPES buffer containing 20 mM molybdate
and 2 mM DTT. Aliquots of cytosol (400 ul) were applied to 5 ml,
prechilled, 5-20% linear sucrose density gradients (made in the presence
of molybdate) and then run in a TV-865 vertical tube rotor at 65,000 rpm
(370,000 x g (average)) for 2 hr. The tubes were then quickly
fractionated into steroid incubation tubes containing 20 nM [3H]TA.
After a 40 hr incubation, bound-free steroid separations were performed
on Sephadex G-25 columns. Fractions are represented as percent of total
counts (12,000 cpm) collected from the gradient. The profile shown here
is representative of 4 independent replications.

189

190
The next step was to determine if the factor(s) were heat stable or
heat labile. For this experiment, brain cytosol was prepared as
previously described in the absence of molybdate. A portion of the
unlabeled cytosol was retained on ice while the remainder was incubated
in a boiling water bath for 10 min. The heat-treated cytosol was then
subjected to centrifugation at 100,000 g for 15 min to remove any
precipitated proteins, etc.. The resulting supernatant was then used to
equilibrate small Sephadex G-25 columns of the same dimensions as those
used in the previous experiment for macromolecular/micromolecular
separation. Other identical columns were equilibrated in either
non-molybdate-containing buffer or molybdate-containing buffer.
Aliquots of the non-heat-treated cytosol were applied to each of these
columns and eluted with the corresponding buffer, heat-treated cytosol
preparation, etc.. The macromolecular fractions were collected from
these columns and either incubated directly with 20 nM [3H]DEX +/- 4 uM
[1H]DEX for 24 hr at 0 C, or first incubated for 30 min at 20 C prior to
the steroid incubation.
The binding of the preparations run through the non-molybdate
columns were, on average, only about 10% of those run through molybdate-
equilibrated columns, whereas those preparations run through columns
equilibrated with heat-treated cytosol retained nearly 70% of the
maximal binding. Of those preparations that were incubated at 20 C
prior to steroid incubation, the molybdate-treated group displayed no
loss in binding, whereas the binding of the non-molybdate treated group
was reduced by more than 80%. Preparations run through columns
equilibrated with heat-treated cytosol were, by comparison,

191
reduced by only 50%, indicating an obvious protective effect of the
heat-treated cytosol fraction.
A more extensive experiment was conducted to further investigate
the ability of these heat-stable factors to stabilize the unoccupied
receptor in vitro. In this experiment, cytosol from both brain and
liver (equal wet weights of each tissue were used) was prepared in the
previously described manner using non-molybdate containing buffer. A
portion of each of these cytosol preparations was incubated in a boiling
water bath for 10 min followed by centrifugation at 100,000 g for 10
min. Non-heat-treated brain cytosol was run on a series of columns
equilibrated and eluted with either molybdate-containing or non-
molybdate-containing buffer. The macromolecular fraction (800 ul) was
collected into tubes containing 800 ul of one of the following:
molybdate-free buffer, molybdate-containing buffer, 100% heat-treated
liver cytosol, 50% heat-treated liver cytosol and 50% molybdate-free
buffer, 25% heat-treated liver cytosol and 75% molybdate-free buffer,
100% heat-treated brain cytosol, 50% heat-treated brain cytosol and 50%
molybdate-free buffer, 25% heat-treated brain cytosol and 75%
molybdate-free buffer, the macromolecular fraction of heat-treated liver
cytosol and the macromolecular fraction of the heat-treated brain
cytosol. The collection tubes were maintained on ice during the
filtration process. An aliquot of the contents of each group was
incubated directly with 20 nM [3H]DEX +/- 4 uM [1H]DEX while a second
aliquot was first incubated for 30 min at 20 C prior to steroid
incubation.
The results from this experiment again indicated a protective
effect of heat-treated cytosol, but not the macromolecular fraction of

192
heat-treated cytosol. Heat-treated liver cytosol was more effective
than heat-treated brain cytosol and the effect appeared to be
concentration dependent. The degree of binding loss was greater in this
experiment when heat-treated cytosol was added to the gel-filtered
macromolecular fraction of non-heat-treated cytosol than it was in the
previous experiment where the Sephadex G-25 columns were actually
pre-equilibrated with the heat-treated cytosol. This difference could
either be attributed to the increased dilution of the heat-stable factor
when added after gel filtration of non-heat-treated cytosol or
degradation occurring in the absence of the heat-stable factor during
the column run itself.
The findings of the previous experiment led to the modification of
the procedure described previously for vertical tube rotor sedimentation
analysis of non-molybdate-stabilized unoccupied glucocorticoid
receptors. Most of the precautionary steps that had been added to
increase receptor stability such as pre-chilling of the rotor and post¬
fractionation addition of molybdate were retained. The sucrose
gradients, however, were made up with 50% heat-treated liver cytosol
prepared as previously described.
The results of this experiment indicated that while the major peak
(the peak previously sedimenting at 9 S) was still apparent in these
gradients, the smaller peak was not, indicating that the presence of the
heat-stable factor(s) may have been having an effect on the presence of
the different sedimenting forms of the unoccupied receptor similar to
the effect of molybdate. The total bound radioactivity resulting from
these gradients was still significantly lower than that obtained with
gradients containing molybdate. Not surprizingly, aggregation appeared

193
to be a little more of a problem than had previously been encountered
with gradients lacking the heat-treated cytosol.
Discussion
Before the discovery that molybdate was an effective inhibitor of
inactivation of unoccupied glucocorticoid receptor binding capacity and
activation of the receptor to a nuclear-binding form (Leach et al.,
1979), characterization of the unoccupied receptor was virtually
impossible because of the greater lability of this form of the receptor.
This was particularly evident after gel filtration or other analytical
procedures that tended to separate the unoccupied receptor from the
cytosolic factor(s) that appear to provide some degree of stabilization.
As a consequence, virtually all reports published on the physicochemical
properties of glucocorticoid receptors employed the use of prelabeled
receptors, which reduced the problem of lability while introducing the
problem of uncontrolled activation of receptors during the often lengthy
analytical procedures. Unoccupied receptors, in the absence or presence
of molybdate, appear incapable of undergoing activation prior to the
binding of a steroid ligand (Atger and Milgrom, 1976; Bailly et al.,
1978; Densmore, unpublished). This implies either that binding of the
steroid illicits conformational changes in the receptor molecule
prerequisite for the subsequent steps of activation or that if the
unoccupied receptor does undergo an activation of sorts, it apparently
renders the receptor inactive and incapable of binding steroid. Strong
evidence for the latter of these possibilities has most recently been
provided with the finding that activated glucocorticoid receptors appear
completely incapable of rebinding steroid after steroid dissociation,
even in the presence of molybdate (Chou and Luttge, unpublished).

194
Although the use of molybdate for receptor stabilization has become
an almost routine part of most steroid receptor studies, the vast
majority of reports still focus on the characteristics of prelabeled
receptors. Only one report has examined the effects of molybdate on the
sedimentation properties of non-liganded steroid receptors. El Dieb et
al. (1983) described the effect of ligand binding on the sedimentation
behavior of cytosolic progestin receptors. They found that when cytosol
was prelabeled, receptors sedimented at 4.4 S and 7.8 S, whereas when
the fractionated gradients were postlabeled, the unoccupied receptors
sedimented at 4.4 S and 9-10 S. They also noted that 20 mM molybdate
blocked the conversion of 9-10 S to 7-8 S receptors. In contrast with
the results for unoccupied glucocorticoid receptors in the present study
wherein the 4-5 S peak for the unoccupied glucocorticoid receptor was
only apparent on molybdate-free gradients, the 4.4 S peak for progestin
receptors was reportedly not eliminated by the presence of molybdate in
the gradient. Despite the similarities in amino-acid structure between
progestin and glucocorticoid receptors (Conneely et al., 1986), other
tissue, species, buffer and methodology differences between the two
studies make a direct comparison of the results difficult.
A more recent study has examined the chromatographic behavior of
non-liganded glucocorticoid receptors in the presence and absence of 20
mM molybdate on Agarose A-1.5 m columns (Radojcic et al., 1986). In the
absence of molybdate, a single peak corresponding to a Stokes radius of
5.7 nM was detected, while addition of molybdate throughout the course
of the chromatographic analysis of non-liganded receptor resulted in the
appearance of a single peak with a Stokes radius of 8.0 nM. While these
authors' results are similar to those obtained in the present study

195
using sedimentation analysis, Radojcic et al. (1986) detected
non-liganded receptors post-procedure with an enzyme-linked
immunosorbent assay (ELISA) based on antibodies raised in rabbits
against the purified activated glucocorticoid receptor. Unfortunately,
these workers did not show whether or not the peaks they detected were
actually still capable of binding steriod, thereby eliminating the
possibility that the antibody was merely recognizing an inactive
degradation product of the receptor.
The ability of heat-stable factors to eliminate the appearance of
the 4.5 S-sedimenting form of the unoccupied glucocorticoid receptor in
the present study is indicative of several possibilities. One or more
heat-stable cytosolic factors may be acting much like molybdate, but in
a more limited fashion, to hold receptor subunits together. If this is
the case, the slow-sedimenting form appearing on gradients not
containing either molybdate or heat-treated cytosol may represent a
nonphysiological artefact of the procedure. Another possibility is that
the slower sedimenting form exists in vivo, but is rapidly inactivated
in vitro by heat-stable factors. An understanding of this phenomenon
may eventually enhance our knowledge of the apparent heterogeneous
subunit makeup of the glucocorticoid receptor in vivo and what role the
individual subunits play in the overall mechanism of glucocorticoid
receptor-mediated action. The recent suggestion that one or more of
these subunits, in particular a 59,000 dalton non-steroid-binding
subunit, is similar or identical to subunits found in unactivated
progestin, estrogen and androgen receptors (Tai et al., 1986), indicates
the significance of the findings of the present study to an
understanding of steroid receptor mechanisms in general.

CHAPTER VI
CONCLUDING REMARKS
The work of this dissertation focussed on a number of different
questions regarding the structure and function of the glucocorticoid
type II receptor in mammalian brain. While some of these questions may
at first seem to be disconnected, it is important to realize the
relation that they have to one another and to an overall understanding
of the genomic mechanisms of glucocorticoid action. A complete
understanding of these genomic mechanisms requires a thorough
understanding of the glucocorticoid receptor, a necessary intermediate
in the overall scheme. In turn, anything that affects the receptor's
essential role in the process must likewise be understood. Clearly,
anything that affects the ability of a receptor to bind steroid, the
rate of steroid association, the rate of receptor activation to its
nuclear binding form, the ability of the activated form to bind to its
nuclear acceptor site(s) or the stability of any or all of these forms
could potentially have an impact on the cell's ability to respond to a
glucocorticoid hormone signal. It may therefore not be completely
sufficient to know only the concentration of glucocorticoid receptors in
a given tissue, in order to predict precisely how sensitive that tissue
will be to glucocorticoids under particular physiological conditions.
Obviously it is impossible in one study to consider every factor that
could impact on receptor-mediated glucocorticoid actions and it is
certainly a difficult task to apply, with a high degree of confidence,
196

197
the knowledge gained from in vitro studies using crude, incomplete and
decompartmentalized preparations to a complex, highly ordered living
system, but such studies are nevertheless essential in order to complete
the framework for such a global understanding of glucocorticoid action.
In addition, because of very recent findings that other steroid
receptors, particularly those binding progesterone (Conneely et al.,
1986), have amino acid sequences that are remarkably similar to that of
the glucocorticoid receptor, it is not difficult to imagine that some,
if not much, of what is learned regarding glucocorticoid receptor
function could be applied to the understanding of other steroid receptor
systems in other tissues and other organisms spanning a vast
phylogenetic realm.
One aspect of the work involved an in depth investigation of the
surface hydrophobicity of the various forms of the glucocorticoid
receptor. It is surprizing that although the intracellular localization
of activated, unactivated and unoccupied glucocorticoid receptors in
vivo is currently a controversial topic, little or no attention has been
paid to possible hydrophobic interactions between various receptor forms
and cellular components. While a great deal of attention has been
focussed on the ionic characteristics of glucocorticoid and other
steroid receptors, there is little doubt that hydrophobic properties may
also play an important role in the cellular compartmentalization of
these receptors. This study found that activation of the glucocorticoid
receptor was associated with a profound increase in surface
hydrophobicity as determined by hydrophobic interaction chromatography.
This increased hydrophobicity could be indicative of changes required
for the glucocorticoid receptor molecule to effectively interact with
the molecular machinery that controls gene expression.

198
Another in vitro phenomenon which could potentially represent a
mechanism by which glucocorticoid action is affected in vivo has to do
with reversible changes in the glucocorticoid receptor's binding status.
The reduction of glucocorticoid binding capacity associated with
sulfhydryl oxidation and the restoration of the lost binding capacity
associated with sulfhydryl reduction was examined in some detail.
Because of the typically nonspecific nature of many sulfhydryl
reactions, the incredibly complex system of intra- and extracellular
thiols and disulfides and the myriad of enzymes that catalyze protein
sulfhydryl oxidation and reduction reactions in a relatively nonspecific
fashion, precise conclusions regarding the role of such a mechanism in
the in vivo up- and down-regulation of glucocorticoid binding capacity
simply cannot be made. For that matter, similar conclusions also cannot
be made regarding the in vivo role of sulfhydryl oxidation and reduction
in regulating the enzyme activity of any thiol-containing enzyme or the
ligand binding activity of any thiol-containing receptor for basically
the same reasons. Never-the-less, there exists a remarkable array of
enzymes and receptors whose activities can be readily turned up or down,
on or off by experimentally altering the thiol/disulfide ratio of the
system, implying the possibility, at least, that many of these
molecules, including glucocorticoid and other steroid receptors, may
exist in vivo in a reversibly inactivated form. Even if these inactive
or "down-regulated” forms exist in some sort of constant equilibrium
with the active or "up-regulated" forms in the absence of an active
mechanism for shifting the equilibrium in response to physiological
needs, the implications for sustained glucocorticoid sensitivity of

199
target tissues during abnormal (or perhaps even normal) exposure to
steroids are obviously worth consideration. Results from this study
have revealed a number of similarities as well as differences in the
biochemical and hydrodynamic properties between the sulfhydryl up- and
down-regulated receptors which could have functional significance if
both forms are someday unambiguously proven to exist in vivo. The
sedimentation properties of the two forms are identical, indicating that
down-regulation of the receptor is not likely to involve mixed disulfide
formation between the receptor and another protein subunit. Hydrophobic
interactions, on the other hand, are increased slightly as a result of
sulfhydryl oxidation. Such differences could potentially affect
subcellular compartmentalization, receptor stability, etc. in vivo.
Biochemical differences include the susceptibility of up-regulated, but
not down-regulated, forms to irreversible inactivation by sulfhydryl
reactive reagents such as NEM, providing a potential means of measuring
the relative populations of up- and down-regulated forms in homogenate
or cytosol with a higher degree of certainty. This is because
subsequent in vitro oxidation or reduction of up- or down-regulated
receptors respectively is prevented during the long steroid incubations
required to determine binding capacity.
Activation of the glucocorticoid receptor is a topic that has
received a great deal of attention in the field for many years, yet the
mechanisms of activation remain shrouded in controversy. This is
partially the result of years of describing activation in terms of
physicochemical changes in receptor properties associated with the
process while not controlling for activation during the analytical
procedures. In addition, activation now appears to be a multistep

200
process dependent, in part, on a number of cytosolic factors or
conditions which typically vary from one lab's preparation to another's.
Although the discovery that molybdate inhibits the process of activation
has greatly aided the study of this phenomenon, there are many in the
field who feel that the molybdate-stabilized receptor may not be
representative of the in vivo unactivated form. Results from this
dissertation have provided some information relevant to both an
understanding of the process of activation as well as an understanding
of the structure of the non-molybdate-stabilized unactivated receptor.
The former was accomplished by perfecting a purification procedure for
the glucocorticoid receptor and the latter involved perfecting
analytical procedures for the highly labile unoccupied receptor not
stabilized by cytosolic factors or molybdate. Whereas heat activation
of the purified glucocorticoid receptor led to changes similar to those
exhibited by unpurified receptors, such as increased hydrophobicity, the
purified receptors showed no concomitant increase in affinity for DNA.
The dependence of the receptor on cytosolic factors in order to achieve
complete and effectual activation introduces yet another possible point
at which the cell can influence genomic glucocorticoid actions. With
regards to the structure of the unactivated receptor, there appears to
exist a slower sedimenting form, when unoccupied receptors are run in
the absence of molybdate. This form disappears, however, if small
molecular weight cytosolic factors are present in the gradient, implying
that the molybdate-stabilized unactivated receptor may be representative
of the in vivo unoccupied and occupied unactivated receptor after all.
In conclusion, the findings presented in this dissertation provide
new details regarding several interrelated steps in the receptor

201
mediated process of glucocorticoid action. Though the work is a
testament to the incredibly complex task of determining potential
mechanisms of steroid receptor regulation and activation, the complexity
of the system makes the eventual elucidation of glucocorticoid receptor
structure, function and regulation an even more important event in the
overall realm of cell biology and biochemistry, not to mention its
importance to understanding the more specific role of glucocorticoids in
the function of the nervous system.

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BIOGRAPHICAL SKETCH
The author was born June 30, 1954 in Birmingham, Alabama, where he
lived for the first five years of his life. At age five, his family
moved to beautiful Huntsville, Alabama, home of the Redstone Arsenal and
the Marshall Space Flight Center, at a time when the U.S. space program
was just developing. The high tech, "space age" environment of
Huntsville played an important role in shaping the dreams and
aspirations of the author during most of the next twenty years. Upon
graduating from S. R. Butler High School in 1972, the author enrolled at
the University of Alabama in Huntsville where he earned his B.S. degree
in biology. Because of the preponderance of aerospace industries and
the author’s general interest in space science, he took a position with
a local aerospace firm upon leaving college. During the next two years
his experience included projects related to the Space Lab/Space Shuttle
program (NASA), early phases of the Strategic Defense Initiative (U. S.
Army), particle accelerator electromagnet construction (Brookhaven
National Laboratory) and numerous other aerospace efforts. During this
time he also started graduate studies in the Department of Biological
Sciences at the University of Alabama in Huntsville under the
supervision of Dr. Harold J. Wilson. He eventually took a graduate
teaching assistantship position with the Department and later earned a
Master of Science degree. In 1979 he married Anita Anna Tiller and in
1980 they moved to Gainesville, Florida, where they both began graduate
studies at the University of Florida. The author conducted his doctoral
233

234
research in the Department of Neuroscience under the direction of Dr.
William G. Luttge and studied the regulation of glucocorticoid receptor
binding in the brain. He left the University of Florida in 1986 to
accept a postdoctoral fellowship with Dr. Bert O'Malley of the
Department of Cell Biology at Baylor College of Medicine in Houston,
Texas.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Robert Associate Professor of Biochemistry
and Molecular Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
.JíÉkklJL
Steven R. Childers
Associate Professor of Pharmacology
and Therapeutics
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
1
IjyUksn r.
Kathleen T. Shiverick
Associate Professor of Pharmacology
and Therapeutics

This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
August, 1987
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA




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