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System A amino acid transport activity in membrane vesicles and reconstituted proteoliposomes

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Title:
System A amino acid transport activity in membrane vesicles and reconstituted proteoliposomes
Creator:
Schenerman, Mark Allen, 1959-
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English
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156 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Amino acids ( jstor )
Cell membranes ( jstor )
Centrifugation ( jstor )
Hepatocytes ( jstor )
Incubation ( jstor )
Liver ( jstor )
Membrane proteins ( jstor )
P branes ( jstor )
pH ( jstor )
Rats ( jstor )
Amino Acid Activation ( mesh )
Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Biological Transport ( mesh )
Cell Membrane ( mesh )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1986.
Bibliography:
Bibliography: leaves 149-155.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Mark Allen Schenerman.

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University of Florida
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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.
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SYSTEM A AMINO ACID TRANSPORT ACTIVITY IN MEMBRANE
VESICLES AND RECONSTITUTED PROTEOLIPOSOMES











By

MARK ALLEN SCHENERMAN


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

1986

























This dissertation is dedicated to my parents, whose constant

love and support made this work possible.
















ACKNOWLEDGMENTS


I would like to thank my mentor, Dr. Michael S.

Kilberg, for his patience and understanding even in the

midst of my impatience. I would also like to thank Dr.

Efraim Racker for his kind advice and hospitality during my

visits to Ithaca. Finally, I would like to thank Mary

Handlogten, Donna Bracy, and Tom Chiles for their friendship

and assistance during the course of my studies.
















TABLE OF CONTENTS


ACKNOWLEDGMENTS.....................................

LIST OF TABLES .....................................

LIST OF FIGURES ....................................

ABBREVIATIONS USED..................................

ABSTRACT............................................

CHAPTERS

I AMINO ACID TRANSPORT IN ANIMAL CELLS.......

II CHARACTERIZATION OF SYSTEM A AMINO ACID
TRANSPORT ACTIVITY IN MEMBRANE VESICLES....

Introduction...............................
Materials and Methods......................
Results....................................
Discussion .. ............. ....... ....... .

III RECONSTITUTION OF SYSTEM A TRANSPORT
ACTIVITY INTO ARTIFICIAL PROTEOLIPOSOMES...

Introduction........................... ....
Materials and Methods.......................
Results ....................................
Discussion.................................

IV FURTHER DISCUSSION ON THE USE OF MEMBRANE
VESICLES AND RECONSTITUTION................


APPENDICES

A ANALYTICAL ASSAYS AND PROCEDURES...........

B ENZYME ASSAYS...............................

C SOLUTIONS FOR THE PREPARATION OF PLASMA
MEMBRANES AND TRANSPORT OF VESICLES........


Page

iii

vi

vii

ix

xi



1


14

14
19
26
70


76

76
82
90
123


128




134

138


146








D SOLUTIONS FOR THE PREPARATION AND
RECONSTITUTION OF EAT CELL MEMBRANE......... 147

BIBLIOGRAPHY........................................ 149

BIOGRAPHICAL SKETCH.................................. 156
















LIST OF TABLES


Page
1-1 Characteristics of Na+-Dependent Neutral
Amino Acid Transport Systems ................... 3

2-1 Enzyme Activities in the Plasma Membrane-
Enriched Fraction ............................... 27

2-2 Alkali-Ion Specificity for Systems A and ASC
in Rat Liver Plasma Membrane Vesicles........... 38

2-3 Effect of Gramicidin or Monensin on AIB Uptake.. 51

2-4 Trans-Inhibition in Isolated Plasma Membrane
Vesicles....................................... 53

2-5 Glucagon Stimulation of System A in Rat
Hepatocytes is Retained in Isolated Plasma
Membrane Vesicles............................... 59

2-6 Enzyme Activities in Membranes from Control
and Glucagon-Treated Hepatocytes............... 61

3-1 Enzyme Marker Activities in EAT Cell Membranes.. 91

3-2 Reconstitution of System A Activity into
Proteoliposomes Following Detergent
Extraction of EAT Cell Membranes................ 105
















LIST OF FIGURES


Page
2-1 Time-Dependent Uptake of AIB by Rat Liver
Plasma Membrane Vesicles...................... 30

2-2 MeAIB Inhibition of Na+-Dependent AIB
Uptake by Isolated Vesicles.................. 33

2-3 Alanine, Cysteine, Histidine and Glycine
Transport by Rat Liver Plasma Membrane
Vesicles..................................... 35

2-4 Effect of pH on Alanine Uptake in Membrane
Vesicles..................................... 42

2-5 Effect of the Extravesicular Osmolarity on
AIB Uptake.................................... 44

2-6 Effect of Incubation Temperature on the
Na -Dependent Uptake of AIB................... 47

2-7 Relation Between Membrane Protein
Concentration and AIB Uptake.................. 49

2-8 System A Activity in Plasma Membrane Vesicles
from Control or Glucagon-Injected Rats....... 57

2-9 Decay of System A Activity in Membrane
Vesicles Incubated at 40C or -70C........... 64

2-10 Decay of Systems A, N and ASC in Vesicles from
Glucagon-Treated and Normal Hepatocytes...... 67

2-11 Flow Chart of HepG2 Membrane Preparation...... 69

3-1 Flow Chart of the Detergent-Extraction and
Reconstitution of System A Transport Activity
Using the Freeze-Thaw Procedure............... 86

3-2 Titration of the Cholate to Protein Ratio for
Reconstitution of System A Amino Acid
Transport in EAT Cell Membranes.............. 94


vii








3-3 Titration of the Lipid to Protein Ratio for
Reconstitution of System A Activity Using
EAT Cell Membranes ............................. 97

3-4 Determination of the Optimal Period of
Sonication for Reconstituted Proteoliposomes
from EAT Cell Membranes...................... 99

3-5 Determination of the Optimal Concentration of
C E for Extraction of EAT Cell
Mebbane Proteins Prior to Reconstitution.... 102

3-6 Temperature Stability of the Membrane Protein
Extract...................................... 108

3-7 Time Course of AIB Uptake into Proteoliposomes
in the Presence and Absence of Valinomycin... 111

3-8 Osmotic Sensitivity of the Reconstituted
Proteoliposomes.............................. 113

3-9 Measurement of the Intravesicular Volume of
the Reconstituted Proteoliposomes Using
3-0-Methyl-Glucose........................... 116

3-10 SDS-Polyacrylamide Gel Electrophoresis of
EAT Cell Membranes and Reconstituted
Proteoliposomes.............................. 122
















ABBREVIATIONS USED


AIB 2-aminoisobutyric acid

C12E9 polyoxyethylene-9-lauryl ether

cAMP adenosine 3':5'-cyclic monophosphate

CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-
1-propanesulfonate
CHX cycloheximide

CMC critical micellar concentration

EAT Ehrlich ascites tumor

EDTA ethylenediamine tetraacetic acid

EGTA ethyleneglycol-bis-(B-amino-ethyl ether)
N, N'-tetraacetic acid

FBS fetal bovine serum

GABA Y-aminobutyric acid

HEPES 4-(2-hydroxyethyl)-l-piperazineethane-
sulphonic acid

HepG2 human hepatoma cell line

MeAIB 2-(methylamino)-isobutyric acid

MEM Eagle's minimal essential medium

NADPH reduced nicotinic dinucleotide phosphate

NaKRB sodium-containing Kreb's-Ringer bicarbonate
buffer

NP-40 non-ionic industrial detergent

NRK-49F normal rat kidney cell line
ix









octyl -
glucoside

osM

Pi

PMSF

R.S.A.

sarc

S.D.

SOS

tau

TCA

PAGE


N-octyl-B-0-glucopyranoside


osmolarity

inorganic phosphate

phenylmethylsulfonyl fluoride

relative specific activity

sarcosine

standard deviation

sodium dodecyl sulfate

taurine

trichloroacetic acid

polyacrylamide gel electrophoresis
















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


SYSTEM A AMINO ACID TRANSPORT ACTIVITY IN MEMBRANE
VESICLES AND RECONSTITUTED PROTEOLIPOSOMES


By


MARK ALLEN SCHENERMAN


May 1986

Chairman: Dr. Michael S. Kilberg
Major Department: Biochemistry and Molecular Biology


System A amino acid transport activity and its regula-

tion has been studied in plasma membrane vesicles isolated

from rat liver. Enzyme markers were used to show that the

membrane preparation was enriched in plasma membrane yet

showed minimal contamination from endoplasmic reticulum mem-

branes and no contamination by mitochondrial membranes. The

plasma membrane vesicles actively accumulated amino acids

through Systems A, ASC, N and Gly, all of which are depen-

dent on an imposed trans-membrane Na+ gradient. Amino

acid uptake was into an osmotically-sensitive space and was

inhibited by ionophores that collapse the transmembrane

Na+ gradient.









Regulation of System A-mediated transport activity was

studied by stimulating transport activity using various

effectors, then isolating membrane vesicles from those

cells. When plasma membrane vesicles were isolated from

either glucagon-treated or normal hepatocytes, it was dis-

covered that the membrane vesicles from cells exposed to the

hormone partially retained the stimulated transport activ-

ity. The lack of retention of all of the enhanced activity

in the membrane vesicles is explained, in part, by a rapid

loss of activity during storage at 40C (half-life = 13 h).

Neither protease inhibitors nor dithiothreitol blocked this

decay.

A procedure for reconstitution of transport activity

was developed using Ehrlich ascites tumor cell membranes.

The membrane proteins were solubilized in cholate and urea

which were exchanged for the non-ionic detergent polyoxy-

ethylene-9-lauryl ether (C12Eg) by dialysis. After

mixing with sonicated asolectin and cholate, the detergent-

extract was reconstituted by a freeze-thaw procedure. The

proteoliposomes were collected by centrifugation and tested

for System A activity by a rapid filtration assay. Reconsti-

tuted transport activity was determined to be optimal when

the cholate to protein ratio (w/w) was 3:1 and the lipid to

protein ratio (w/w) was 20:1. The development of this pro-

cedure for reconstitution will facilitate further studies

toward purifying the protein(s) associated with System A

because reconstitution can be used to monitor the increase

in specific activity of the carrier during purification.
xii








The non-ionic nature of the detergent C12E9 will

also facilitate the separation of proteins in the detergent-

extract according to their charge.


xiii
















CHAPTER I
AMINO ACID TRANSPORT IN ANIMAL CELLS


Amino acids are pivotal contributors to many metabolic

pathways in cells including gluconeogenesis, glycolysis, and

the Kreb's cycle. For example, amino acids released from

muscle provide the carbon skeletons for de novo glucose

synthesis in the liver through the glucose-alanine cycle

(Felig, 1973). The supply of amino acid precursors for glu-

coneogenesis has long been considered to be a control point

for the entire pathway (Exton et al., 1970). It has been

shown more recently that the rate-limiting step in alanine

metabolism in rat hepatocytes is the transport of alanine

into the cells (Sips et al., 1980a). In this way, the intra-

cellular concentration of amino acids is tightly regulated

according to the nutritional needs of the cell.

Amino acids can be transported into cells through two

types of carrier-mediated pathways. Eukaryotic cell trans-

port can either be by active accumulation, that is, coupled

to a trans-membrane Na gradient (Na -dependent), or

by facilitated transport not driven by ion fluxes (Na -

independent). Neutral amino acid transport was first

observed as two distinct pathways in Ehrlich ascites tumor

(EAT) cells and the pathways were designated by Oxender and








Christensen (1963) as System A (Na+-dependent) and

System L (Na -independent). Since that time, numerous

other amino acid transport systems have been defined and

characterized in animal cells (Christensen, 1984). The

present work will focus on Na -dependent neutral amino

acid transport systems in isolated membranes derived from

rat hepatocytes and EAT cells.

At least five Na+-dependent neutral amino acid

transport systems have been described in animal cells. Some

of the characteristics of these systems are summarized in

Table 1-1. System A prefers amino acids having short,

polar, or linear sidechains such as alanine, serine, methio-

nine, and glycine. Two non-metabolizable amino acids have

proven to be particularly useful for characterizing System A

activity. The Na+-dependent uptake of 2-aminoisobutyric

acid (AIB) and its N-methylated derivative, 2-(methylamino)-

isobutyric acid (MeAIB), have proven to be highly specific

for System A activity, particularly in rat hepatocytes

(Kilberg et al., 1985a). System A is stereospecific for L-

amino acids and is strongly inhibited as the pH of the

medium is lowered (Kilberg et al., 1980). The alkali-ion

specificity of System A depends somewhat on the cell line

tested because Li is not acceptable as a substitute for

Na in rat hepatocytes (Edmondson et al., 1979), but

Li -for-Na substitution is well tolerated by the

activity in EAT cells (Christensen and Handlogten, 1977).

System A activity has been observed in all nucleated cells

tested.





















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System ASC was also originally characterized in EAT

cells (Christensen et al., 1967). It was detected because

the System A-specific probe, MeAIB, did not completely inhi-

bit Na -dependent alanine, serine, or cysteine uptake.

System ASC prefers neutral amino acids with small, polar

sidechains with particular affinity for those containing

oxygen or sulfur atoms, such as serine, threonine, and cys-

teine. In rat hepatocytes, Na -dependent cysteine

uptake was observed to be totally insensitive to MeAIB inhi-

bition and cysteine is considered, therefore, to be a selec-

tive probe for hepatic System ASC activity (Kilberg et al.,

1979). System ASC is not as sensitive as System A to lower-

ing the pH of the medium and, in liver tissue, it readily

accepts Li -for-Na substitution. System ASC also

shows a high degree of stereospecificity for L-amino acids

and appears to be ubiquitous, as it has been found in all

eukaryotic cells so far tested (Shotwell et al., 1983).

System Gly is a Na -dependent glycine-specific

transport system first described in rabbit reticulocytes

(Winter and Christensen, 1965), but is also present in

pigeon erythrocytes, rat hepatocytes, and hepatoma cells.

Sodium-dependent glycine uptake is not restricted entirely

to System Gly in rat hepatocytes because MeAIB inhibits a

portion of the transport (Christensen and Handlogten, 1981).

System 3 is a Na -dependent transport system which

supports the uptake of B-amino acids. Taurine has served

as a model substrate for System B, but B-alanine can also

be used as a selective substrate. System B has been well








characterized in EAT cells (Christensen, 1964) and rat hepa-

tocytes.

System N was first described in rat hepatocytes as a

system which was specific for Na -dependent asparagine,

glutamine, and histidine uptake (Kilberg et al., 1980).

System A mediates a portion of the transport of these sub-

strates in rat hepatocytes, but this component can be elimin-

ated by measuring uptake in the presence of an excess of the

System A-specific probe, MeAIB. System N is highly sensi-

tive to decreasing the pH of the medium and also exhibits a

high degree of specificity for L-amino acids. System N has

also been shown to occur in fetal rat hepatocytes and the

rat hepatoma cell line H4-II-EC3 (Vadgama and Christensen,

1983).

Neutral amino acid transport activity is regulated by

three different processes: trans-effects, hormones, and sub-

strate availability. The transport systems affected by

these factors are indicated in Table 1-1. Combined, the

characteristics of specificity and regulation of each trans-

port system make it possible to uniquely identify a given

transport system operating in a particular cell line.

Regulation of transport activity by trans-effects is an

acceleration or deceleration of amino acid uptake caused by

an elevated concentration of substrates inside the cell. An

acceleration of the transport rate, or trans-stimulation, is

a common characteristic of System ASC. In other words, when

cells are pre-loaded with a System ASC substrate then washed

free of external substrate and tested for System ASC uptake,








the activity is increased (Gazzola et al., 1980). A deceler-

ation of the transport rate, or trans-inhibition, is a

common characteristic of System A. In this case, when cells

are loaded with a System A substrate, washed free of exter-

nal substrate, and tested for System A uptake, the activity

is lower (Kelley and Potter, 1978). Trans-inhibition of

System A is cycloheximide-insensitive indicating de novo

protein synthesis is not required (Kilberg et al., 1985b).

Regulation of transport activity by amino acid depriva-

tion, sometimes called adaptive regulation, has been

observed for Systems A and N. The increase in activity is

detected after a time lag of 1-2 h, is blocked by cyclohexi-

mide, and is kinetically-defined as an increase in Vmax

(Kilberg et al., 1985b). The implication is that the

increased transport activity caused by the response to amino

acid deprivation is due to a de novo synthesis and inser-

tion of new carriers into the plasma membrane. A lowering

of the Km of the carrier for Na+ or amino acid is not

consistent with the data collected from several different

laboratories (Guidotti et al., 1978; Shotwell et al., 1983).

A wide variety of hormones affect the activity of

System A. Other systems, such as System N, are also

affected by hormones but no transport systems other than

System A show such a large response after hormone treatment

(Kilberg, 1982; Shotwell et al., 1983; Kilberg et al.,

1985a). Some of the hormones which induce hepatic System A

activity include growth hormone, growth factors, glucocorti-

coids, catecholamines, glucagon, and insulin. The








stimulatory effects of most of the hormones tested were

kinetically-defined as an increase in Vmax (Shotwell et al.,

1983). Insulin- and glucagon-dependent stimulation of

System A is also blocked by cycloheximide, actinomycin, and

tunicamycin indicating that the continuous synthesis of RNA

and glycoprotein is required to express glucagon-stimulated

activity (Kilberg et al., 1985a). The increase in Vmax and

the dependence on de novo glycoprotein synthesis has led

to the proposal that hormonal induction of System A trans-

port results in the insertion of a greater number of active

carrier molecules in the plasma membrane.

The first 15-30 min after exposure of freshly isolated

hepatocytes to glucagon is characterized by a stimulation of

System A activity which is independent of de novo pro-

tein synthesis. After 30 min, cycloheximide eliminates any

further increase in transport activity (Edmondson and

Lumeng, 1980). There are several possible explanations for

the protein synthesis-independent portion of the hormone-

stimulated transport activity. One possibility is that glu-

cagon is causing a redistribution of the trans-membrane ion

gradients resulting in increased electrogenic transport.

Friedmann and Dambach (1980) showed that glucagon-treatment

of rat liver resulted in a hyperpolarization of the cells

and an increase in the membrane potential from 39.0 mV to

47.2 mV. Bradford et al. (1985) showed that the membrane

potential increased in isolated hepatocytes after treatment

with dibutyryl cAMP using a 35C1 uptake procedure to

measure the potential. The increase in potential was








maintained for at least 40 min and could be partly

responsible for the corresponding increase in alanine uptake

induced by glucagon during the first 15-30 min of exposure.

Presumably, a change in membrane potential would affect all

Na+-dependent electrogenic transport systems and this

has not been reported, implying that the change in membrane

potential is not the only factor contributing to the

increased System A activity.

Another possible explanation for the protein synthesis-

independent increase in System A activity after glucagon-

treatment would be migration of cryptic carriers to the

plasma membrane as has been described for the glucose

carrier (Simpson and Cushman, 1985). Convincing evidence

supporting this model has not as yet been provided and

awaits adequate methods of subcellular fractionation as well

as methods for testing transport activity in various mem-

brane fractions. Reconstitution of glucose transport was

used by Suzuki and Kono (1980) to show the insulin-

stimulated movement of glucose carriers from an intra-

cellular compartment to the plasma membrane. Similar

studies could be undertaken for System A once a reliable

reconstitution process has been developed.

Post-translational modifications could also be respon-

sible for regulating the protein synthesis-independent por-

tion of the hormone-stimulated activity. Nilsen-Hamilton

and Hamilton (1979) observed that treatment of 3T3-fibro-

blast membrane vesicles with cAMP resulted in increased AIB

uptake. However, when the vesicles were treated under








hypotonic conditions with cAMP-deoendent protein kinase, an

inhibition of AIB uptake was observed. As expected, the
32P incorporation was increased in many proteins after

the addition of protein kinase and no conclusion could be

reached correlating specific protein phosphorylation and the

System A carrier protein.

Amino acid and hexose transport have frequently been

observed to be increased in tumorigenic cells (Parnes and

Isselbacher, 1978). In particular, it has been reported

that the Na -dependent uptake of AIB, a specific probe

of System A activity in most cells, is increased in trans-

formed cells (Isselbacher, 1972; Foster and Pardee, 1969).

Increased rates of glycolysis and System A transport activ-

ity were detected when "normal cells" were transformed

either by transfection with specific oncogenes or by expo-

sure of the cells to the low molecular weight polypeptides

called "transforming growth factors" (TGF). Exposure of rat

fibroblasts (rat-l) or myc-transformed rat-1 cells to the

23 kDa polypeptide called TGF-B greatly increased the rate

of System A transport (Racker et al., 1985).

Stimulation of amino acid transport and glycolysis was also

observed when a normal rat kidney cell line (NRK-49F) was

exposed to TGF-B (Boerner et al., 1985). The increased

activity reported in the experiments where TGF-B was used

to transform the cells was blocked by cycloheximide. The

cycloheximide sensitivity of the transformation-stimulated

System A activity appeared to be analogous to the cyclohexi-

mide sensitivity detected during the hormone- and








starvation-induced increase in transport activity. These

results raise the possibility that the same stimulatory mech-

anism (i.e., increased number of carriers being inserted

into the plasma membrane) may be operating to execute the

hormone-, starvation-, and transformation-induced System A

activity.

The relationship between growth factors, which stimu-

late System A activity, and tumorigenesis is beginning to be

understood through several different observations reviewed

recently by Weinberg (1985). When the sequence of the

platelet-derived growth factor, which is known to stimulate

System A activity (Owen et al., 1982), was compared to the

sequence of the v-sis oncogene product, considerable homol-

ogy was observed (Doolittle et al., 1983). Epidermal growth

factor (EGF) is another peptide which has been reported to

stimulate amino acid transport activity in human fibroblasts

(Hollenberg and Cuatrecasas, 1975). The cDNA clone for the

epidermal growth factor receptor was recently sequenced and

has been found to be homologous to a large portion of the

v-erbB oncogene product (Ullrich et al., 1984). It would

seem that certain growth factors, which are known to stimu-

late System A activity or the receptors for those growth

factors, have homology to oncogene products. This may imply

some unknown link between transforming-ability and the abil-

ity to stimulate System A activity which still remains to be

elucidated.

Glucagon- or starvation-induced transport activity has

been observed to decay if the stimulated cells are exposed






11

to amino acids. In fact, certain amino acids, such as aspar-

agine, cause a protein synthesis-dependent repression of

activity. Other amino acids, such as AIB, cause a rapid

decline in transport activity which is not protein

synthesis-dependent and is probably attributable to trans-

inhibition (Handlogten et al., 1985). The decay process is

blocked by cycloheximide and actinomycin (Handlogten and

Kilberg, 1984) implying that de novo protein and RNA syn-

thesis are required for the decay to occur. Interestingly,

the decay process is also blocked by a-amanitin, an inhibi-

tor of RNA polymerase II at the concentrations used in the

study, but not by cordycepin or ara-A, inhibitors of poly(A)

polymerase (Handlogten et al., 1985). These results suggest

that a mRNA must be synthesized de novo for the decay

process to operate and that the mRNA has the unusual feature

of lacking a poly(A) extension. The major group of mRNA

molecules known to lack poly(A) code for the histone pro-

teins. The role of these proteins in controlling the decay

process is, at present, unclear. What process is actually

responsible for causing the loss in System A activity is

unknown also. Whether it is a protease, post-translational

modification, internalization, or some other process remains

to be elucidated.

The purpose of this study is to learn more about the

hormonal and adaptational regulation of System A, and to

learn about some of the molecular aspects of System A-asso-

ciated proteins. At present, we can only examine the regula-

tion of transport activity in whole cells by observing the








effects of inhibitors of protein and RNA synthesis on trans-

port activity; therefore, many questions remain as to the

exact mechanisms that exist for the regulation of System A

transport. For example, how does a hormone like glucagon

activate transport activity in a protein synthesis-indepen-

dent manner? What other evidence besides kinetics and

cycloheximide-sensitivity can be collected to support the

notion that hormone-induction, amino acid-deprivation, or

cellular transformation causes an increased number of System

A carrier molecules to be inserted into the plasma membrane?

How does the amino acid-dependent decay process relate to

the regulation of transport activity? How is transport regu-

lated by trans-effects?

The strategy we have used to begin our study of these

questions is to prepare isolated membrane vesicles which

mediate System A transport. These membrane vesicles can be

used in studies of trans-effects, hormonal, and adaptational

control to establish if these modes of regulation are associ-

ated with the plasma membrane. For example, the argument

that hormones, amino acid-deprivation, or transformation

causes an increased number of System A carriers to be inser-

ted into the plasma membrane would be strengthened if it

could be determined that transport activity was increased in

membrane vesicles prepared from those induced cells. Like-

wise, if trans-inhibition could be demonstrated in isolated

membrane vesicles, it would support the notion that trans-

inhibition is a membrane-associated phenomenon.








Having perfected this artificial system for assaying

the transport of amino acids, the next part of the strategy

is to reconstitute System A activity after extracting it

from its native membrane environment and replacing it into

artificial lipid bilayers. If this assay system could be

developed, it would then be possible to perform several puri-

fication steps on the membrane-extract and assay purifica-

tion by reconstitution. Purification and identification of

System A-associated proteins would be an important prelude

to the elucidation of the mechanism of amino acid transport

because studies could be done on the carrier in an isolated

state in an artificial membrane. The consequences of reveal-

ing the regulation and mechanism of System A transport activ-

ity would be medically relevant because its activity is

increased not only in tumor cells but also in hepatic cells

from diabetic animals.
















CHAPTER II
CHARACTERIZATION OF SYSTEM A AMINO ACID TRANSPORT
ACTIVITY IN MEMBRANE VESICLES



Introduction

Use of Membrane Vesicles for Study of Nutrient Transport

Membrane vesicles from various cell types and tissues

have proven to be extremely useful for studying nutrient

transport processes. The earliest studies of active trans-

port systems in membrane vesicles were reported using bacter-

ial membranes (Kaback, 1960). Transport systems for various

sugars and amino acids were characterized by using bacterial

vesicles prepared by osmotic lysis and testing uptake using

a rapid filtration system (Kaback, 1974). The development

of methods for preparation of vesicles from bacterial cells

led other groups to develop similar techniques for eukaryo-

tic cells.

Nutrient transport in membrane vesicles from eukaryotic

cells and tissues has been reviewed recently in several ex-

cellent articles (Lever, 1980; Murer and Kinne, 1980; Sachs

et al., 1980). Sodium-dependent transport systems for D-

hexoses, L-amino acids, bile acids, and various ions have

been demonstrated in vesicles from brush border as well as

basolateral membranes. Most transport systems tested show








similar specificities in membrane vesicles when compared to

intact cells as has been demonstrated for the Na -

dependent glucose transporter in intestinal brush border

vesicles (Kessler and Semenza, 1983).

Several criteria have been applied to membrane vesicles

to determine decisively if solute is accumulated in the

intravesicular space via a carrier-mediated process: 1)

exchange diffusion of the solute with an internal solute, 2)

temperature dependence of influx and efflux, 3) osmotic sen-

sitivity of the vesicles for accumulation of solute, 4) sat-

uration kinetics of the substrate and, 5) selective effects

on ion-dependent transport caused by ionophores.



Advantages of Studying Transport in Membrane Vesicles

There are several advantages to studying transport phe-

nomena in isolated membrane vesicles versus intact cells.

Membrane vesicles offer the possibility of examining trans-

port processes in the absence of intracellular metabolism.

Many nutrients, such as amino acids or glucose, are trans-

ported into a cell and then rapidly metabolized, complica-

ting transport studies considerably. Membrane vesicles also

allow one to introduce defined media to either side of the

membrane, thus facilitating carrier function studies from

the cis or trans side. Various electrochemical driving

forces can be introduced and the effects on the carrier

readily observed. Vesicles are, additionally, ideal systems

for monitoring electrochemical potential-sensitive and pH-

sensitive dyes. Membrane vesicles which show transport








activity are also often useful as starting material for

reconstitution studies.

Drawbacks to the use of membrane vesicles for transport

studies include the following: 1) the orientation of the

carrier is not always known and, 2) the proteins necessary

for activity may be partially or completely degraded during

the course of vesicle isolation, making transport studies

difficult. In many instances, the disadvantages of using

membrane vesicles are greatly outweighed by the amount of

information that can be obtained through their use.


Homogeneity of Plasma Membrane Vesicles

The hepatocyte is a polarized cell, that is, its plasma

membrane is separated into functional domains with special-

ized functions. The bile canalicular domain is the barrier

between the cell and the bile-collecting ducts. It is

through this membrane surface that bile acids produced in

the hepatocyte are secreted into the bile duct. The sinusoi-

dal or blood-domain is the border between the cell and the

blood-carrying vessels. It is through this membrane surface

that peptide hormones such as insulin or glucagon interact

with the hepatocyte. The final domain, referred to as the

contiguous surface, is the region of hepatocyte plasma mem-

brane which is found between two hepatocyte cells. It is in

this domain that cell-cell communication occurs through gap

junctions.

The most commonly used technique for determining homoge-

neity in membrane vesicle preparations is through the use of








enzyme markers. The level of subcellular contamination can

also be quantitated using enzyme marker assays (Evans,

1980). Indicators of plasma membrane purity include 5'-

nucleotidase, Na+,K+-ATPase, alkaline phosphatase,

leucine aminopeptidase, and adenylate cyclase. Endoplasmic

reticulum contamination can be quantitated through the use

of glucose-6-phosphatase or NADPH:cytochrome c reductase

assays. Mitochondrial contamination can be determined using

either succinate:cytochrome c reductase as an indicator of

the inner mitochondrial membrane or monoamine oxidase as an

indicator of the outer mitochondrial membrane. Contamina-

tion by Golgi remnants can be quantitated using galactosyl

transferase and lysosomal contamination can be determined

using acid phosphatase. Nuclear contamination can be deter-

mined using DNA as a marker and cytoplasmic contamination

can be quantitated using latent lactate dehydrogenase activ-

ity. A qualitative determination of contamination by sub-

cellular organelles can also be performed using transmission

electron microscopy.

Enzyme markers can also be used to distinguish the func-

tional domains of the hepatocyte plasma membrane. The bile-

canalicular surface contains most of the activity for leu-

cine aminopeptidase and 5'-nucleotidase (Roman and Hubbard,

1983), whereas glucagon-activated adenylate cyclase is found

primarily on the blood-sinusoidal surface (Wisher and Evans,

1975) and Na+,K -ATPase is located primarily on the

contiguous and sinusoidal surfaces of the rat liver plasma

membrane (Poupon and Evans, 1979).










Isolation of Membrane Vesicles from Rat Liver

Several groups have succeeded in isolating plasma

membrane-enriched vesicles from rat liver (Neville, 1968;

Ray, 1970; Touster et al., 1970). One group in particular

has addressed the functional polarity of the hepatocyte and

has developed a technique for separating the canalicular por-

tion of the plasma membrane from the blood-sinusoidal and

contiguous membrane surfaces (Wisher and Evans, 1975). More

recently, membranes from the three separate domains have

been prepared to even greater homogeneity through the use of

sucrose step-gradients (Hubbard et al., 1983). The best

separation of plasma membrane domains reported so far used

rate-zonal centrifugation and resulted in a 64-fold enrich-

ment of canalicular membrane markers and a 34-fold enrich-

ment of basolateral (a mixture of sinusoidal and contiguous

membrane surfaces) membrane markers (Meier et al., 1984b)

over the activity detected in the homogenate. Centrifuga-

tion through a Percoll gradient has also proven useful in

purifying plasma membrane vesicles from rat liver (Prpic et

al., 1984).

Amino acid transport has been studied to a limited

extent in membrane vesicles prepared from rat liver. The

first report of Na -dependent amino acid transport

assayed alanine uptake after isolation of membranes by dis-

continuous sucrose gradient centrifugation (Van Amelsvoort

et al., 1978). Another group (Meier et al., 1984a), has

shown that about equal amounts of sodium-dependent amino








acid transport are found in the basolateral and the canalicu-

lar surfaces. Increased Na -dependent alanine transport

has been reported in rat liver vesicles after starvation of

the animals (Quinlan et al., 1982) or after treatment of

hepatocytes with dibutyryl cAMP (Samson and Fehlmann, 1982).

In our work, we have demonstrated that membrane vesi-

cles from rat liver can be prepared and these vesicles activ-

ely accumulate amino acids. The vesicles were also prepared

from glucagon-treated hepatocytes as well as amino acid-

starved hepatocytes in order to show that increased System A

transport observed in the intact cells was retained in the

membrane vesicles. Vesicles were also prepared from a human

hepatoma cell line (HepG2) and those vesicles retained

increased System A activity as is observed in intact cells.



Materials and Methods


Materials

The radio-labelled compounds used were [carboxyl-

14C] inulin, [methyl-3H1 2-aminoisobutyric acid (AIB),

ICN Pharmaceuticals; L-[ H] cystine, Schwarz/Mann; L-[2,5-

3H] histidine, [2-3 H glycine, L-[2,3-3H] alanine, and

3-O-methyl-D-[U14C] glucose, Amersham. Filters used for

transport assays were either Millipore type HAWP (0.45 pm)

or Gelman type GN-6 (0.45 pm). Highly purified glucagon

was a generous gift from Dr. Mary Root of Lilly Laborator-

ies. Fetal bovine serum (FBS) and Eagle's Minimal Essential

Medium (MEM) were obtained from Flow Laboratories. All






20

other chemicals were reagent grade or better and were

obtained from Sigma Chemical Company. Rats were from a col-

ony maintained by the University of Florida Animal Resources

facility.



Hepatocyte Isolation and Transport Assay

Hepatocytes were isolated from male Sprague-Dawley rats

(100-200 g) as described previously (Kilberg et al., 1983).

Usually, more than 90% of the cells were viable as deter-

mined by the trypan blue exclusion assay. Both the control

and the hormone-treated rats were fasted overnight prior to

cell isolation. The experimental animals were injected with

1 mg of glucagon per 100 g body weight 4 h before surgery.

A small portion of both control and hormone-treated hepato-

cytes was suspended in Na -containing Kreb's-Ringer bi-

carbonate buffer (NaKRB) containing 0.1 mM cycloheximide

(CHX) and placed in monolayer culture. Following a 2 h cul-

ture period, the activity of System A was determined by

assaying the Na -dependent transport of 50 pM AIB for 1

min at 370C as described by Kilberg et al. (1983). The

remaining cells were resuspended in 40 ml of ice-cold Buffer

A (0.25 M sucrose, 0.2 mM MgC12, 10 mM HEPES-KOH, pH 7.5)

for preparation of a plasma membrane-enriched subcellular

fraction.








Rat Liver Plasma Membrane Isolation

Plasma membrane vesicles were prepared as described by

Van Amelsvoort et al. (1978). The liver of a 24 h-fasted

male Sprague-Dawley rat (150 to 200 g) was perfused with an

iso-osmotic homogenization buffer consisting of 0.25 M

sucrose, 0.2 mM MgCl2, 10 mM HEPES-KOH, pH 7.5 (Buffer

A). All subsequent procedures were carried out at 40C.

The blanched liver was removed and homogenized with 22

strokes using a Potter-Elvehjem homogenizer with a motor-

driven, loose-fitting teflon pestle. After the addition of

EDTA to a final concentration of 1 mM, the homogenate was

forced through a nylon screen (75 pm) and then centrifuged

at 1000xg for 10 min. The supernatant and the loose upper-

layer of the pellet were saved and the remaining solid

pellet was resuspended in Buffer A containing 1 mM EDTA.

This suspension was centrifuged again at l000xg for 10 min.

The resulting supernatant was collected as before, pooled

with the previous one, and then centrifuged at 20,000xg for

30 min. The loose upper-layer of the pellet was collected

and resuspended in Buffer A containing 1 mM EDTA by passing

the material through a 19 ga. needle six times. This mem-

brane fraction was purified further by placing it on a dis-

continuous sucrose gradient composed of 20%(w/v) and

39.5%(w/v) sucrose each containing 10 mM HEPES-KOH, pH 7.5.

The gradients were centrifuged at 50,000xg for 2.5 h. Vesi-

cles enriched in plasma membrane were collected from the

20%/39.5% sucrose interface and diluted 1:1 with 0.2 mM

MgC12, 10 mM HEPES-KOH, pH 7.5. These membranes were








then pelleted by centrifugation at 100,000xg for 40 min.

The resulting pellet was resuspended by vortexing in Buffer

A to a final concentration of approximately 10 mg pro-

tein/ml. The overall yield of the procedure was about 2-3

mg protein per g liver (wet weight). Vesicles could be

stored for up to one month at -700C with minimal loss of

transport activity.


Preparation of Membrane Vesicles from Cultured Hepatocytes

When membrane vesicles were prepared from cultured

cells following substrate starvation, the freshly isolated

hepatocytes were placed in 150 mm collagen-coated dishes

(Kilberg et al., 1983) in NaKRB (amino acid-free medium) or

NaKRB containing 20 mM asparagine (amino acid-supplemented

medium) at a density of 27 million viable cells per dish.

The cells were incubated at 370C in a humidified atmosphere

of 5% C02/95% air for 6 h and then each dish was rinsed

with 10 ml of phosphate buffered saline (154 mM NaCl, 10 mM

Na2HPO4, brought to pH 7.5 with HC1). The cells were

scraped into 5 ml of Buffer A and homogenized by 25 strokes

with the Potter-Elvehjem homogenizer with a tight-fitting

teflon pestle. Membrane vesicles were isolated as described

above. The total yield from 10 dishes of cultured cells was

approximately 3 mg of membrane protein.








Isolation of Membrane Vesicles from Human Hepatoma Cells

A human hepatoma cell line (HepG2) was grown to confluence

in fourteen 150 mm Petri dishes in MEM supplemented with 5%

FBS The dishes were rinsed twice with PBS and then

the cells were scraped into a total of 30 ml of Buffer A.

The suspension was homogenized using 10 strokes of a

tight-fitting Potter-Elvehjem homogenizer and the homogenate

was brought to 1 mM in EDTA. The homogenate was centrifuged

at 1000xg for 10 min. The supernatant (Sl) and the pellet

(PI) were both saved. The S1 fraction was centrifuged at

45,000xg for 30 min, and the resulting supernatant (S2) was

discarded. The corresponding pellet (P2) was resuspended in

1 ml of Buffer A and saved. Membranes contained in the

nuclear pellet were prepared using the initial pellet (P1)

which was placed on top of two sucrose step gradients

containing 39.5% sucrose as the lower layer and 20% sucrose

as the middle layer. The gradients were centrifuged at

50,000xg for 2.5 h. The white, fluffy material at 20%/39.5%

interface was removed and was diluted 1:1 with 0.2 mM

MgC12, 10 mM HEPES, pH 7.5. The suspension was

centrifuged at 100,000xg for 1 h to pellet the vesicles.

The final pellet (P3) was resuspended in 1 ml of Buffer A.

The protein content of the fractions was measured by a Lowry

assay. The total protein yield of P2 was approximately 7 mg

and that of P3 was approximately 3 mg.



Cells could also be grown in roller culture (2 L) in
HEPES-MEM, pH 7.45 containing 5% FBS. The cells were
removed by treating with 2 mM EDTA for 2 h at 370C then
scraping with a rubber policeman into 40 ml of PBS.










Vesicle Transport Assay

Just prior to use, the membrane vesicles were diluted

with Buffer A to a final concentration of 2.5 mg protein per

ml and then incubated at 220C for 15 min. To initiate

amino acid uptake, 20 pl (50 pg protein) of the vesicle

suspension was added to 20 pl of Buffer A supplemented with

10 mM MgC12, 120 mM of either NaSCN or KSCN, and 200 pM

radioactively-labelled amino acid. These two solutions will

be referred to as Na+- and K -uptake buffers, respective-

ly. Where indicated in the figure legends, 60 mM Na2SO4

or K2SO4 was used to replace the corresponding thiocyan-

ate salts. Uptake was terminated by the addition of 1 ml of

ice-cold Buffer A containing 100 mM NaC1 (stop-buffer). The

mixture was vortexed immediately and passed over a 0.45 pm

nitrocellulose filter. The filter was washed with another 3

ml of ice-cold stop-buffer and then analyzed for trapped

radioactivity in 5 ml of Bray's scintillation cocktail

(Bray, 1960). Unless otherwise indicated in the figure

legends, the results were from a single membrane preparation

and the S.D. of triplicate assays was less than 10% of the

mean.


Enzyme Marker Assays

The activities of 5'-nucleotidase (Morre, 1971), glu-

cose-6-phosphatase (Swanson, 1955), succinate:cytochrome c

reductase (Kilberg and Christensen, 1979), and cytochrome ox-

idase (Kilberg and Christensen, 1979) were assayed by








previously described methods. Inorganic phosphate was

determined by the method of Fiske and Subbarow (1925).

Fluoride-stimulated (10 mM NaF) adenylate cyclase activity

was measured by a modification of the procedure described by

Wisher and Evans (1975). The cAMP produced was detected by

a protein-binding assay supplied as a kit by Amersham Corp.

Tests for contamination of the final membrane fraction by

intracellular membranes showed a similar profile to that

obtained by Van Amelsvoort et al. (1978).


Determination of the Extravesicular Volume

The extravesicular volume of pelleted membrane vesicles
14
was determined using 14C-inulin. Before use, the

14C-inulin stock (1 mg inulin/ml; 7 pCi/ml) was fil-

tered through a Gelman filter (0.22 pm) to remove particu-

lates and the filtrate was counted for radioactivity.

Vesicles (2.48 mg/500 pl) were mixed with 400 pl of Buffer

A and 100 pl of 14C-inulin and the mixture was vor-

texed. After 10 pl was removed for determination of the

total radioactivity, the suspension was centrifuged at

100,000xg for 1 h and 100 pl of the supernatant was removed

for determination of the total radioactivity. The sides of

the centrifuge tube and the surface of the pellet were

washed 3 times with 3 ml aliquots of ice-cold Buffer A,

then the pellet was resuspended in 1 ml of Buffer A and 200

pl of the suspension was removed for determination of the

total radioactivity. The total dpm remaining in the pellet

after the washes was divided by the total dpm present in the








initial suspension before centrifugation. This value was

divided by the total mg of protein present in the suspension

to give 23.2 pl of extravesicular volume per mg protein for

the 100,000xg pellet.


Protein Determination

Vesicle protein was determined by a modification of the

method of Bensadoun and Weinstein (1976). Approximately

10-50 pg of membrane protein was suspended in 1 ml of 0.1%

sodium dodecyl sulfate (SDS). Following a 10 min incubation

at 220C, the protein was precipitated by adding 750 pl of

ice-cold 24%(w/v) trichloroacetic acid (TCA) and then

pelleted by centrifugation at 12,000xg for 20 min. The pro-

tein content of the pellet was measured by a modification of

the Lowry technique as described previously (Kilberg et al.,

1983). Bovine serum albumin (5 to 100 pg) was used as the

standard.



Results


Enzyme Marker Activities in Rat Liver Membrane Vesicles

Enzyme marker analysis of the membrane vesicles pre-

pared from rat liver revealed that the vesicles are enriched

approximately 10-fold in the plasma membrane marker enzymes

5'-nucleotidase and adenylate cyclase (Table 2-1). A marker

enzyme for microsomal contamination, glucose-6-phosphatase,

showed 3-fold enrichment. There was a 10-fold reduction in

the level of the mitochondrial enzyme marker activities,








TABLE 2-1
Enzyme Activities in the Plasma Membrane-Enriched Fraction

Membrane vesicles were tested for the presence of particular
enzyme markers for plasma membrane, endoplasmic reticulum, and
mitochondrial inner membrane. The activities of 5'- nucleotidase
and glucose-6-phosphatase are expressed in terms of pmol Pi
formed per mg protein per h. Adenylate cyclase activity is
expressed as pmol cAMP formed per mg protein per h. Succin-
ate:cytochrome c reductase and cytochrome c oxidase activities
are expressed as nmol cytochrome c reduced per mg protein per
min. Relative specific activity (R.S.A.) is determined by divi-
ding the specific activity of the enzyme in the plasma membrane-
enriched fraction by the specific activity in the homogenate.
The data are the averages + S.D. of triplicate determina-
tions.


Enzyme Activity Homogenate Vesicles R.S.A.


5'-nucleotidase 4.1 + 0.2 38.9 + 1.8 9.4

Adenylate cyclase 9.1 + 3.6 94.2 + 8.9 10.4

Glucose-6-phosphatase 10.2 + 1.5 31.9 + 1.6 3.1

Succinate:cytochrome c 14.0 + 0.31 1.32 + 0.09 0.1
reductase

Cytochrome c 13.2 + 0.35 1.01 + 0.18 0.1
oxi dase








succinate:cytochrome c reductase and cytochrome c oxidase

(Table 2-1). These enzyme marker activities are consistent

with those observed by Sips et al. (1980b).


Time-Dependent Uptake of AIB

Fig. 2-1 depicts the time-course of AIB transport by

isolated membrane vesicles. AIB uptake showed a Na -

dependent overshoot in the presence of a Na+ gradient

with maximal transport at 3 min. By 40 min, the accumula-

tion of AIB reached a steady-state level, presumably because

the Na+ gradient had been dissipated. The uptake in the

absence of Na+ was essentially hyperbolic in nature, and

reached a plateau after 15 min (Fig. 2-1).

The presence of a Na -dependent overshoot suggests
transport of the amino acid against a concentration gradi-

ent. To confirm this hypothesis, the intravesicular water

space was estimated. A value of 1.2 pl/mg protein was

determined by the 3-0-methyl-glucose method of Kletzien et

al. (1975). Using this value, it was calculated that the

steady state distribution ratio for AIB (AIBin/AIBout

in the absence of Na was slightly less than one.

Hence, an accumulated level of the amino acid greater than

240 pmol per mg protein represents transport against a con-

centration gradient.




















Fig. 2-1. Time-Dependent Uptake of AIB by Rat Liver Plasma
Membrane Vesicles. Membrane vesicles (?.5 mg protein/mi)
were diluted into either Na () or K (*) uptake
buffer containing 200 pM radioactively-labelled AIB. After
incubation at 220C for the time indicated, an aliquot (50
pg) was removed and assayed for trapped radioactivity as
described in Methods section. The difference between the
transport in the presence of NaSCN or KSCN is shown as the
Na -dependent uptake (A). The data are the averages +
S.D. of three determinations.
















400

& 320
a.

E 240

S160

1 80


0 10 20 30 40 50 60

Minutes






31

MeAIB Inhibition of AIB Uptake

Fig. 2-2 illustrates the inhibition of Na+-dependent AIB

uptake by MeAIB, a System A-specific analog (Kilberg et al.,

1981). The inhibition was concentration dependent yielding

an apparent Ki of 0.6 + 0.2 mM. This value is in good

agreement with unpublished data from our laboratory indi-

cating that the Km for the high-affinity Na -dependent

component of MeAIB uptake by isolated hepatocytes is about

0.3 mM. Although an excess of MeAIB does not completely

block AIB uptake (Fig. 2-2), it is clear that most (> 85%)

of the Na -dependent AIB uptake occurs by the MeAIB-

inhibitable route (i.e., System A). These data support the

use of AIB as a selective substrate for System A in isolated

rat liver plasma membrane vesicles.


Sodium-Dependent Transport of Naturally-Occurring Amino
Acids

The uptake of four individual amino acids was measured

to obtain evidence for the presence of Systems A, ASC, N,

and Gly in the isolated membrane vesicles. The rate of

alanine transport in the presence of a Na+ gradient was

considerably greater than that in the absence of Na+ (Fig.

2-3A). The time-course of alanine uptake showed a rapid

Na -dependent overshoot, about 50% of which was not inhibi-

table by an excess of MeAIB. These data, in agreement with

results from intact hepatocytes (Kilberg et al., 1979) and

isolated liver membranes prepared in other laboratories

(Samson and Fehlmann, 1982; Sips et al., 1980b), indicate





















Fig. 2-2. MeAIB Inhibition of Na -Dependent AIB Uptake
by Isolated Vesicles. Rat liver plasma membranes were
assayed for Na -dependent AIB transport in the presence of
the System A-specific probe, MeAIB, at the indicated
concentrations. The Na -dependent uptake of AIB was
measured as described in the Methods section. Computer
analysis of the data by a FORTRAN program as described by
Kilberg et al. (1983) yielded an estimated Ki of 0.6 +
0.2 mM for MeAIB. The results are reported as the averages
+ S.D. of assays in triplicate.

















:200 ,
o

o
S160

.120 app. Ki =0.6*0.2mM.

E so
o

80

IL I

0 5 10 15 20 25

CMeAIB] mM





















Fig. 2-3. Alanine, Cysteine, Histidine and Glycine Trans-
port by Rat Liver Plasma Membranes. Membrane vesicles were
used to measure the transport of the indicated substrate.
The transport of alanine (A), cysteine (plus 1 mM dithio-
threitol) (B), and histidine (C) was tested in the presence
or absence of 5 mM MeAIB. Glycine (0) uptake was assayed in
the presence or absence of 5 mM threonine. The substrate
concentration in each case was 200 pM and transport was
measured at 220C. .The difference between uptake iq Na
( ) and that in K ( ) was taken as the total Na -
dependent transport (A). The Na -dependent component in
the presence of the inhibitor (MeAIB or threonine) is also
shown (V). See the text for interpretation of these data as
evidence for the specific transport systems indicated. The
results are presented as the averages + S.D. of three
determinations.





























500


400


300


200

100

o
0

S 1100




660


440


220


2 3 4 5 0 1 2 3 4

Minutes








that the hepatic Na -dependent uptake of alanine, at a sub-

strate concentration of 200 pM, is about equally divided

between Systems A and ASC.

Additional evidence for the presence of System ASC was

obtained by examining cysteine transport. The Na+depen-

dent uptake rate for cysteine was considerably less than

that seen for alanine (e.g., 150 versus 450 pmol per mg pro-

tein per 15 s). As reported previously for intact hepato-

cytes (Kilberg et al., 1979), the Na+-dependent transport

of cysteine by the membrane vesicles was not inhibited by an

excess of MeAIB (Fig. 2-3B). These results demonstrate the

ability to monitor specifically hepatic System ASC in rat

liver vesicles by measuring Na -dependent cysteine

uptake.

The Na+-dependent transport of glutamine and histi-

dine by intact hepatocytes is mediated to a large extent by

System N (Kilberg et al., 1980). In the absence of MeAIB,

histidine uptake by the vesicles (Fig. 2-3C) showed a rapid

Na+-dependent overshoot that decayed at a slow rate. When

Na -dependent histidine uptake was assayed in the presence

of an excess of MeAIB, conditions that provide a specific

test for System N activity (Kilberg et al., 1980), a rapid

overshoot was observed with an accumulation of more than 400

pmol per mg protein at 15 s. These results suggest the pre-

sence of System N activity in the isolated vesicles, but

also indicate that histidine transport in this membrane pre-

paration is not restricted entirely to System N.








Hepatic Na -dependent glycine transport is mediated

by Systems A, ASC, and Gly (Christensen and Handlogten,

1981). System Gly activity can be assayed selectively by

measuring Na -dependent glycine uptake in the presence of

an amino acid that can inhibit efficiently the other two sys-

tems. We have chosen threonine for this purpose because, in

cultured hepatocytes, this amino acid is transported effec-

tively by both Systems A and ASC (Kilberg et al., 1985a).

When Na -dependent glycine uptake was measured in the ves-

icles, approximately 40% escaped inhibition by threonine

(Fig. 2-30). Although these data show that a significant

portion of glycine uptake occurs by Systems A and ASC, they

also demonstrate that System Gly activity can be measured

readily in isolated liver plasma membrane vesicles.


Further Evidence for Heterogeneity of Alanine Transport

For isolated rat hepatocytes, transport by System ASC

is partially retained when Li+ replaces Na+ as the

extracellular alkali ion, whereas little or no System A-

mediated uptake occurs in the presence of lithium (Edmondson

et al., 1979). Such system selectivity for Li is just

the reverse in the Ehrlich ascites tumor cell (Christensen

and Handlogten, 1977). To determine whether this cell-spe-

cific property of System A was observed in isolated liver

membranes, Na and Li -dependent amino acid trans-

port was measured. Li+ substitution for Na+ caused

an 80% decrease in AIB uptake (Table 2-2). Lithium-

dependent transport depends to some degree on amino acid








TABLE 2-2

Alkali-Ion Specificity for Systems A and ASC
in Rat Liver Plasma Membrane Vesicles

Membrane vesicles (2.5 mg protein/ml) were diluted into
Buffer A containing 10 mM MgC1l and one of the following:
120 mM NaC1, 120 mM LiC1, or 120 mM KC1. Incubation of the
vesicles at 22C in the presence of 200 pM substrate was
for 1 min (alanine) or 5 min (AIB). All other assay condi-
tions were the same as described in the text. The veloci-
ties are expressed in terms of pmol amino acid accumulated
per mg protein per unit time. In parentheses, the Li -
dependent data are shown as a percent of control values
determined in Na The results are reported as the aver-
ages + S.D. of three determinations.


Substrate Alkali ion Velocity Na or Li -
Tested dependent uptake

AIB Na+ 371 + 12 196 (100)

Li+ 217 + 7 42 (21)

K+ 175 + 16 -


Alanine Na+ 654 + 37 542 (100)

Li+ 526 + 57 414 (76)

K+ 112 + 10


Alanine Na+ 405 + 55 293 (100)

+ 5 mM MeAIB Li+ 438 + 42 326 (111)

K+ 112 + 10 -








structure (Christensen and Handlogten, 1977); therefore,

alanine was chosen as an additional test substrate.

Lithium-dependent alanine transport via System ASC has been

demonstrated for isolated rat hepatocytes (Kilberg et al.,

1981; Kilberg et al., 1979; Edmondson et al., 1979),

although Quinlan et al. (1982) reported that the rate of

Li -dependent alanine uptake by liver plasma membrane

vesicles was only 15% of the corresponding rate in Na.

Alanine uptake mediated by System A, estimated by subtrac-

ting the alkali-ion dependent velocity in the presence of

MeAIB from that seen in the absence of the inhibitor, was

249 and 88 pmol per mg protein per min in Na and Li ,

respectively (Table 2-2). In contrast, the rates of alanine

transport by System ASC, as monitored by MeAIB-insensitive

uptake, were the same in the presence of Li+ and Na+

(Table 2-2). These data are in agreement with those

obtained with intact hepatocytes and demonstrate that in iso-

lated vesicles System ASC accepts Li+ to a greater degree

than does System A. They also provide additional evidence

for the heterogeneity of alanine transport in the isolated

plasma membrane vesicles.

One of the established tests for distinguishing between

Systems A and ASC is the greater degree of inhibition of the

former system by increased H+ concentration in the uptake

buffer (LeCam and Freychet, 1977). To further demonstrate

the heterogeneity of Na+-dependent alanine transport in

isolated liver membrane vesicles, we assayed alanine uptake

in the presence or absence of MeAIB at pH values between 5.5








and 8.0 (Fig. 2-4). Decreasing the pH caused an inhibition

of System A-mediated Na+-dependent alanine transport by

about 67% over the pH range tested, whereas Na -

dependent alanine uptake via System ASC remained relatively

constant (Fig. 2-4).



Characterization of Amino Acid Uptake into Membrane Vesi-
cles

An important criterion of carrier-mediated transport is

the stereospecificity of the process. When the Na -

dependent uptake of radioactively-labelled 200 pM L-alanine

was measured in the presence of 5 mM of unlabelled alanine,

the L-isomer caused a 54% inhibition of transport (control =

553 + 39, plus inhibitor = 256 + 12 pmol per mg pro-

tein per min), whereas the D-isomer caused only a 17% inhibi-

tion (control = 553 + 39, plus inhibitor = 457 + 36

pmol per mg protein per min). Kinetic analysis of L-alanine

transport is consistent with the observed level of inhibi-

tion by the L-isomer. Likewise, experiments testing inhibi-

tion of Na -dependent L-histidine uptake by the corre-

sponding L- or D-isomers showed that they inhibited trans-

port by 94% and 17%, respectively. These results demon-

strate the stereospecificity of amino acid transport by the

isolated membranes.

To determine if the plasma membrane vesicles were tight-

ly sealed, the intravesicular space was altered by changing

the osmolarity of the incubation medium. Fig. 2-5 illus-

trates that the uptake of AIB in sodium or in potassium was





















Fig. 2-4. Effect of pH on Alanine Uptake in Membrane
Vesicles. Na -dependent alanine uptake was assayed in the
presence (V) or absence (A) of 5 mM MeAIB. The pH of the
uptake buffers was varied from 5.9 to 8.1. All other assay
conditions were the same as those described in the Methods
section. Data are presented as the averages + S.D. for
triplicate determinations of the Na -dependent transport.


















S550
0
S440


e 330
9L A + ASC
I.B
E 220


110 -



5.5 6.1 6.7 7.3 7.9 8.5
pH





















Fig. 2-5. Effect of the Extravesicular Osmolarity on AIB
Uptake. Membrane vesicles were diluted into Na ( )
or K -( *) uptake buffer containing sucrose from 0.2 to
0.9 M and radioactively-labelled AIB (200 pM). Each of
these suspensions was divided into 3 tubes (50 pg each) and
incubated at 40C for 1 h. The accumulation of the amino
acid was assayed as described in the Methods section. The
data are the averages + S.D. of three determinations.





44










400

. 320
0
a 240

E
J 160 ,-

o 80 --
E


0 1 2 3 4 5

[sucrose] M"1








decreased when the concentration of sucrose in the incuba-

tion medium was increased. The inversely linear relation

between the sucrose concentration and the steady state amino

acid accumulation indicates that AIB is being transported

into an osmotically-sensitive compartment, presumably the

intravesicular space. Extrapolation of the data to infinite

sucrose concentration suggests that some leakage of sucrose

or some non-specific binding to the vesicles does occur

(Fig. 2-5).

The Na+-dependent uptake of AIB into vesicles was

dependent on temperature changes and protein concentration.

Sodium-dependent uptake of AIB after a 6 min incubation at

15C was only 78% of that at 22C, whereas the uptake at

4C was only 11% of the uptake observed at 220C (Fig.

2-6). To provide evidence that the uptake of amino acids

was mediated by a membrane-bound protein or protein-complex,

the effect of increasing the concentration of protein con-

tent in each assay was measured. Fig. 2-7 shows that,

between 10 and 200 pg of protein, the rate of AIB uptake

varied linearly with the amount of vesicle protein included

in the assay. This relation was observed for both total and

sodium-dependent transport.

Two lines of evidence support the conclusion that the

Na+-dependent uptake of amino acids by the membranes is

the result of an imposed trans-membrane Na+ gradient.

First, when the extravesicular Na concentration is

increased the rate of transport also increases. Secondly,

if the sodium gradient is dissipated by incubation of the





















Fig. 2-6. Effect of Incubation Temperature on the
Na -Dependent Uptake of AIB. Membrane vesicles (2.5 mg
protein/ml) were incubated at either 220C (* ), 150C, ( )
or 4C (A) for 15 min prior to assay of System A activity
as described in the Methods section. The uptake was per-
formed as usual except that the uptake buffer was pre-
incubated at the indicated temperature and the uptake was
performed for the indicated period of time at the appropri-
ate temperature.

















* 160
0


E
' 80
o

E 40
I_


0 2 4 6
Minutes





















Fig. 2-7. Relation Between Membrane Protein Concentration
and AIB Uptake. Membrane vesicles were diluted into Na -
or K -uptake buffer containing 200 pM AIB. The amount
of membrane protein in the assay was varied from 10 to 200
pg. Uptake of the amino acid was measured for 6 min at
22C in Na containing buffer( 0). The Na -dependent
AIB uptake is also shown (A). The data are the averages
+ 5.0. of triplicate determinations. Where not shown,
The standard deviation bars are within the symbol.


















50 -


40


30


E

10



0 40 80 120 160 200

ug vesicle protein








vesicles with a Na -selective ionophore such as gramici-

din or monensin (Pressman, 1976), the rate of Na -

dependent transport is decreased significantly (Table 2-3).

To demonstrate that the membrane vesicles retained

their native permeability with respect to anions, and to

show that the transport occurred by an electrogenic process,

Na+-dependent alanine uptake was measured in the presence

of different counter-anions. If Na -dependent uptake of

200 pM alanine in NaSCN-containing buffer was set equal to

100% (698 pmol per mg protein per min), the Na -dependent

transport rates in NaCl or Na2S04 were 86% and 73%,

respectively. The ability of an anion to cross a lipid

bilayer depends on the lipophilic nature of the anion; order-

ing the anions used from most lipophilic to least lipophilic

is as follows: thiocyanate > chloride > sulfate. Hence, the

order of effectiveness for these anions in increasing trans-

port activity parallels their relative lipophilic properties

and illustrates the electrogenic nature of the process (more

lipophilic anions permeate the membrane more rapidly causing

an increase in membrane potential).


Trans-Inhibition of System A in Membrane Vesicles

The activity of System A is decreased considerably when

the cytoplasmic concentration of its substrates is elevated

(Kelley and Potter, 1978). This phenomenon, referred to as

"trans-inhibition", is cycloheximide-insensitive and gener-

ally is thought to occur because the amino acids bind to the

carrier and "lock" it in the cytoplasmic orientation (White






51

TABLE 2-3

Effect of Gramicidin or Monensin on AIB Uptake

Membrane vesicles (2.5 mg protein/mi) were incubated at
220C for 15 min with either 2% ethanol, 20 pg/ml gramici-
din in 2% ethanol, or 20 pg/ml monensin in 2% ethanol. The
suspensions were then diluted (1:1) into either Na or
K -uptake buffer containing 200 pM of radioactively-
labelled AIB. The transport was measured in the usual
manner as described in the text. The velocities are
expressed as pmol AIB per mg protein per 6 min and are the
averages + S.D. of three individual assays.



Additions Alkali ion Velocity Na+-dependence

Ethanol Na+ 232 + 8 118

Ethanol K+ 114 + 16


Gramicidin Na+ 146 + 3 29

Gramicidin K+ 117 + 13 ---


Monensin Na+ 130 + 5 8

Monensin K+ 122 + 6 ---








and Christensen, 1983; Kilberg et al., 1985a). An alternate

explanation for trans-inhibition is possible. In response

to increased cellular levels of System A substrates, the

carrier may be rapidly internalized by a process analogous

to the response observed for the adipocyte glucose carrier

after removal of insulin from the medium (Lienhard, 1983;

Simpson and Cushman, 1985). Likewise, if the elevated amino

acid levels were decreased, the carrier would be shuttled

back to the plasma membrane location resulting in a protein

synthesis-independent increase in transport. The latter

response, referred to as "release from trans-inhibition",

has been demonstrated with intact hepatocytes by several

laboratories. This "shuttle hypothesis" is made even more

plausible by recent evidence for a serum-dependent shuttling

of System A carriers in human fibroblasts (Gazzola et al.,

1984).

Isolated plasma membrane vesicles provide an excellent

model system to test whether trans-inhibition involves inter-

nalization of endocytic vesicles, and therefore requires in-

tact cells. If the phenomenon can be demonstrated in plasma

membrane vesicles, the results would argue that trans-

inhibition is, indeed, a membrane-associated response and

not dependent on extensive cellular machinery or architec-

ture. The results of Table 2-4 show that vesicles previ-

ously loaded with MeAIB exhibited a 26% reduction in measur-

able System A activity. When 0-glutamine was used, an amino

acid with little or no saturable transport in hepatocytes,

no decrease in activity was observed. These data indicate








TABLE 2-4

Trans-Inhibition in Isolated Plasma Membrane Vesicles

Membrane vesicles (5 mg of protein) were incubated at 4C
for 2 h in Buffer A containing either no additions (con-
trol), 25 mM D-glutamine, or 25 mM MeAIB. After the incuba-
tion period, 0.5 ml of ice-cold Buffer A was added to each
condition. The samples were immediately vortexed and centri-
fuged in a microcentrifuge (15,000xg) for 5 min at 40C.
The supernatants were removed and 1 ml of ice-cold Buffer A
was added back without disturbing the pellet. The tubes
were centrifuged again in the microcentrifuge at 40C for 2
min. The supernatants were removed and the pellets were
resuspended in 200 pl of ice-cold Buffer A. The uptake of
200 pM MeAIB for 1 min at 220C was immediately tested as
described in the Methods section. The velocities are
expressed as pmol MeAIB per mg protein per min and are repor-
ted as the averages + S.D. of triplicate determinations.
The values in parentheses are the percent of control for
the Na -dependent velocity. Theoretical calculations
indicate that leakage of MeAIB into the extravesicular space
could not account for the degree of inhibition observed.
Actual determinations of the extravesicular concentration of
MeAIB were not performed and the MeAIB trapped in the extra-
vesicular space may contribute to the inhibition observed.

Loading Alkali Total Na+-dependent
Condition Ion velocity velocity



Control Na+ 173 + 4 136 (100)

K+ 37 + 3


25 mM D-glutamine Na+ 166 + 14 136 (100)

K+ 30 + 8 --


25 mM MeAIB Na+ 130 + 9 101 (74)*

K+ 29 + 7 ---


* p < 0.025








that trans-inhibition can occur in isolated membrane

vesicles and probably does not depend on internalization of

the System A carrier as is observed for the down-regulation

of the insulin receptor. It is important to note that these

data could also be explained by competitive inhibition of

AIB uptake caused by MeAIB trapped in the extravesicular

volume. The data should be regarded with caution until the

concentration of MeAIB in the extravesicular volume is

actually determined.


Effect of Glucagon-Treatment in vivo and Amino Acid-
Starvation in vitro on System A Activity in Plasma Membrane
Vesicles

It is well documented that glucagon-treatment, either

in vivo (Handlogten and Kilberg, 1984) or in vitro

(LeCam and Freychet, 1976; Pariza et al., 1976), causes a

protein synthesis-dependent increase in hepatic System A

transport activity. Our laboratory has provided evidence

that the hormone-induced molecule responsible for the stim-

ulation of System A activity is a glycoprotein (Barber et

al., 1983). The stimulation by glucagon is generally consid-

ered to be the result of de novo synthesis of carriers or

carrier-associated molecules and their subsequent insertion

into the plasma membrane. To determine whether the System

A-associated glycoprotein induced by glucagon is also

located in the plasma membrane, the System A activity in

membrane vesicles from hepatocytes taken from either normal

or glucagon-treated rats was first tested using thiocyanate

as the counter-anion in the uptake buffer, but the








Na+-dependent overshoot was too rapid to measure. In

order to demonstrate the peak of Na -dependent AIB uptake,

sulfate was used to replace thiocyanate as the counter-anion

in the uptake buffers. The lower lipophilicity of the

sulfate anion slowed the Na -dependent uptake so that the

peak of the Na+-dependent overshoot could be measured

accurately.

Fig. 2-8 illustrates the time course for the Na -

dependent uptake of AIB in normal or glucagon-treated vesi-

cles*. It is evident that the Na+-dependent AIB

uptake in the glucagon-stimulated vesicles is increased at

least 2-fold over the normal vesicles. The System A activ-

ity peaks at approximately 3 min in the control membranes,

whereas the maximal uptake occurs at only 1 min in the

glucagon-treated vesicles. The higher rate of decline of

the Na -dependence in the glucagon-treated vesicles

compared to the normal vesicles is probably due to either a

more rapid dissipation of the trans-membrane Na+ gradi-

ent or a Faster equilibration of AIB across the membrane.

All subsequent assays of AIB transport that were designed to

make comparisons between control and hormone-treated vesi-

cles were performed for 15 s in sulfate-containing buffers.

These data indicate that the glucagon-induced activity is

retained in the membrane vesicles isolated from those same

cel s.


For brevity, the vesicles prepared from the hepato-
cytes that were isolated from the glucagon-injected rats
will be referred to as "glucagon-treated vesicles".





















Fig. 2-8. System A Activity in Plasma Membrane Vesicles
from Control or Glucagon-Injected Rats. Membrane vesicles
were isolated from control rats (A) or rats injected with 1
mg glucagon/100 g body weight 4 h prior to membrane isola-
tion ( V). Following the isolation, the vesicles were
immediately tested for Na -dependent AIB transport as
described in the section on Materials and Methods using
either 60 mM Na SO4 or K SO4 uptake buffer.
The time of uptake at 22 C was varied from 10 s to 30 min.
The results are expressed as the averages of triplicate
determinations and the standard deviations, omitted for
clarity, were generally less than 10%.















.5 300

2 250

a 200
E
A 150

o 100
E
50


0
0


Minutes








The System A activity in primary cultures of rat hepato-

cytes is also induced if the cells are incubated in an amino

acid-free medium (Kelley and Potter, 1978). Like glucagon-

dependent stimulation, this process, referred to as adaptive

regulation, is thought to result from increased synthesis of

a System A-associated glycoprotein located in the plasma mem-

brane (Barber et al., 1983). To test this hypothesis, cul-

tured hepatocytes were incubated for 6 h in amino acid-free

medium (NaKRB) or NaKRB supplemented with 20 mM asparagine

and then membrane vesicles were prepared from those cells.

The System A activity was enhanced nearly 6-fold in the vesi-

cles from the starved cells. The rate of Na -dependent

AIB uptake in the vesicles from starved (no amino acid) and

fed (20 mM asparagine) cells was 189 + 14 and 33 + 6

pmol per mg protein per min, respectively. The degree of

induction for System A transport activity measured in intact

cells is generally 5- to 10-fold.

After several different membrane preparations had been

tested for the level of glucagon-dependent stimulation of

Na -dependent AIS transport, it appeared as though the

amount of hormone-induced activity for intact cells was not

reflected in the membrane vesicles. To monitor the degree

of hormone stimulation, hepatocytes from glucagon-injected

rats were assayed for System A activity and then plasma mem-

brane vesicles were isolated from the same preparation of

cells. The data shown in Table 2-5 are representative of

many experiments. In this instance, the intact cells showed

a 30-fold increase in System A transport following glucagon






























c E 0 +u =
SIU C- 4-

S- L 0 > 0 *04-

.> /) ( >L > 0



+ I- O ) >C 0

A- .0 in f Q .0
CC0 a L L O



i ITto< ONC:,
o C- m CS m C

i I,- *-

o-'1 E 0 r- L -



w Iom ouo
0S >CL L.C ( CL C






Cl V C O Ea -C **
SCv S0 ) C 3 -


MC W v





0 .E* *- E (0 0- 4 0
E CO C 4- +' 4)
4- E U CO er ,
CL 0 0U C L O4 VI C-
0I L3 CM 0 ) LO U



3 00 C 0 J O 0



C 4L" L 0 C C C 0
w- O U U C C U
O UO CO0 4




= QC'o = 0CC


C L C ( +O o0
*- 3 Oy C-r-- (D ^ "I O


S- oa o E c o 0) C
C E L. S E- S C

(0 **- Q.r- L. **~ C ET0 .) 40
4- 4- l.. OT 0 0 C t0
C j c 1) 4-' C' c





o U I C- Ca 3 o








f 4-) 4.4 +/1 l fl' 4
C J t 43 C c '- )4 o

S r -*r- Lcon o 4,

+U > (J T3 u
C/ < CC *~ *C- C .
+ CE C1)0 0 1.. 3 -
f0 4 L. U *C .C to


oCE*-r +> */ cu>
.C '4- CI 4-)Ct)3 30)


4J

u
0

r_


0











0(
u

CO >
I-









'I4-
0
C















Ct
0











E
I-









C
O











C
O3


e-
LD


a)1 V
C: 01


)- .0 ,-
4- EV
=oM oE :
- U








treatment, whereas the membrane vesicles showed a 13-fold

change.

The apparent incomplete retention of stimulated System

A activity in the membrane vesicles compared to the intact

cells may be due to a difference in the composition of the

control and glucagon-treated vesicles with respect to the

three domains known to exist on the hepatocyte surface

(Wisher and Evans, 1975; Hubbard et al., 1983). It has been

reported that Na+-dependent alanine transport activity is

about equally distributed on the cell surface (Van

Amelsvoort et al., 1980; Meier et al., 1984b), but these

studies were not performed on hormone-induced membranes.

We assayed plasma membrane marker enzymes to determine

if the amount of canalicular and basolateral membrane in the

control and glucagon-treated vesicles were similar. The

control vesicles showed an enrichment of 6-fold for 5'-

nucleotidase canalicularr) and 2-fold for fluoride-stimula-

ted adenylate cyclase (basolateral) when compared to the

homogenate, whereas the vesicles from glucagon-treated rats

showed a 9-fold enrichment of 5'-nucleotidase activity and a

5-fold enrichment of fluoride-stimulated adenylate cyclase

(Table 2-6). These data indicate that there are differences

in the absolute content of canalicular and basolateral mem-

brane between the two vesicle populations, but the glucagon-

treated vesicles actually show a higher degree of enrichment

for both markers. The ratio of the 5'-nucleotidase activity

to the fluoride-stimulated adenylate cyclase activity

appears to be similar for both preparations (control = 2.5,






61

TABLE 2-6
Enzyme Activities in Membranes from Control and
Glucagon-Treated Hepatocytes
Membrane protein (100 pg) was analyzed for 5'-nucleotidase
and adenylate cyclase activity as described in the Methods
section. Values for 5'-nucleotidase activity are expressed
as pmol P formed per mg protein per h, whereas those
for adenylate cyclase activity are expressed as nmol cAMP
formed per mg protein per h. Adenylate cyclase was stimula-
ted using 10 mM NaF. The non-stimulated level of adenylate
cyclase ranged from 0.02-0.05 and 0.13-0.29 in the homogen-
ate and membrane vesicles, respectively, and has been sub-
tracted from the results to yield the data shown. The
results are given as the averages of three determinations
and the standard deviations were typically less than 15%.

Donor Rat Homogenate Membrane Enrichment
Enzyme Tested Vesicles

Control

5'-nucleotidase 2.10 12.0 5.7


F--stimulated
adenylate cyclase 0.26 0.60 2.3

Glucagon-treated

5'-nucleotidase 1.90 17.3 9.1


F--stimulated
adenylate cyclase 0.22 1.11 5.1






62

glucagon-treated = 1.8). Hence, a difference in the compo-

sition with respect to plasma membrane domains is probably

not responsible for the decreased glucagon-dependent stimula-

tion reported in Table 2-6.


Inactivation of Glucagon-Stimulated System A Activity

The decreased retention of stimulated System A activity

in membrane vesicles might also be explained if the System A

carrier complex is irreversibly inactivated during the iso-

lation procedure. In order to test for inactivation,

freshly isolated membrane vesicles from normal or glucagon-

treated rats were incubated at 4*C for 48 h. At specific

intervals, the System A activity was assayed in both vesicle

preparations. The results indicate that at 40C there is a

time-dependent loss of System A activity in the glucagon-

treated vesicles (Fig. 2-9). The decay of the hormone-

stimulated transport consisted of a single exponential compo-

nent. The preparation of the membranes requires about 6 h

and we assume that the decay was ongoing during that time.

The value for the half-life of the decay was approximately

13 h. In several studies, maintenance of the glucagon-

stimulated vesicles at -70C for 48 h protected most, if

not all, of the initial activity (Fig. 2-9). In contrast,

the basal rate of System A-mediated uptake seen in the con-

trol vesicles did not change during a 24 h incubation at

4C (Fig. 2-9).

A mixture of 2 mM PMSF, 2 mM EDTA, trasylol (30 trypsin

inhibitor units/ml), 0.1 mM leupeptin and 0.5 mM bacitracin





















Fig. 2-9. Decay of System A Activity in Membrane Vesicles
Incubated at 40C or -70C. Isolated membrane vesicles
either from glucagon-treated (*,V) or control rats(A) were
incubated at 40C (AV) or -70 ( ) for the indicated
times. Vesicles stored at -70C were in aliquots and were
thawed only once prior to assay. At the indicated times,
Na -dependent AIB uptake was measured as described in
Materials and Methods using either 60 mM Na SO or
K SO uptake buffer. The results are expressed as a
percent of the initial Na -dependent uptake in the
glucagon-treated vesicles (423 + 42 pmol AIB accumulated
per mg protein per 15 s) and are averages of triplicate
determinations. Standard deviations are typically less than
20% of the mean.



















100
o

u 75

0.
c
e 50


25



0 10 20 30 40
Hours








added to the vesicle suspension did not slow the decay of

System A activity in the membrane vesicles maintained at

4C. Dithiothreitol (1 mM) also did not have any effect on

the decay of transport activity. Similar results were

obtained if the vesicles were subjected to a freeze-thaw

cycle in the presence of the inhibitors to trap them inside

the intravesicular space.

To determine if the degradative process observed was

specific for System A, we tested the stability of Systems N

and ASC in control and glucagon-treated vesicles during incu-

bation at 40C. The glucagon-treated vesicles showed simi-

lar patterns of decay for Systems A and N (Fig. 2-10A).

Transport by System A and System N was enhanced in the

glucagon-treated vesicles when compared to the control mem-

branes. Interestingly, these data are consistent with ear-

lier observations in cultured hepatocytes suggesting that

System N is stimulated by glucagon in vivo (Barber et al.,

1983) but not in vitro (Kilberg et al., 1980). The esti-

mated half-life of the decay of the hormone-stimulated activ-

ity of System A and N was approximately 13 h. It is diffi-

cult to be certain if System ASC is decaying either in the

glucagon-treated vesicles or in the control vesicles due to

the low basal rate of activity. Membrane vesicles isolated

from control hepatocytes also appeared to show decay of

Systems A and N (Fig. 2-10B). The half-life value for

System N was calculated to be about 13 h; however, in the

case of Systems A and ASC these measurements were too impre-

cise to report due to the low level of activity present.





















Fig. 2-10. Decay of Systems A, N, and ASC in Vesicles from
Glucagon-Treated and Normal Hepatocytes. Membrane vesicles
from glucagon-treated (A) or normal control rats (B) were
incubated at 4*C for the indicated times. After each period
of incubation, the vesicles were tested for amino acid
transport activity as described in Fig. 2-3. System A (*),
System N (a), and System ASC (A) were tested as described
in the Methods section. The results are expressed as the
mean + S.D. of triplicate determinations of the
Na -dependent pmol of amino acid accumulated per mg pro-
tein per 15 s. Where not indicated, the error bars are con-
tained within the symbols.


























S800
Pn

o 600

0
S400oo
E


I 200




0 5 10 15 20


0 5 10 15 20
Hours








System A Transport Activity in Membrane Vesicles from Human
Hepatoma Cells
Several investigators have reported that glycolysis and

nutrient transport activities are increased in transformed

cell lines (Parnes and Isselbacher, 1978). Amino acid trans-

port, in particular, is increased in membrane vesicles pre-

pared from SV40-transformed 3T3 cells (Lever, 1976). Our

laboratory has studied System A activity in a number of hepa-

toma cell lines from both human and rat. For example, a

human hepatoma cell line (HepG2) shows an increased basal

transport rate of nearly 12-fold when compared to the normal

hepatocyte (38.6 + 6.1 versus 444 + 22 pmol AIB per

mg protein per min for the normal and hepatoma cells, respec-

tively). To test whether or not the increased System A-

mediated transport activity observed in the human hepatoma

cells is retained in isolated membranes prepared from HepG2

cells, several membrane vesicle fractions were prepared as

described in the Methods section. Each of the subcellular

membrane fractions tested displayed Na -dependent AIB

transport activity (Fig. 2-11). The P1 fraction (l000xg

pellet) showed a sodium-dependence of 137 + 18 pmol AIB

per mg protein per min, while the P2 (45,000xg pellet) and

the P3 fractions (discontinuous sucrose gradient pellet)

showed 614 + 24 and 631 + 18 pmol AIB per mg protein

per min, respectively (Fig. 2-11). It appears, from these

results, that either sucrose-gradient centrifugation or a

differential centrifugation procedure at 45,000xg will

result in a similar level of specific activity for System A-

mediated transport. The data from either of these two






HOMOGENATE (NO)




II
1000XG CENTRIFUGATION



S1 (ND) P1 (137 + 18)



45,000XG CENTRIFUGATION



S2 (0) P2 (614 + 24) SUCROSE-STEP
GRADIENT
50,000XG
CENTRIFUGATION


I
P3 (631 + 18)

Fig. 2-11. Flowchart of HepG2 Membrane Preparation. HepG2
membranes were prepared as shown above and described in the
Methods section. The supernatant fractions (S) and the
pellet fractions (P) are indicated for each step. Fractions
which were tested for System A transport activity are indi-
cated in parentheses as the Na -dependent pmol of AIB
accumulated per mg protein per min + S.D. for triplicate
determinations. Fractions which were not tested for activity
are indicated by NO.








fractions indicate that System A activity is greater in

membrane vesicles from human hepatoma cells when compared to

the transport observed in vesicles from normal rat

hepatocytes (Fig. 2-8). When vesicles from the HepG2 cells

were prepared by similar centrifugation methods, but using

either bath sonication or nitrogen cavitation for cellular

disruption, the results were the same. Furthermore,

membrane vesicles from a rat hepatoma, H4-II-EC3, also

retained elevated rates of System A activity (data not

shown). These data argue that the increased transport

activity observed in transformed liver cell lines is

retained in membrane vesicles prepared from those cells.



Discussion


Membrane vesicles have been prepared from either rat

liver tissue or isolated rat hepatocytes. These vesicles

actively accumulated AIB and more than 85% of the AIB accumu-

lation was inhibited by the System A-specific analog, MeAIB.

These data indicate that AIB is useful as a selective probe

for System A activity in isolated membrane vesicles. The

accumulation of AIB was energized by the imposed trans-

membrane Na gradient. The uptake of AIB was against a

concentration gradient as indicated by the distribution

ratio which was greater than one at the maximal overshoot

point.

The vesicles also actively accumulated typical sub-

strates of the other sodium-dependent amino acid transport








systems found in rat liver. Alanine, cysteine, histidine,

and glycine were used as model substrates for Systems A,

ASC, N, and Gly, respectively. The specificities of these

amino acids for their respective transport systems were simi-

lar to those observed in intact hepatocytes (Kilberg, 1982).

The only notable difference was that Na -dependent hist-

idine uptake showed a greater degree of inhibition by MeAIB

in membrane vesicles than in whole cells (Kilberg et al.,

1980).

Several of the criteria that Lever (1980) has applied

to establish active transport of nutrient molecules by mem-

brane vesicles were tested in this system. Uptake of alan-

ine and histidine was stereospecific and AIB was actively

accumulated into an enclosed, osmotically-sensitive space.

AIB uptake was also dependent on temperature and protein con-

centration. AIB uptake was inhibited by the ionophores gram-

icidin and monensin which collapse the trans-membrane

Na+ gradient by either forming channels for Na+ ions

gramicidinn) or carrying Na+ ions across the membrane

(monensin). The electrogenicity of transport was demonstra-

ted by observing that the use of less permeant counter-

anions in the buffer resulted in lower transport velocities.

The membrane vesicle preparation was shown to be

enriched in plasma membrane through measurements of 5'-

nucleotidase and adenylate cyclase activities. Enzyme

marker studies indicated that the vesicles had some contamin-

ation by microsomes, but little or no contamination by mito-

chondria. Collectively, the results demonstrate that these






72

membrane vesicles are useful for amino acid transport stu-

dies by providing a system free of many of the restrictions

and complications imposed by intact cells. The charac-

teristics that we have described for this vesicle system are

consistent with those of other amino acid transport studies

using isolated rat liver membranes (Sips et al., 1980b;

Samson and Fehlmann, 1982; Quinlan et al., 1982; Meier et

al., 1984b). The demonstration of active amino acid trans-

port and its properties represents the first step toward

using isolated rat liver membranes to study the regulation

of System A by trans-effects, hormones, and adaptive con-

trol.

Membrane vesicles facilitate the study of trans-

inhibition of System A because, presumably, endocytosis and

recycling cannot occur due to the removal of intracellular

ultrastructures such as the microtubular network. Using

these membrane vesicles, trans-inhibition was detected when

the vesicles were loaded with AIB but not when the vesicles

were loaded with D-glutamine, an amino acid with no satur-

able Na -dependent uptake. Our results support the hypo-

thesis that the System A carrier is locked into an internal

orientation by binding of AIB (or a Na /AIB complex) at

the intracellular surface of the plasma membrane.

It is well established that glucagon treatment and star-

vation of hepatocytes for amino acids results in a protein

synthesis-dependent increase in System A activity (Kilberg,

1982; Shotwell et al., 1983). The increase observed in both

cases is kinetically defined as an increase in the Vmax of








the carrier rather than a change in the Km for the test sub-

strate (Shotwell et al., 1983). Taken together, the kinetic

effects and the protein synthesis-dependency are interpreted

to indicate that the stimulated transport activity is due to

a greater number of active carrier molecules in the plasma

membrane. We have utilized isolated membrane vesicles to

provide additional support to that proposal. Vesicles iso-

lated from amino acid-starved hepatocytes displayed elevated

System A transport at a similar magnitude to that observed

in intact cells, a result that is consistent with the hypo-

thesis.

A large number of experiments were performed in which

rats were treated with glucagon in vivo and then membrane

vesicles were prepared from isolated hepatocytes. Increased

transport activity was always detected in the freshly iso-

lated vesicles and the level of stimulation paralleled, but

typically did not equal, the degree of induction observed in

the intact hepatocytes. Several explanations are possible

for the apparent incomplete retention of glucagon-stimulated

activity. We have eliminated some of these through experi-

mentation including differences in membrane composition of

the vesicles preparations from normal and glucagon-treated

cells and differences in the time at which the maximal

Na+-dependent transport is observed. It is also clear

from our studies that irreversible inactivation of System A

activity in the membrane vesicles from hormone-treated hepa-

tocytes contributes in a significant way to the loss of ele-

vated transport activity in the isolated membranes.








When Systems N and ASC were tested for inactivation in

membrane vesicles, System N decayed with a half-life similar

to that for System A. It could not be determined accurately

if System ASC was decaying at a significant rate. These

data imply that the inactivation process affects other trans-

porters as well as System A and may, in fact, result from a

non-specific effect on vesicle integrity. It cannot be

determined accurately if the inactivation process is also

occurring in vesicles from normal cells because the data at

each time point are not statistically different from each

other. Another unknown is the status of the membrane perme-

ability with respect to Na An increase in permeabil-

ity to Na+ ions could result in an apparent inactivation

of the carrier.

Membrane vesicles isolated from human hepatoma cells

(HepG2) showed increased System A activity when compared to

normal rat hepatocytes. Such a comparison is difficult

because we do not know the basal activity of transport in

normal human liver tissue. However, it is interesting to

note that the level of Na -dependent AIB uptake in HepG2

vesicles is comparable to the transport activity in

glucagon-induced rat liver vesicles. Our laboratory has

made direct comparisons between normal rat hepatocytes in

culture and several rat hepatoma cell lines. In every case,

the hepatoma cells contain enhanced transport activity. The

data obtained with isolated membrane vesicles are consistent

with the hypothesis that transformation induces new System A

carriers to be inserted into the plasma membrane, similar to






75

the proposed mechanism for glucagon-induction and adaptive

control of System A. Ideally, one would like to test System

A activity in a particular cell line before and after trans-

formation. Such an experiment was done using Rat-1 cells

transformed by the myc oncogene (Racker et al., 1985) and

NRK-49F cells transformed by transforming growth factor-B

(Boerner et al., 1985). Each cell line exhibited increased

System A activity following transformation; the induction

required de novo protein synthesis.
















CHAPTER III
RECONSTITUTION OF SYSTEM A TRANSPORT ACTIVITY INTO
ARTIFICIAL PROTEOLIPOSOMES



Introduction

Reconstitution of ion transport systems has proven to

be a powerful method of resolving the transporters into

their functional components. The elegance of taking a

multi-protein complex apart, separating it into its indivi-

dual components, and then recombining the components to

regain activity is apparent for the mitochondrial ATPase

(Racker, 1976). A number of comprehensive reviews have

appeared over the last few years that discuss various

methods of performing reconstitution of ion transporters or

membrane receptors (Levitzki, 1985; Eytan, 1982; Hokin,

1981; Racker, 1979).

Reconstitution of transport processes is a technique

that must be developed in a series of stages. The first

stage is the establishment of a membrane source that con-

tains large quantities of activity. The second stage is the

demonstration of transport activity in an artificial proteo-

liposome system by combining membrane fragments and sonica-

ted liposomes (membrane-fragment reconstitution). The final

stage requires removal of the transporter from its native

76








membrane by detergent-extraction. Reconstitution is

achieved by combining the extracted proteins with lipid and

then removing the detergent. Recovery of activity from an

inactive detergent-extract is a convincing argument for

reconstitution.

Several methods of reconstitution after detergent-

extraction of membrane proteins have proven to be highly

successful. The first reconstitution of oxidative phosphor-

ylation was performed using a cholate-dialysis technique

(Kagawa and Racker, 1971). The technique slowly removes

detergent by dialysis against detergent-free buffers which

facilitates the insertion of proteins into liposomes. Since

1971, a wide variety of transport systems have been reconsti-

tuted using detergent-dialysis techniques (Racker, 1979).

Although these methods have been useful for many systems,

they require the proteins to be in contact with detergent

for long periods of time (18-98 h) often resulting in inacti-

vation.

To circumvent the inactivation problem caused by long

periods of dialysis, rapid reconstitution techniques were

devised. One example is the detergent-dilution procedure

which has been useful for the reconstitution of cytochrome

oxidase activity (Racker, 1972) and the reconstitution of

coupled oxidative phosphorylation (Racker, 1975). The tech-

nique is simple and rapid in that it only requires dilution

of the detergent-extract so as to lower the detergent concen-

tration below its critical micellar concentration (CMC)








followed by centrifugation to collect the resulting

proteoliposomes.

Another rapid technique to remove detergents with

micelle molecular weights less than 10 kDa, such as cholate

and deoxycholate, was described by Brunner et al. (1976).

The procedure requires passing the detergent-extract over a

Sephadex G-50 column. The gel filtration matrix retards the

small detergent molecules and allows the large lipid and pro-

tein molecules to fuse into proteoliposomes and pass through

in the void volume. McCormick et al. (1984) used this proce-

dure prior to a freeze-thaw step to achieve reconstituted

amino acid transport activity from EAT cell membranes.

A procedure which has shown great success for the glu-

cose transporter from red blood cells (Kasahara and Hinkle,

1976) requires a freeze-thaw step coupled with sonication.

This technique involves combining sonicated lipid with solu-

bilized membrane proteins followed by rapid freezing in a

dry-ice-ethanol bath. After thawing at room temperature,

the mixture is sonicated for short periods of time resulting

in a 3-fold greater activity than without the sonication.

Alfonso et al. (1981) have observed that the activity of the

mitochondrial proton-pump is increased even further when

small amounts of cholate are added before the freeze-thaw

step. Pick (1981) suggests that the fusion process which

occurs during the rapid freezing and thawing is a result of

the formation of crystalized water molecules in two frozen

planes separated by the membrane domain. The bilayer is








easily fractured, exposing hydrophobic regions which can

fuse to form large liposomes.

Another technique used successfully to achieve recon-

stitution of transport processes is the Ca+ -fusion pro-

cedure. This alternate procedure capitalizes on the observa-

tion that liposomes which contain phosphatidylethanolamine

and phosphatidylserine or cardiolipin rapidly fuse in the

presence of Ca+2 (Miller and Racker, 1976). This tech-

nique has proven useful for reconstitution of cytochrome

oxidase activity from mitochondria and has also found wide

use in drug delivery systems (Szoka and Papahadjopoulos,

1980).

Reconstitution of Na -dependent amino acid trans-

port activity has been reported in kidney brush border mem-

branes (Kinne and Faust, 1977; Takahashi et al., 1985),

SV-40-transformed 3T3 fibroblasts (Nishino et al., 1978),

and EAT cell membranes (McCormick et al., 1984; Cecchini et

al., 1977). Reconstitution of amino acid transport activity

from kidney brush border membranes has been achieved,

although the level of Na -dependence was low. Kinne and

Faust (1977) extracted membranes with Triton X-100, then

removed excess Triton by passing the detergent-extract over

Bio-Bead SM-2 columns. The detergent-extract was reconsti-

tuted by combining it with lipid and sonicating the mixture.

Sodium-dependent alanine uptake was quantitated, but less

than a 2-fold difference was reported for uptake in Na+

versus K -containing buffers. The authors did not dis-

cuss the margin of error present in the assays so the data






80

may not be statistically different. The activity reported

may not be representative of System A activity because

Na+-dependent alanine uptake may be through several dif-

ferent systems including System ASC.

Reconstitution of System A from SV-40-transformed mouse

fibroblasts membranes, extracted with 2% cholate was accom-

plished by Nishino et al. (1978), by passing the extracted

proteins over a Sephadex G-50 column, and then subjecting

them to a freeze-thaw step. Sodium-dependent AIB uptake was

determined by a rapid filtration technique and was reported

to be at least 3-fold higher in sodium thiocyanate than in

choline chloride.

Reconstitution of System A in EAT cell membranes has

been reported after cholate-extraction followed by detergent

dilution (Cecchini et al., 1977) or cholate-extraction fol-

lowed by dialysis (Bardin and Johnstone, 1978). Later

reports from Johnstone's group demonstrated the existence of

Na -dependent AIB uptake after extraction of EAT mem-

brane proteins in 2.5% cholate/4 M urea followed by dialysis

to 0.25% cholate, Sephadex G-50 chromatography, and a

freeze-thaw step (McCormick et al., 1984).

Little progress has been made toward the purification

of the System A carrier using reconstitution as an assay.

Cecchini et al. (1978) reported that an EAT cell membrane-

extract retained Na -dependent alanine uptake after

ammonium sulfate fractionation, Biogel P-60 chromatography

and DEAE-cellulose chromatography. Polyacrylamide gel elec-

trophoresis of the final extract showed only 15 or so








Coomassie-staining bands. McCormick et al. (1985) reconsti-

tuted Na+-dependent AIB transport from EAT cell mem-

branes that had been extracted with cholate/urea as

described above and showed an apparent enrichment in a pro-

tein of 125 kDa in the proteoliposomes when compared to the

native plasma membranes. Using reconstitution as an assay,

Takahashi et al. (1985) have determined by radiation-

inactivation studies that the target size for the Na -

dependent glucose transporter from kidney brush border mem-

branes is one-million daltons and 1.2 million daltons for

the Na+-dependent alanine transporter.

The object of the present studies was to devise a

rapid, efficient system to reconstitute System A activity

from EAT cell membranes. Techniques for reconstitution of

System A described by McCormick et al. (1984) and Cecchini

et al. (1978) were attempted in our laboratory and in the

laboratory of Dr. Efraim Racker but no reproducible activity

could be obtained. In collaboration with Dr. Racker, we

developed a novel reconstitution technique for System A

activity which was rapid and reproducible. Having estab-

lished the technique, future research in the laboratories of

both Dr. Kilberg and Dr. Racker will focus on the purifica-

tion of the transporter using reconstitution as an assay.






82

Materials and Methods


Materials

Cholic acid was obtained from Sigma Chemical Company

and was recrystallized three times in ethanol as described

by Kagawa and Racker (1971). Ultrapure urea was obtained

from Pierce. Protein concentration by ultrafiltration in a

stirred cell was performed using an Amicon model 8010 or

8050 cell fitted with a YM-30 membrane. Asolectin was

obtained from Associated Concentrates and was stored at

-200C. (U-14C)-sucrose in 20% ethanol was obtained

from ICN. The detergent NP-40 was obtained from Particle

Data Laboratories, Inc. and octyl-glucoside was supplied by

Calbiochem. Hydroxylapatite (Bio-Gel HTP) and all reagents

for electrophoresis were obtained from Bio-Rad. All experi-

ments requiring sonication were performed at 220C in a bath

sonicator supplied by Laboratory Supplies Co., Inc. All

other reagents were obtained from Sigma Chemical Company.


Preparation of EAT Cell Membrane

EAT cell membranes were prepared as described by Racker

et al. (1984). 75 Swiss white mice (Charles River--19-21 g)

were injected intraperitoneally with 0.25 ml of EAT cell sus-

pension which had been removed from mice and filtered

through two layers of cheesecloth. After 8-10 days, the

mice were sacrificed by cervical dislocation, the intraperi-

toneal cavity was carefully opened, and the ascites fluid

was collected and filtered through two layers of cheese-








cloth. The ascites fluid was typically whitish-yellow in

color and was not tinged at all with blood. Any bloody

fluid was discarded. The cells were diluted in 10 volumes

of wash buffer (140 mM NaCl, 5 mM KC1, 1 mM MgCl2, 10 mM

HEPES, pH 7.4) and placed in GSA rotor bottles. The cell

suspension was centrifuged at 650xg in a GSA rotor for 5

min. The supernatant was decanted and the pellets were

resuspended in a small volume of wash buffer. The pellet

suspension was centrifuged again at 650xg for 5 min (table-

top centrifuge) in order to pack the cells. The cells were

frozen in 45 ml centrifuge tubes and stored at -70C. Fro-

zen cells (150 g) were used for each preparation of mem-

branes. The frozen cells were thawed in warm water and

poured into 1.5 L of 10 mM CaC12 (dihydrate; Fisher ACS,

pH 8.0) at room temperature. The cells were stirred slowly

at 40C for 5 h and were homogenized by rapidly forcing

through a Logeman homogenizer with the nozzle half closed

(this and all subsequent steps were done at 40C). The homo-

genate was poured into 6 GSA rotor bottles and centrifuged

at 650xg for 5 min. The pellets were discarded and the

supernatant was collected and centrifuged at 16,000xg for 40

min in the GSA rotor. The pellets were gently homogenized

in 30 ml of sucrose-EDTA-PMSF buffer (0.3 M sucrose, 1 mM

EDTA, 1 mM PMSF, pH 7.4) using a typewriter brush and a

loose-fitting Potter-Elvehjem homogenizer. The suspension

was incubated on ice for 1 h then it was centrifuged at

27,000xg for 30 min. The supernatant was decanted and the

pellet was resuspended in 30 ml of sucrose-EDTA-PMSF as








before. The pellet was gently homogenized and centrifuged

again at 27,000xg for 30 min. The pellet was resuspended as

before and centrifuged at 27,000xg for 30 min. The final

pellet was resuspended in a small volume of sucrose-EDTA-

PMSF and frozen at -700C in 0.5 ml aliquots (total yield =

200-300 mg per 150 g of packed cells).


Detergent-Extraction of EAT Cell Membrane

EAT cell membrane was extracted with detergent as

described in the figure legends or in the text. The details

of the protocol for optimized extraction of the EAT mem-

branes using cholate and urea by the technique of McCormick

et al. (1984) is described here. Twenty mg of EAT cell mem-

brane protein was mixed with 10 ml of solubilization buffer

(2.5% cholic acid, 4 M urea, 0.1 mM EDTA, 100 mM NaCl, 5 mM

Tris-HC1, pH 7.4) at a protein concentration of 2 mg/ml at

4C for 30 min. The mixture was centrifuged at 125,000xg

for 45 min. The supernatant was removed and placed in a dia-

lysis bag, then the suspension was dialyzed overnight

against 100 volumes of dialysis buffer (0.2% C12E9,

5 mM Tris-HCl, 0.1 mM MgCl2, 0.1 mM CaCl2, 100 mM

KC1, 1 pM PMSF, pH 7.45) at 40C. The dialysate could be

stored at -70C for up to one month with minimal loss of

activity. After the determination of protein content by a

modified Lowry procedure (Bensadoun and Weinstein, 1976),

the suspension was prepared for reconstitution.








Freeze-Thaw Reconstitution and Transport Assay

Reconstitution of transport activity was performed by

mixing 0.5 mg of protein from either the membrane-fragment

preparation or the detergent-extract, 10 mg of sonicated aso-

lectin (stock = 40 mg/ml) and 1.5 mg of K+ cholate

(stock = 10% w/v). The final volume of the mixture ranged

from 0.7-1.0 ml. The mixture was frozen in liquid nitrogen

and thawed at room temperature, then the suspension was

diluted with 2 ml of K+-uptake buffer (120 mM KC1, 10 mM

MgCl2, 10 mM HEPES, pH 7.45) and centrifuged at

125,000xg for 45 min. The pellet was resuspended in 200 pl

of K+-uptake buffer with a stirring rod and gentle vor-

texing. A summary of the reconstitution procedure, inclu-

ding further details, is shown in Fig. 3-1.

Amino acid transport buffers were prepared as 2X stocks

using NaCl (120 mM NaC1) or KCI (120 mM KC1) uptake buffer

and 200 pM AIB. To initiate uptake, 20 p1 of vesicles

were added to 20 pl of uptake buffer. The mixture was vor-

texed and incubated at 22C for 1 min. To stop the uptake,

1 ml of ice-cold stop buffer (154 mM NaCl, 10 mM

Na2HPO4, adjusted to pH 7.45 with HC1) was added and

the suspension was vortexed. The mixture was immediately

filtered over a Gelman nitrocellulose filter (25 mm diameter

and 0.45 pm pore size) and the filter was washed once with

3 ml of ice-cold stop buffer. The filter was placed into a

15 ml plastic scintillation vial and the radioactivity

trapped on the filter was determined after adding 5 ml of

Bray's scintillation cocktail. Transport activity is





86

ISOLATED MEMBRANE FRAGMENTS

C12E9 EXTRACTION CHOLATE/UREA EXTRACTION
I I
MEMBRANES STIRRED WITH MEMBRANES STIRRED WITH
0.2% C E 2.5% CHOLATE & 4 M UREA
FOR 30 MI~2A0 40C FOR 30 MIN AT 40C
\ I
CENTRIFUGE AT 125,000XG FOR 1 H
I I
SUPERNATANT (DETERGENT-EXTRACT) SUPERNATANT (DETERGENT-EXTRACT)
CONTAINS 20-30% OF THE PROTEIN CONTAINS > 85% OF THE PROTEIN

DIALYZE AGAINST 0.2% C12E
(DIALYSATE) 9

COMBINE:
DETERGENT-EXTRACT OR DIAYLSATE (0.5 MG PROTEIN)
ASOLECTIN (10 MG)
CHOLATE (1.5 MG)

FREEZE-THAW AND THEN DILUTE 1:10 WITH K+-UPTAKE BUFFER

WASH PROTEOLIPOSOMES AND THEN ASSAY SYSTEM A ACTIVITY

Fig. 3-1. Flowchart of the Procedure for Detergent-
Extraction and Reconstitution of System A Transport Activity
Using the Freeze-Thaw Technique.








expressed as pmol of AIB trapped per mg protein per min.

Unless otherwise indicated in the figure legends, the

results were from a single membrane preparation and the S.D.

of triplicate determinations was less than 10% of the mean.

Concentration of Detergent-Extracts Using Ultrafiltration

Detergent-extracts (1-2 mg protein/ml) were placed in

an Amicon ultrafiltration cell (10 ml or 50 ml size) fitted

with a YM-30 membrane. The apparatus was immersed in a tray

of ice water and the tray was placed on a magnetic stirring

plate. Nitrogen gas was introduced into the cell at 60 psi

while stirring was continued on ice. Concentration was

allowed to continue until the volume was reduced to 1-2 ml.

The concentrated extract was removed with a Pasteur pipette.


Sucrose Trapping in Reconstituted Proteoliposomes

Detergent-extract (0.5 mg protein/413 pl), 10 mg of

asolectin (250 pl) and 1.5 mg (15 pl) of K+ cholate

were combined with 5 pl (8.28 x 106 dpm) of 14C-

sucrose (0.746 pCi/pl; 1.21 mM) and then frozen in liquid

nitrogen. After thawing, the suspension was diluted with 2

ml of K+-uptake buffer and 100 u1 was removed for deter-

mination of radioactivity. A control sample was prepared in

which the 1C-sucrose was added after the freeze-thaw

step. Each suspension (100 pl) was passed over a Sephadex

G-50 column (1x16 cm; flow rate = 1.33 ml/min) equilibrated

with K+-uptake buffer. Fractions (0.22 ml) were col-

lected in plastic scintillation mini-vials and then analyzed




Full Text
Fig. 2-5. Effect of the Extravesicular Osmolar^ty on AIB
Uptake. Membrane vesicles were diluted into Na ()
or K -() uptake buffer containing sucrose from 0.2 to
0.9 M and radioactively-1abel1ed AIB (200 pM). Each of
these suspensions was divided into 3 tubes (50 pg each) and
incubated at 4C for 1 h. The accumulation of the amino
acid was assayed as described in the Methods section. The
data are the averages + S.D. of three determinations.


61
TABLE 2-6
Enzyme Activities in Membranes from Control and
Glucagon-Treated Hepatocytes
Membrane protein (100 pg) was analyzed for 5'-nuceoti dase
and adenylate cyclase activity as described in the Methods
section. Values for 51-nuc1eoti dase activity are expressed
as pmol P. formed per mg protein per h, whereas those
for adenylate cyclase activity are expressed as nmol cAMP
formed per mg protein per h. Adenylate cyclase was stimula
ted using 10 mM NaF. The non-sti mu 1ated level of adenylate
cyclase ranged from 0.02-0.05 and 0.13-0.29 in the homogen
ate and membrane vesicles, respectively, and has been sub
tracted from the results to yield the data shown. The
results are given as the averages of three determinations
and the standard deviations were typically less than 15%.
Donor Rat
Enzyme Tested
Homogenate
Membrane
Vesicles
Enrichment
Control
5'-nuceoti dase
2.10
12.0
5.7
F~-stimulated
adenylate cyclase
0.26
0.60
2.3
G1ucagon-treated
5'-nucleotidase
1.90
17.3
9.1
F-stimulated
adenylate cyclase
0.22
1.11
5.1


Fig, 2-2. MeAIB Inhibition of Na -Dependent AIB Uptake
by Isolated Vehicles. Rat liver plasma membranes were
assayed for Na -dependent AIB transport in the presence of
the System A-specific p^obe, MeAIB, at the indicated
concentrations. The Na -dependent uptake of AIB was
measured as described in the Methods section. Computer
analysis of the data by a FORTRAN program as described by
Kilberg et al. (1983) yielded an estimated Ki of 0.6 +
0.2 mM for MeAIB. The results are reported as the averages
+ S.D. of assays in triplicate.


28
succinate:cytochrome c reductase and cytochrome c oxidase
(Table 2-1). These enzyme marker activities are consistent
with those observed by Sips et al. (1980b).
Time-Dependent Uptake of AIB
Fig. 2-1 depicts the time-course of AIB transport by
isolated membrane vesicles. AIB uptake showed a Na + -
dependent overshoot in the presence of a Na+ gradient
with maximal transport at 3 min. By 40 min, the accumula
tion of AIB reached a steady-state level, presumably because
the Na+ gradient had been dissipated. The uptake in the
absence of Na+ was essentially hyperbolic in nature, and
reached a plateau after 15 min (Fig. 2-1).
The presence of a Na+-dependent overshoot suggests
transport of the amino acid against a concentration gradi
ent. To confirm this hypothesis, the intraves i cu 1ar water
space was estimated. A value of 1.2 pl/mg protein was
determined by the 3-0-methyl-glucose method of Kletzien et
al. (1975). Using this value, it was calculated that the
steady state distribution ratio for AIB (AIB^ n/AI)
in the absence of Na+ was slightly less than one.
Hence, an accumulated level of the amino acid greater than
240 pmol per mg protein represents transport against a con
centration gradient.


109
Characterization of the Reconstituted Proteoliposomes
Several characteristics of the reconstituted proteo-
liposomes were examined to be certain of their integrity and
transport competence. Fig. 3-7 depicts a time course of AIB
uptake into the reconstituted proteo 1iposomes In contrast
to the rapid overshoot of amino acid uptake observed in rat
liver vesicles (see Fig. 2-8), the proteoliposomes did not
show any apparent overshoot. This difference between mem
brane vesicles and reconstituted proteoliposomes may be
explained in part by a slower dissipation of the trans-mem
brane Na+ gradient. Although we have no direct evi
dence for this interpretation, several other groups have
observed similar uptake curves for Na+-dependent glucose
uptake (Koepsell et al., 1983) and Na+-dependent AIB
uptake (McCormick et al., 1985) in reconstituted proteolipo-
somes. Treatment of the reconstituted proteoliposomes
(K+-loaded) with valinomycin resulted in a higher rate
of AIB uptake both in the presence and in the absence of
N a + supporting the notion that AIB transport into recon
stituted proteoliposomes is electrogenic.
When the proteoliposomes were allowed to accumulate AIB
to a steady state in media of increasing osmolarity, the AIB
uptake was decreased (Fig. 3-8). The AI3 accumulation
showed an inversely linear relationship with respect to
medium osmolarity. Results such as these are generally
interpreted as indicating that transport of the substrate is
into a sealed, osmotica11y-sensitive compartment. Extrapola
tion of the data so as to intersect the Y-axis gives an indi
cation of the level of non-specific binding and/or sucrose


CHAPTER III
RECONSTITUTION OF SYSTEM A TRANSPORT ACTIVITY INTO
ARTIFICIAL PROTEOLIPOSOMES
Introduction
Reconstitution of ion transport systems has proven to
be a powerful method of resolving the transporters into
their functional components. The elegance of taking a
muti-protein complex apart, separating it into its indivi
dual components, and then recombining the components to
regain activity is apparent for the mitochondrial ATPase
(Racker, 1976). A number of comprehensive reviews have
appeared over the last few years that discuss various
methods of performing reconstitution of ion transporters or
membrane receptors (Levitzki, 1985; Eytan, 1982; Hokin,
1981; Racker, 1979).
Reconstitution of transport processes is a technique
that must be developed in a series of stages. The first
stage is the establishment of a membrane source that con
tains large quantities of activity. The second stage is the
demonstration of transport activity in an artificial proteo-
liposome system by combining membrane fragments and sonica
ted liposomes (membrane-fragment reconstitution). The final
stage requires removal of the transporter from its native
76


31
Me AIB Inhibition of AIB Uptake
Fig. 2-2 illustrates the inhibition of Na+-dependent AIB
uptake by MeAIB, a System A-specific analog (Ki1 berg et al.t
1981). The inhibition was concentration dependent yielding
an apparent Ki of 0.6 + 0.2 mM. This value is in good
agreement with unpublished data from our laboratory indi
cating that the Km for the high-affinity Na+-dependent
component of MeAIB uptake by isolated hepatocytes is about
0.3 mM. Although an excess of MeAIB does not completely
block AIB uptake (Fig. 2-2), it is clear that most (> 85%)
of the Na+-dependent AIB uptake occurs by the MeAIB-
inhibitable route (i.e., System A). These data support the
use of AIB as a selective substrate for System A in isolated
rat liver plasma membrane vesicles.
Sodium-Dependent Transport of Naturally-Occurring Amino
Acids
The uptake of four individual amino acids was measured
to obtain evidence for the presence of Systems A, ASC, N,
and Gly in the isolated membrane vesicles. The rate of
alanine transport in the presence of a Na+ gradient was
considerably greater than that in the absence of Na+ (Fig.
2-3A). The time-course of alanine uptake showed a rapid
Na + -dependent overshoot, about 50% of which was not i n hi bi
ta b 1 e by an excess of MeAIB. These data, in agreement with
results from intact hepatocytes (Kilberg et al., 1979) and
isolated liver membranes prepared in other laboratories
(Samson and Fehlmann, 1982; Sips et al., 1980b), indicate


108


133
and how do these changes in transport activity modulate the
overall metabolism of the cell? Perhaps, during the next
several years of study on eukaryotic amino acid transport,
reconstitution systems such as the one developed in our
laboratory will help answer such questions.


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
SYSTEM A AMINO ACID TRANSPORT ACTIVITY IN MEMBRANE
VESICLES AND RECONSTITUTED PROTEOLIPOSOMES
By
MARK ALLEN SCHENERMAN
May 1986
Chairman: Dr, Michael S. Kilberg
Major Department: Biochemistry and Molecular Biology
System A amino acid transport activity and its regula
tion has been studied in plasma membrane vesicles isolated
from rat liver. Enzyme markers were used to show that the
membrane preparation was enriched in plasma membrane yet
showed minimal contamination from endoplasmic reticulum mem
branes and no contamination by mitochondrial membranes. The
plasma membrane vesicles actively accumulated amino acids
through Systems A, ASC, N and Gly, all of which are depen
dent on an imposed trans-membrane Na+ gradient. Amino
acid uptake was into an osmoti cal 1y-sensitive space and was
inhibited by ionophores that collapse the transmembrane
Na+ gradient.
x 1


Fig. 3-4. Determination of the Optimal Period of Sonica-
tion for Reconstituted Proteoliposomes from EAT Cell Mem
branes. Membrane fragments (0.5 mg protein) were mixed with
sonicated asolectin (10 mg) and cholate (1.5 mg) and the
suspensions were frozen in liquid nitrogen. After thawing
at room temperature and diluting with K -uptake buffer,
the suspensions were sonicated in a bath sonicator for
varying periods of time from 10 s to 2 min. Following soni-
cation, the proteoliposomes were collected by centrifuga
tion and tested for System A activity as described in the
Methods section.+ The results are reported as the averages
+ S.D. of the Na -dependent AIB uptake for tripli
cate determinations.


80
may not be statistically different. The activity reported
may not be representative of System A activity because
Na+-dependent alanine uptake may be through several dif
ferent systems including System ASC.
Reconstitution of System A from SV-40-transformed mouse
fibroblasts membranes, extracted with 2% cholate was accom
plished by Nishino et al. (1978), by passing the extracted
proteins over a Sephadex G-50 column, and then subjecting
them to a freeze-thaw step. Sodium-dependent AIB uptake was
determined by a rapid filtration technique and was reported
to be at least 3-fold higher in sodium thiocyanate than in
choline chloride.
Reconstitution of System A in EAT cell membranes has
been reported after cholate-extraction followed by detergent
dilution (Cecchini et al., 1977) or cholate-extraction fol
lowed by dialysis (Bardin and Johnstone, 1978). Later
reports from Johnstone's group demonstrated the existence of
Na+-dependent AIB uptake after extraction of EAT mem
brane proteins in 2 5% cholate/4 M urea followed by dialysis
to 0.25% cholate, Sephadex G-50 chromatography, and a
freeze-thaw step (McCormick et al., 1984).
Little progress has been made toward the purification
of the System A carrier using reconstitution as an assay.
Cecchini et al. (1978) reported that an EAT cell membrane-
extract retained Na+-dependent alanine uptake after
ammonium sulfate fractionation, Biogel P-60 chromatography
and DEAE-cel 1ulose chromatography. Polyacry1 amide gel elec
trophoresis of the final extract showed only 15 or so


36
that the hepatic Na+-dependent uptake of alanine, at a sub
strate concentration of 200 pM, is about equally divided
between Systems A and ASC.
Additional evidence for the presence of System ASC was
obtained by examining cysteine transport. The Na+depen-
dent uptake rate for cysteine was considerably less than
that seen for alanine (e.g., 150 versus 450 pmol per mg pro
tein per 15 s). As reported previously for intact hepato-
cytes (Kilberg et al,, 1979), the Na+-dependent transport
of cysteine by the membrane vesicles was not inhibited by an
excess of MeAIB (Fig. 2-3B). These results demonstrate the
ability to monitor specifically hepatic System ASC in rat
liver vesicles by measuring Na+-dependent cysteine
uptake.
The Na+-dependent transport of glutamine and histi
dine by intact hepatocytes is mediated to a large extent by
System N (Kilberg et al., 1980). In the absence of MeAIB,
histidine uptake by the vesicles (Fig. 2-30 showed a rapid
Na+-dependent overshoot that decayed at a slow rate. When
Na+-dependent histidine uptake was assayed in the presence
of an excess of MeAIB, conditions that provide a specific
test for System N activity (Kilberg et al., 1980), a rapid
overshoot was observed with an accumulation of more than 400
pmol per mg protein at 15 s. These results suggest the pre
sence of System N activity in the isolated vesicles, but
also indicate that histidine transport in this membrane pre
paration is not restricted entirely to System N.


ni


Fig. 2-9. Decay of System A Activity in Membrane Vesicles
Incubated at 4C or -70C. Isolated membrane vesicles
either from glucagon-treated (,) or control rats (A) were
incubated at 4C (A>T) or -70C () for the indicated
times. Vesicles stored at -70C were in aliquots and were
thawed only once prior to assay. At the indicated times,
Na -dependent AI8 uptake was measured as described in
Materials and Methods using either 60 mM Na^SO. or
KS0. uptake buffer. Th| results are expressed as a
percent of the intial Na -dependent uptake in the
glucagon-treated vesicles (423 + 42 pmol AIB accumulated
per mg protein per 15 s) and are averages of triplicate
determinations. Standard deviations are typically less than
20% of the mean.


6
the activity is increased (Gazzola et al.f 1980). A deceler
ation of the transport rate, or trans-inhibition, is a
common characteristic of System A. In this case, when cells
are loaded with a System A substrate, washed free of exter
nal substrate, and tested for System A uptake, the activity
is lower (Kelley and Potter, 1978). Trans-inhibition of
System A is eye 1 ohex i mi de-i nsensitive indicating de novo
protein synthesis is not required (Kilberg et al., 1985b).
Regulation of transport activity by amino acid depriva
tion, sometimes called adaptive regulation, has been
observed for Systems A and N. The increase in activity is
detected after a time lag of 1-2 h, is blocked by cyclohexi-
mide, and is kinet i cal 1y-defined as an increase in Vmax
(Kilberg et al., 1985b). The implication is that the
increased transport activity caused by the response to amino
acid deprivation is due to a de novo synthesis and inser
tion of new carriers into the plasma membrane. A lowering
of the Km of the carrier for Na+ or amino acid is not
consistent with the data collected from several different
laboratories (Guidotti et al., 1978; Shotwell et al., 1983).
A wide variety of hormones affect the activity of
System A. Other systems, such as System N, are also
affected by hormones but no transport systems other than
System A show such a large response after hormone treatment
(Kilberg, 1982; Shotwell et al., 1983; Kilberg et al.,
1985a). Some of the hormones which induce hepatic System A
activity include growth hormone, growth factors, glucocorti
coids, catecholamines, glucagon, and insulin. The


16
activity are also often useful as starting material for
reconstitution studies.
Drawbacks to the use of membrane vesicles for transport
studies include the following: 1) the orientation of the
carrier is not always known and, 2) the proteins necessary
for activity may be partially or completely degraded during
the course of vesicle isolation, making transport studies
difficult. In many instances, the disadvantages of using
membrane vesicles are greatly outweighed by the amount of
information that can be obtained through their use.
Homogeneity of Plasma Membrane Vesicles
The hepatocyte is a polarized cell, that is, its plasma
membrane is separated into functional domains with special
ized functions. The bile canalicular domain is the barrier
between the cell and the bile-collecting ducts. It is
through this membrane surface that bile acids produced in
the hepatocyte are secreted into the bile duct. The sinusoi
dal or blood-domain is the border between the cell and the
blood-carrying vessels. It is through this membrane surface
that peptide hormones such as insulin or glucagon interact
with the hepatocyte. The final domain, referred to as the
contiguous surface, is the region of hepatocyte plasma mem
brane which is found between two hepatocyte cells. It is in
this domain that cell-cell communication occurs through gap
junctions.
The most commonly used technique for determining homoge
neity in membrane vesicle preparations is through the use of


137
Preparation of Recrystallized Cholate
Mater i a 1s:
1. Cholic acid (Sigma-free acid) 100 g
2. 70% ethanol 700 ml/L
3. activated charcoal
Procedure:
1. Weigh 100 g of cholic acid into a 1000 ml side-arm flask.
Add 300 mg of activated charcoal followed by the
addition of 600 ml of 70% ethanol (6 ml ethanol per g
cholic acid; 0.5 mg charcoal per ml of ethanol).
2. Gently heat the mixture with stirring until all of the
cholic acid dissolves (the ethanol will be near boiling).
3. Filter the hot mixture through a Buchner funnel with a #1
Whatman filter in place to remove the charcoal.
4. Allow the filtrate to cool at room temperature. White
crystals should begin to form almost immediately. When
cool, place at 4C overnight.
5. Pour off the supernatant without disturbing the crystals
on the bottom.
6. Dissolve the crystals again by heating and stirring in
500 ml of 70% ethanol. Cool and place at 4C overnight.
7. Pour off the supernatant without disturbing the crystals
on the bottom.
8. Repeat steps 5, 6, and 7.
9. Place the crystals onto watch glasses and heat in an
80C oven until completely dry.
11. The crystals should be white and cuboidal. The yield
should equal 50-75 g.
Reference: Kagawa, Y., Packer, E. (1971) J. Biol. Chem.
246, 5477-5487.


86
ISOLATED MEMBRANE FRAGMENTS
c,,eq extraction cholate/urea extraction
I I
MEMBRANES STIRRED WITH MEMBRANES STIRRED WITH
0.2* C,,Eq 2.5* CHOLATE & 4 M UREA
FOR 30 MI Iv At 4C FOR 30 MIN AT 4C
\ I
CENTRIFUGE AT 125.000XG FOR 1 H
I i
SUPERNATANT (DETERGENT-EXTRACT) SUPERNATANT (DETERGENT-EXTRACT)
CONTAINS 20-30* OF THE PROTEIN CONTAINS > 85* OF THE PROTEIN
DETERGENT-EXTRACT OR DIAVLSATE (0.5 MG PROTEIN)
ASOLECTIN (10 MG)
CHOLATE (1.5 MG)
1
FREEZE-THAW AND THEN DILUTE 1:10 WITH K+-UPTAKE BUFFER
1
WASH PROTEOLIPOSOMES AND THEN ASSAY SYSTEM A ACTIVITY
Fig. 3-1. Flowchart of the Procedure for Detergent-
Extraction and Reconstitution of System A Transport Activity
Using the Freeze-Thaw Technique.


77
membrane by detergent-extraction. Reconstitution is
achieved by combining the extracted proteins with lipid and
then removing the detergent. Recovery of activity from an
inactive detergent-extract is a convincing argument for
reconst itution.
Several methods of reconstitution after detergent-
extraction of membrane proteins have proven to be highly
successful. The first reconstitution of oxidative phosphor
ylation was performed using a cholate-dialysis technique
(Kagawa and Racker, 1971). The technique slowly removes
detergent by dialysis against deter gent-free buffers which
facilitates the insertion of proteins into liposomes. Since
1971, a wide variety of transport systems have been reconsti
tuted using detergent-dialysis techniques (Racker, 1979).
Although these methods have been useful for many systems,
they require the proteins to be in contact with detergent
for long periods of time (18-98 h) often resulting in inacti
vat i on.
To circumvent the inactivation problem caused by long
periods of dialysis, rapid reconstitution techniques were
devised. One example is the deter gent-di1ution procedure
which has been useful for the reconstitution of cytochrome
oxidase activity (Racker, 1972) and the reconstitution of
coupled oxidative phosphorylation (Racker, 1975). The tech
nique is simple and rapid in that it only requires dilution
of the detergent-extract so as to lower the detergent concen
tration below its critical micellar concentration (CMC)


10
starvation-induced increase in transport activity. These
results raise the possibility that the same stimulatory mech
anism (i.e., increased number of carriers being inserted
into the plasma membrane) may be operating to execute the
hormone-, starvation-, and transformation-induced System A
activity.
The relationship between growth factors, which stimu
late System A activity, and tumorigenesis is beginning to be
understood through several different observations reviewed
recently by Weinberg (1985). When the sequence of the
platelet-derived growth factor, which is known to stimulate
System A activity (Owen et al., 1982), was compared to the
sequence of the v-sis oncogene product, considerable homol
ogy was observed (Doolittle et al., 1983). Epidermal growth
factor (EGF) is another peptide which has been reported to
stimulate amino acid transport activity in human fibroblasts
(Hollenberg and Cuatrecasas, 1975). The cDNA clone for the
epidermal growth factor receptor was recently sequenced and
has been found to be homologous to a large portion of the
v-erbB oncogene product (Ullrich et al., 1984). It would
seem that certain growth factors, which are known to stimu
late System A activity or the receptors for those growth
factors, have homology to oncogene products. This may imply
some unknown link between transforming-ability and the abil
ity to stimulate System A activity which still remains to be
elucidated .
Glucagon- or starvation-induced transport activity has
been observed to decay if the stimulated cells are exposed


135
5. Add 1 ml of the molybdate reagent to all of the tubes.
6. Add 0.5 ml of the reducing agent to all of the tubes and
vortex.
7. Incubate all of the tubes at room temperature for 30 min.
8. Record the absorbance of each sample at 660 nm.
9. Determine the P. present in the sample tubes by a
linear regression analysis of the standard curve.
Reference: Fiske, C. H., Subbarow, Y. (1925) J. Biol. Chem.
66, 375-400.


75
the proposed mechanism for glucagon-induction and adaptive
control of System A. Ideally, one would like to test System
A activity in a particular cell line before and after trans
formation. Such an experiment was done using Rat-1 cells
transformed by the myc oncogene (Racker et al., 1985) and
NRK-49F cells transformed by transforming growth factor-0
(Boerner et al., 1985). Each cell line exhibited increased
System A activity following transformation; the induction
required de novo protein synthesis.


105
TABLE 3-2
Reconstitution of System A Activity into Proteoliposomes
Following Detergent Extraction of EAT Cell Membranes
EAT cell membranes were extracted for 30 min at 4C using
either 0.2% C^Eg or 2.5% cholate/4 M urea. After
centrifugation at 100,000xg, the detergent-extracts were dia
lyzed against 100 volumes of either 0.2% C-^Eg, 5 mM
Tris-HCl, 0.1 mM MgC 12, 0.1 mM CaCl2> 100 mM KC1, 1
pM PMSF, pH 7.45 or 0.25% cholate, 5 mM Tris-HCl, 0.1 mM
MgCl£, 0.1 mM CaCl2, 100 mM KC1, 1 pM PMSF, pH 7.45
for 18 h at 4C. The dialysates were reconstituted as
described in the Methods section. System A activities are
expressed as the means + S.O. of the Na+-dependent
pmol of AIB accumulated per mg protein per min.
Detergent-extract Dialysis Buffer System A Activity
0.2% C12Eg
None
1094 + 128
2.5% cho1 ate/ 0.25% cholate 51 + 115
4 M urea
2.5% cholate/ 0.20% C1C)EQ 857 + 43
4 M urea i y


125
Reconstitution of Na+-dependent AIB uptake was
obtained after extraction of membrane proteins by the non
ionic detergent C^Eg, but the efficiency of solubil
ization was low. Since it was the overall goal of this
research to attempt purification of the System A-associated
protein(s) and because it is likely that the actual quantity
of carrier molecules in the membranes is small, it became
clear that a more efficient method for membrane solubiliza
tion had to be found. The method of McCormick et al. (1984)
for extraction of EAT cell membranes with 2.5% cholate and 4
M urea resulted in the solubilization of greater than 85% of
the total proteins. Although the cholate/urea extract did
not yield transport activity following dialysis against
0.25% cholate, exchanging the cholate and urea for 0.2%
C12^9 ^ dialysis resulted in a restoration of
System A activity. The reconstituted activity in the pro
tein extract decayed at a slow, but constant rate during
storage at 4C, but was relatively stable during storage at
-70C. The protein extract also appeared to be moderately
sensitive to dialysis against the low-salt buffer (10 mM
potassium phosphate) but higher salt concentrations (500 mM
potassium phosphate) caused considerable apparent inactiva
tion. The inability to reconstitute transport activity was
probably not due to the salt alone because dialysis of the
high-salt extract against the low-salt buffer did not
restore the Na+-dependent transport.
Several methods of purification of the System A activ
ity were attempted to determine if the specific activity


Fig. 3-9. Measurement of the Intraves i cu 1ar Volume of the
Reconstituted Proteoliposomes Using 3-0-Methyl-G1ucose.
Reconstituted proteoliposomes were incubated with the indi
cated concentrations of [iaC]-3-0-methyl-glucose for 2 h
at 4C. At the end of the incubation period, aliquots of
the proteoliposomes (10.5 pg of protein in 20 pi) were
diluted into 1 ml of ice-cold PBS, and then the mixture was
filtered. The filter was washed twice with 3 ml aliquots of
ice-cold PBS and analyzed for trapped radioactivity as
described in the Methods section.


129
System A. In a series of unpublished experiments performed
by Donna Bracy in our laboratory, we find that rat liver
plasma membrane vesicles can be isolated rapidly using a
Fercoll gradient as described by Prpic et al ( 1984). She
has shown that these vesicles retain glucagon-stimulated
activity as did the liver-derived vesicles described in the
present work. This newer, relatively rapid procedure for
vesicle isolation will facilitate studies of regulatory phe
nomena, particularly after long periods of amino acid depri
vation of cultured cells. These hepatic vesicles may also
provide valuable clues to the structural aspects of the
System A transporter by urea- or sa1t-extracting the mem
brane proteins, or by testing transport activity after pro
tease treatment. These studies may eventually indicate
whether the transporter is a multi-subunit protein and if
the external face of the carrier is sensitive to prote
olysis.
The evidence obtained thus far with the hepatic vesi
cles strongly supports the notion that hormone treatment,
adaptive regulation, and cellular transformation all stimu
late System A activity by similar mechanisms (i.e., increase
the number of active carriers in the plasma membrane). The
intriguing possibility remains that there is some common
step among all of these regulatory processes that results in
the overall control of cellular metabolism. The liver mem
brane vesicles were a valuable tool to show that trans
inhibition is a membrane-associated phenomenon consistent
with the theory that high concentrations of substrate inside


protein.15s**
67
800
600
400
o
E
a
a
0>
5
0
Z
200
200
150
100
50


_u|ui*u¡40Jd .Bui'iouid
99
0
20 40 60 80 100 120
Sonication time (sec)


The non-ionic nature of the detergent will
also facilitate the separation of proteins in the detergent-
extract according to their charge.


65
added to the vesicle suspension did not slow the decay of
System A activity in the membrane vesicles maintained at
4C. Oithiothreitol (1 mM) also did not have any effect on
the decay of transport activity. Similar results were
obtained if the vesicles were subjected to a freeze-thaw
cycle in the presence of the inhibitors to trap them inside
the intraves i cu 1ar space.
To determine if the degradative process observed was
specific for System A, we tested the stability of Systems N
and ASC in control and glucagon-treated vesicles during incu
bation at 4C. The glucagon-treated vesicles showed simi
lar patterns of decay for Systems A and N (Fig. 2-10A).
Transport by System A and System N was enhanced in the
glucagon-treated vesicles when compared to the control mem
branes. Interestingly, these data are consistent with ear
lier observations in cultured hepatocytes suggesting that
System N is stimulated by glucagon in vivo (Barber et al.,
1983 ) but not in vitro (Ki1 berg et al., 1980). The esti
mated half-life of the decay of the hormone-stimulated activ
ity of System A and N was approximately 13 h. It is diffi
cult to be certain if System ASC is decaying either in the
glucagon-treated vesicles or in the control vesicles due to
the low basal rate of activity. Membrane vesicles isolated
from control hepatocytes also appeared to show decay of
Systems A and N (Fig. 2-10B). The half-life value for
System N was calculated to be about 13 h; however, in the
case of Systems A and ASC these measurements were too impre
cise to report due to the low level of activity present.


Fig. 3-2. Titration of the Cholate to Protein Ratio for
Reconstitution of System A Amino Acid Transport in EAT Cell
Plasma Membranes. EAT cell membranes were isolated as
described in the Methods section. Membrane fragments (0.5
mg protein) were mixed with sonicated asolectin (5 mg) and
potassium cholate was added from a 10% (w/v) stock, pH 7.5
so that the cholate to protein ratios (w/w) varied from 1 to
6. After mixing the suspensions, each mixture was frozen in
liquid nitrogen and thawe< at room temperature. The suspen
sions were diluted with K -containing buffer, centri
fuged, resuspended in KC1 uptake buffer and tested for
System A transport as described in the Methods section. The
System A transport activity of Ehrlich membranes which have
been put through the freeze-thaw cycle without added lipid
or cholate is shown (). Th$ velocities are expressed as
the averages + S.O. of the Na -dependent AIB uptake
measured in triplicate.


23
Isolation of Membrane Vesicles from Human Hepatoma Cells
A human hepatoma cell line (HepG2) was grown to confluence
in fourteen 150 mm Petri dishes in MEM supplemented with 5%

FBS The dishes were rinsed twice with PBS and then
the cells were scraped into a total of 30 ml of Buffer A.
The suspension was homogenized using 10 strokes of a
tight-fitting Pot ter-Elvehjem homogenizer and the homogenate
was brought to 1 mM in EDTA. The homogenate was centrifuged
at lOOOxg for 10 min. The supernatant (SI) and the pellet
(PI) were both saved. The SI fraction was centrifuged at
45,000xg for 30 min, and the resulting supernatant (S 2) was
discarded. The corresponding pellet (P 2) was resuspended in
1 ml of Buffer A and saved. Membranes contained in the
nuclear pellet were prepared using the initial pellet (PI)
which was placed on top of two sucrose step gradients
containing 39.5% sucrose as the lower layer and 20% sucrose
as the middle layer. The gradients were centrifuged at
50,000xg for 2.5 h. The white, fluffy material at 20%/39.5%
interface was removed and was diluted 1:1 with 0.2 mM
MgCl?, 10 mM HEPES, pH 7.5. The suspension was
centrifuged at 100,000xg for 1 h to pellet the vesicles.
The final pellet (P3) was resuspended in 1 ml of Buffer A.
The protein content of the fractions was measured by a Lowry
assay. The total protein yield of P2 was approximately 7 mg
and that of P3 was approximately 3 mg.
*
Cells could also be grown in roller culture (2 L) in
HEPES-MEM, pH 7.45 containing 5% FBS. The cells were
removed by treating with 2 mM EDTA for 2 h at 37C then
scraping with a rubber policeman into 40 ml of PBS.


18
Isolation of Membrane Vesicles from Rat Liver
Several groups have succeeded in isolating plasma
membrane-enriched vesicles from rat liver (Neville, 1968;
Ray, 1970; Touster et al., 1970). One group in particular
has addressed the functional polarity of the hepatocyte and
has developed a technique for separating the canalicular por
tion of the plasma membrane from the blood-sinusoidal and
contiguous membrane surfaces (Wisher and Evans, 1975). More
recently, membranes from the three separate domains have
been prepared to even greater homogeneity through the use of
sucrose step-gradients (Hubbard et al., 1983). The best
separation of plasma membrane domains reported so far used
rate-zonal centrifugation and resulted in a 64-fold enrich
ment of canalicular membrane markers and a 34-fold enrich
ment of basolateral (a mixture of sinusoidal and contiguous
membrane surfaces) membrane markers (Meier et al., 1984b)
over the activity detected in the homogenate. Centrifuga
tion through a Percoll gradient has also proven useful in
purifying plasma membrane vesicles from rat liver (Prpic et
al., 1984).
Amino acid transport has been studied to a limited
extent in membrane vesicles prepared from rat liver. The
first report of Na -dependent amino acid transport
assayed alanine uptake after isolation of membranes by dis
continuous sucrose gradient centrifugation (Van Amelsvoort
et al., 1973). Another group (Meier et al., 1984a), has
shown that about equal amounts of sodium-dependent amino


152
Kinne, R. and Faust, R. G. (1977) Biochem. J. 168,
311-314
Kletzien, R. F., Pariza, M. W., Becker, J. E. and Potter, V.
R. (1975) Anal. Biochem. 68, 537-544
Koepsell, H., Menuhr, H., Ducis, I. and Wissmuller, T. F.
(1983) J. Biol. Chem. 258, 1888-1894
Laemmli, U. K. ( 1970 ) Nature 22_7, 680-685
LeCam, A. and Freychet, P. (1976) Biochem. Biophys. Res.
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LeCam, A. and Freychet, P. (1977) J. Biol. Chem. 252,
143-156
Lever, J. E. (1976) Proc. Natl. Acad. Sci. _73, 2614-2618
Lever, J. E. (1980) CRC Crit. Rev. Biochem. 2* 187-246
Levitzki, A. (1985) Biochim. Biophys. Acta 822, 127-153
Lienhard, G. E. (1983) Trends Biochem. Sci. 8, 125-127
McCormick, 0. I., Silvius, J. R. and Johnstone, R. M. (1985)
J. Biol. Chem. 260, 5706-5714
McCormick, J. I., Tsang, D. and Johnstone, R. M. (1984)
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Meier, P. J., St. Meier-Abt, A., Barrett, C. and Boyer, J.
L. (1984a) J. Biol. Chem. 259, 10614-10622
Meier, P. J., Sztul, E. S., Reuben, A. and Boyer, J. L.
(1984b) J. Cell Biol. 98, 991-1000
Miller, C. and Racker, E. (1976) J. Memb. Biol. 26,
319-325
Morre, D. J. (1971) Meth. Enzymol. 22, 130-148
Murer, H. and Kinne, R. ( 1980 ) J. Memb. Biol. 5j>, 81-95
Neville, D. M. ( 1968 ) Biochim. Biophys. Acta ,154, 540-552
Newman, M. J., Foster, D. L., Wilson, T. H. and Kaback, H.
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Ni 1sen-Hami1 ton, M. and Hamilton, R. T. ( 1979 ) Biochim.
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75, 3856-3858


Fig. 3-8. Osmotic Sensitivity of the Reconstituted Pro-
teoliposomes. Reconstituted proteoliposomes were diluted
into NaCl uptake buffer containing various concentrations of
sucrose so that the final osmolarity of the solution ranged
from 0.22 to 0.72 osM. The mixtures were incubated on ice
for 2 h then aliquots (11.7 pg protein in 20 pi) were
removed, diluted into 1 ml of ice-cold PBS, and then the
mixture was filtered. The filter was washed twice with 3 ml
aliquots of ice-cold PBS and analyzed for radioactivity as
described in the Methods section. The results are expressed
as the mean of the pmol AIB accumulated per mg protein per 2
h for triplicate determinations and the standard deviations
were less than 10% of the means.


78
followed by centrifugation to collect the resulting
proteoliposomes.
Another rapid technique to remove detergents with
micelle molecular weights less than 10 kDa, such as cholate
and deoxycho1 ate, was described by Brunner et al. ( 1976 ).
The procedure requires passing the detergent-extract over a
Sephadex G-50 column. The gel filtration matrix retards the
small detergent molecules and allows the large lipid and pro
tein molecules to fuse into proteoliposomes and pass through
in the void volume. McCormick et al. (1984) used this proce
dure prior to a freeze-thaw step to achieve reconstituted
amino acid transport activity from EAT cell membranes.
A procedure which has shown great success for the glu
cose transporter from red blood cells (Kasahara and Hinkle,
1976) requires a freeze-thaw step coupled with sonication.
This technique involves combining sonicated lipid with solu
bilized membrane proteins followed by rapid freezing in a
dry-ice-ethanol bath. After thawing at room temperature,
the mixture is sonicated for short periods of time resulting
in a 3-fold greater activity than without the sonication.
Alfonso et al. (1981) have observed that the activity of the
mitochondrial proton-pump is increased even further when
small amounts of cholate are added before the freeze-thaw
step. Pick (1981) suggests that the fusion process which
occurs during the rapid freezing and thawing is a result of
the formation of crystalized water molecules in two frozen
planes separated by the membrane domain. The bilayer is


APPENDIX C
SOLUTIONS FOR THE PREPARATION OF PLASMA MEMBRANES AND
TRANSPORT OF VESICLES
Preparation of Membrane Vesicles from Rat Liver
1. Homogenization buffer (Buffer
A)
0.25 M sucrose
171.2
g/2 L
0.2 mM MgC1,
0.0813 g/2 L
10 mM HEPE
4.76
g/2 L
Adjust the pH of the
solution
to
7.
5
us i ng
KOH.
2. 39.5% (w/v) sucrose
79.0
g/200 ml
10 mM HEPES
0.476
g/200 ml
Adjust the pH of the
solution
to
7.
5
using
KOH.
3. 20* (w/v) sucrose
40.0
g/200 ml
10 mM HEPES
0.476
g/200 ml
Adjust the pH of the
solution
to
7.
5
using
KOH.
4. 100 mM EDTA
3.72
g/100 ml
Transport of Rat Liver Membrane Vesicles
1.NaCl uptake buffer
120 mM NaCl
10 mM
10 mM
Adjust the pH of the solution to 7.45
frozen in aliquots.
MgC 1 0
HEPES
1.404 g/200 ml
0.406 g/200 ml
0.477 g/200 ml
using 4 N KOH. Store
2. KC1 uptake buffer
120 mM KC1 1.789 g/200 ml
10 mM MgCl2 0.406 g/200 ml
10 mM HEPES 0.477 g/200 ml
Adjust the pH of the solution to 7.45 using 4 N KOH. Store
frozen in aliquots.
3. Stop buffer
Add 1.168 g of NaCl to 200 ml of Buffer A and adjust the pH
of the solution to 7.45. PBS can also be used as a "Stop
Buffer".
146


131
in rat liver. The liver reconstitution system may also be
useful in detecting intracellular pools of carrier molecules
which are present during various metabolic states of the
cell. The cells could be separated into Golgi-, endoplasmic
reticulum-, lysosome-, and plasma membrane-enriched frac
tions and each fraction reconstituted to test for transport
activity. Reconstitution may also be useful in character
izing the decay process seen in intact cells. If reconsti
tuted proteoliposomes do not show a rapid rate of decay for
System A activity, it would be possible to add individual
subcellular fractions or even cytoplasmic elements to deter
mine which components would restore the decay process.
The work described here, along with the work by others,
indicates that hormone-, starvation-, and transformation-
induced transport activity occurs because a greater number
of carriers are being synthesized and inserted into the
plasma membrane. These observations suggest that it should
be feasible to identify proteins related to the regulation
of System A by examining the protein synthetic rates of
individual rat liver membrane proteins in the presence or
absence of induction by hormones or starvation. These
studies, currently underway in our laboratory, are based on
culturing treated or normal hepatocytes in the presence of a
radiolabel led amino acid and then resolving the labelled mem
brane proteins by 2D-PAGE. The changes in synthetic rates
of individual proteins can be quantitated by determining the
relative incorporation of radiolabel into proteins from
induced and normal cells.


122


85
Freeze-Thaw Reconstitution and Transport Assay
Reconstitution of transport activity was performed by
mixing 0.5 mg of protein from either the membrane-fragment
preparation or the detergent-extract, 10 mg of sonicated aso-
lectin (stock = 40 mg/ml) and 1.5 mg of K+ cholate
(stock = 10% w/v). The final volume of the mixture ranged
from 0.7-1.0 ml. The mixture was frozen in liquid nitrogen
and thawed at room temperature, then the suspension was
diluted with 2 ml of K+-uptake buffer (120 mM KC1, 10 mM
MgCljp, 10 mM HEPES, pH 7.45) and centrifuged at
125,000xg for 45 min. The pellet was resuspended in 200 pi
of K+-uptake buffer with a stirring rod and gentle vor-
texing. A summary of the reconstitution procedure, inclu
ding further details, is shown in Fig. 3-1.
Amino acid transport buffers were prepared as 2X stocks
using NaCl (120 mM NaCl) or KC1 (120 mM KC1) uptake buffer
and 200 pM AIB. To initiate uptake, 20 pi of vesicles
were added to 20 pi of uptake buffer. The mixture was vor-
texed and incubated at 22C for 1 min. To stop the uptake,
1 ml of ice-cold stop buffer (154 mM NaCl, 10 mM
Na^HPO^, adjusted to pH 7.45 with HC1) was added and
the suspension was vortexed. The mixture was immediately
filtered over a Gelman nitrocellulose filter (25 mm diameter
and 0,45 pm pore size) and the filter was washed once with
3 ml of ice-cold stop buffer. The filter was placed into a
15 ml plastic scintillation vial and the radioactivity
trapped on the filter was determined after adding 5 ml of
Bray's scintillation cocktail. Transport activity is


LIST OF FIGURES
Page
2-1 Time-Dependent Uptake of AIB by Rat Liver
Plasma Membrane Vesicles 30
2-2 MeAIB Inhibition of Na+-Dependent AIB
Uptake by Isolated Vesicles 33
2-3 Alanine, Cysteine, Histidine and Glycine
Transport by Rat Liver Plasma Membrane
Vesicles 35
2-4 Effect of pH on Alanine Uptake in Membrane
Ves i cl es 42
2-5 Effect of the Extravesicular Osmolarity on
AIB Uptake 44
2-6 Effect of Incubation Temperature on the
Na -Dependent Uptake of AIB 47
2-7 Relation Between Membrane Protein
Concentration and AIB Uptake 49
2-8 System A Activity in Plasma Membrane Vesicles
from Control or G1ucagon-Injected Rats 57
2-9 Decay of System A Activity in Membrane
Vesicles Incubated at 4C or -70C 64
2-10 Decay of Systems A, N and ASC in Vesicles from
Glucagon-Treated and Normal Hepatocytes 67
2-11 Flow Chart of HepG2 Membrane Preparation 69
3-1 Flow Chart of the Detergent-Extraction and
Reconstitution of System A Transport Activity
Using the Freeze-Thaw Procedure 86
3-2 Titration of the Cholate to Protein Ratio for
Reconstitution of System A Amino Acid
Transport in EAT Cell Membranes 94
vi i


154
Samson, M. and Fehlmann, M. (1982) Biochim. Biophys. Acta
687, 35-41
Shotwell, M. A., Kilberg, M. S. and Oxender, D. L. (1983)
Biochim. Biophys. Acta 737, 267-284
Simpson, I. A. and Cushman, S. W. (1985) Curr. Top. Memb.
Transport 24_, 459-503
Sips, H. J., Groen, A. K. and Tager, J. M. (1980a) FEBS
Lett. U9, 271-274
Sips, H. J., Van Amelsvoort, J. M. M. and van Dam, K.
(1980b) Eur. J. Biochem. 105, 217-224
Suzuki, K. and Kono, T. (1980) Proc. Natl. Acad. Sci .
77, 2542-2545
Swanson, M. A. (1955) Meth. Enzymol. 2, 541-543
Szoka, F. and Papahadjopoulos, D. (1980) Ann. Rev. Biophys.
Bioeng. j), 467-508
Takahashi, M,, Malathi, P., Preiser, H. and Jung, C. Y.
(1985) J. Biol. Chem. 260, 10551-10556
Touster, 0., Aronson, N. N., Dulaney, J. T. and Hendrickson,
H. (1970) J. Cell Biol. 47, 604-618
Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J.,
Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libermann, T.
A. and Schlessinger, J. (1984) Nature 309, 418-425
Unger, R. H. (1978) Metab. Clin. Exp. 27, 1691-1706
Vadgama, J. V. and Christensen, H. N. (1983) J. Biol. Chem.
258, 6422-6429
Van Amelsvoort, J. M. M., Sips, H. J., Apitule, M. E. A. and
van Dam, K. (1980) Biochim. Biophys. Acta 600,
950-960
Van Amelsvoort, J. M. M., Sips, H. J. and van Dam, K. (1978)
Biochem. J. r74f 1083-1086
Weinberg, R. A. (1985) Science 230, 770-776
White, M. F. and Christensen, H. N. (1983) J. Biol. Chem.
258, 8028-8038
Winter, C. G. and Christensen, H. N. (1965) J. Biol. Chem.
240, 3594-3600
Wisher, M. H. and Evans, W. H. (1975) Biochem. J. 146,
375-388


3-3
Titration of the Lipid to Protein Ratio for
Reconstitution of System A Activity Using
EAT Cell Membranes
97
3-4
Determination of the Optimal Period of
Sonication for Reconstituted Proteoliposomes
from EAT Cell Membranes
99
3-5
Determination of the Optimal Concentration of
Ci2E9 for Extraction of EAT Cell
Membrane Proteins Prior to Reconstitution....
102
3-6
Temperature Stability of the Membrane Protein
Extract
108
3-7
Time Course of AIB Uptake into Proteo 1iposomes
in the Presence and Absence of Val inomycin...
111
3-8
Osmotic Sensitivity of the Reconstituted
Proteo 1iposomes
113
3-9
Measurement of the Intraves i cu 1ar Volume of
the Reconstituted Proteoliposomes Using
3-0-Methyl-Glucose
116
3-10
SDS-Polyacrylamide Gel E1ectrophoresis of
EAT Cell Membranes and Reconstituted
Proteoliposomes
122
v i i i


APPENDIX D
SOLUTIONS FOR THE PREPARATION AND RECONSTITUTION
OF EAT CELL MEMBRANE
Isolation of Plasma Membranes from EAT Cells
1. Cell wash buffer
140 mM NaCl
40.95 g/5L
5 mM K C1
1.865 g/5L
1 mM MgCl -
10 mM HEPES
1.017 g/5L
11.92 g/5L
Adjust the pH of the solution to
7.4 using
4 N KOH.
2. 10 mM CaCl
2.94 g/2 L
(dihydrate; Fisher ACS, pH 8.0)
3. 100 mM phenylmethylsulfonyl
0.0348 g/2 ml
fluoride (PMSF) stock
of DMSO
4. sucrose/EDTA/PMSF
0.3 M sucrose
10.269 g/100 ml
1 mM EDTA
1 ml of 100 mM
1 mM PMSF
stock, pH 7.4
1 ml of 100 mM
Adjust the pH of the solution to
7.4 using
PMSF stock
HC1
Solubilization and Reconstitution of System A
Transport Using EAT Membranes
1. Solubilization buffer
2.5%(w/v) cholic acid (recrystalized )
4 M urea (Pierce-u11rapure )
0.1 mM EDTA
0.5 g/20 ml
4.80 g/20 ml
20 pi of 100 mM
EDTA stock
100 mM NaCl 0.117 g/20 ml
5 mM Tris-HCl 0.015 g/20 ml
Adjust the pH of the solution to 7.4 using 2 N NaOH while
heating gently. Allow the solution to cool and then
recheck the pH.
2. 20*(w/v) C12E^ stock 2Q ml/100 ml
Stir the detergent in distilled water until dissolved.
147


Fig- 2-8- System A Activity in Plasma Membrane Vesicles
from Control or Glucagon-Injected Rats. Membrane vesicles
were isolated from control rats () or rats injected with 1
mg glucagon/100 g body weight 4 h prior to membrane isola
tion (). Following the + isol ation, the vesicles were
immediately tested for Na -dependent AIB transport as
described in the section on Materials and Methods using
either 60 mM Na?S0. or K^SO. uptake buffer.
The time of uptake^at 22*C was varied from 10 s to 30 min.
The results are expressed as the averages of triplicate
determinations and the standard deviations, omitted for
clarity, were generally less than 10%.


62
glucagon-treated = 1.8). Hence, a difference in the compo
sition with respect to plasma membrane domains is probably
not responsible for the decreased glucagon-dependent stimula
tion reported in Table 2-6.
Inactivation of Glucagon-Stimulated System A Activity
The decreased retention of stimulated System A activity
in membrane vesicles might also be explained if the System A
carrier complex is irreversibly inactivated during the iso
lation procedure. In order to test for inactivation,
freshly isolated membrane vesicles from normal or glucagon-
treated rats were incubated at 4C for 48 h. At specific
intervals, the System A activity was assayed in both vesicle
preparations. The results indicate that at 4C there is a
time-dependent loss of System A activity in the glucagon-
treated vesicles (Fig. 2-9). The decay of the hormone-
stimulated transport consisted of a single exponential compo
nent. The preparation of the membranes requires about 6 h
and we assume that the decay was ongoing during that time.
The value for the half-life of the decay was approximately
13 h. In several studies, maintenance of the glucagon-
stimulated vesicles at -70C for 48 h protected most, if
not all, of the initial activity (Fig. 2-9). In contrast,
the basal rate of System A-mediated uptake seen in the con
trol vesicles did not change during a 24 h incubation at
4C (Fig. 2-9).
A mixture of 2 mM PMSF, 2 mM EDTA, trasylol (30 trypsin
inhibitor units/ml), 0.1 mM leupeptin and 0.5 mM bacitracin


Fig. 2-1. Time-Dependent Uptake of AIB by Rat Liver Plasma
Membrane Vesicles. Membran§ vesicles (J.5 mg protein/ml)
were diluted into either Na ( ) or K () uptake
buffer containing 200 pM radioacti ve 1y-1abel1ed AIB. After
incubation at 22C for the time indicated, an aliquot (50
pg) was removed and assayed for trapped radioactivity as
described in Methods section. The difference between the
transport in the presence of NaSCN or KSCN is shown as the
Na -dependent uptake (A). The data are the averages +
S.D. of three determinations.


8
maintained for at least 40 min and could be partly
responsible for the corresponding increase in alanine uptake
induced by glucagon during the first 15-30 min of exposure.
Presumably, a change in membrane potential would affect all
Na+-dependent electrogenic transport systems and this
has not been reported, implying that the change in membrane
potential is not the only factor contributing to the
increased System A activity.
Another possible explanation for the protein synthesis-
independent increase in System A activity after glucagon-
treatment would be migration of cryptic carriers to the
plasma membrane as has been described for the glucose
carrier (Simpson and Cushman, 1985). Convincing evidence
supporting this model has not as yet been provided and
awaits adequate methods of subcellular fractionation as well
as methods for testing transport activity in various mem
brane fractions. Reconstitution of glucose transport was
used by Suzuki and Kono (1980) to show the insulin-
stimulated movement of glucose carriers from an intra
cellular compartment to the plasma membrane. Similar
studies could be undertaken for System A once a reliable
reconstitution process has been developed.
Post-translational modifications could also be respon
sible for regulating the protein synthesis-independent por
tion of the hormone-stimulated activity. Nilsen-Hamilton
and Hamilton (1979) observed that treatment of 313-fibro
blast membrane vesicles with cAMP resulted in increased AIB
uptake. However, when the vesicles were treated under


83
cloth. The ascites fluid was typically whitish-yel1ow in
color and was not tinged at all with blood. Any bloody
fluid was discarded. The cells were diluted in 10 volumes
of wash buffer (140 mM NaCl, 5 mM KC1, 1 mM MgCl2> 10 mM
HEPES, pH 7.4) and placed in GSA rotor bottles. The cell
suspension was centrifuged at 650xg in a GSA rotor for 5
min. The supernatant was decanted and the pellets were
resuspended in a small volume of wash buffer. The pellet
suspension was centrifuged again at 650xg for 5 min (table-
top centrifuge) in order to pack the cells. The cells were
frozen in 45 ml centrifuge tubes and stored at -70C. Fro
zen cells (150 g) were used for each preparation of mem
branes. The frozen cells were thawed in warm water and
poured into 1.5 L of 10 mM CaC1^ (dihydrate; Fisher ACS,
pH 8.0) at room temperature. The cells were stirred slowly
at 4C for 5 h and were homogenized by rapidly forcing
through a Logeman homogenizer with the nozzle half closed
(this and all subsequent steps were done at 4C). The homo
genate was poured into 6 GSA rotor bottles and centrifuged
at 650xq for 5 min. The pellets were discarded and the
supernatant was collected and centrifuged at 16,000xg for 40
min in the GSA rotor. The pellets were gently homogenized
in 30 ml of sucrose-EDTA-PMSF buffer (0.3 M sucrose, 1 mM
EDTA, 1 mM PMSF, pH 7.4) using a typewriter brush and a
loose-fitting Potter-Elvehjem homogenizer. The suspension
was incubated on ice for 1 h then it was centrifuged at
27,000xg for 30 min. The supernatant was decanted and the
pellet was resuspended in 30 ml of sucrose-EDTA-PMSF as


141
Na+ J<+ -ATPase Assay
Materials:
1. 10X buffer
250 mM Tris base
50 mM MgC19
5 mM EGTA
3 g/100 ml
1 g/100 ml
0.190 g/100 ml
Adjust the pH of the solution to 7.4 using concentrated HC1.
2. 10X ATP stock
50 mM Na -ATP (Sigma grade I) 0.0254 g/ml
Adjust the pH of the solution to 7-7.5 using 0.2 N NaOH.
This mixture is prepared just prior to use.
3. 10X NaCl-KCl stock
1 M NaCl 0.0585 g/ml
300 mM KC1 0.0224 g/ml
4. 10X ouabain stock
10 mM ouabain (octahydrate) 0.0729 g/10 ml
5. 10%(w/v) TCA
Procedure:
1. Place the buffer, salt stock, and ATP in a 37C water
bath and allow the mixture to come to equilibrium.
2. Into 15 ml plastic conical centrifuge tubes, place 100
pi of buffer, 100 pi salt stock and/or 100 pi of
ouabain stock, 100 pi of ATP stock and 500 pi of water
(600 pi water in the absence of ouabain) in the sample
tubes at 37C. The sample blank contains all the same
components with 100 pi water instead of ATP and the
substrate blank contains all the same components except,
100 pi of water is added instead of 100 pi of membrane.
3. The membrane fraction to be tested is diluted to 1 mg
protein/ml and warmed to 37C.
4. To initiate the assay, add 100 pi of membrane (100 pg
protein) to each mixture (except the substrate blank) and
incubate at 37C for 15 min.
5. Terminate the assay by adding 1 ml of ice-cold 10% TCA
and vortexing. Centrifuge the mixtures at 10,000 RPM for
20 min in a Sorvall SM-24 rotor.
6. Remove 0.5 ml of the supernatant from each sample and use
for the determination of P. as described in Appendix
A. 1


74
When Systems N and ASC were tested for inactivation in
membrane vesicles, System N decayed with a half-life similar
to that for System A. It could not be determined accurately
if System ASC was decaying at a significant rate. These
data imply that the inactivation process affects other trans
porters as well as System A and may, in fact, result from a
non-specific effect on vesicle integrity. It cannot be
determined accurately if the inactivation process is also
occurring in vesicles from normal cells because the data at
each time point are not statistically different from each
other. Another unknown is the status of the membrane perme
ability with respect to Na+. An increase in permeabil
ity to Na+ ions could result in an apparent inactivation
of the carrier.
Membrane vesicles isolated from human hepatoma cells
(HepG2) showed increased System A activity when compared to
normal rat hepatocytes. Such a comparison is difficult
because we do not know the basal activity of transport in
normal human liver tissue. However, it is interesting to
note that the level of Na+-dependent AIB uptake in HepG2
vesicles is comparable to the transport activity in
glucagon-induced rat liver vesicles. Our laboratory has
made direct comparisons between normal rat hepatocytes in
culture and several rat hepatoma cell lines. In every case,
the hepatoma cells contain enhanced transport activity. The
data obtained with isolated membrane vesicles are consistent
with the hypothesis that transformation induces new System A
carriers to be inserted into the plasma membrane, similar to


24
Vesicle Transport Assay
Oust prior to use, the membrane vesicles were diluted
with Buffer A to a final concentration of 2.5 mg protein per
ml and then incubated at 22C for 15 min. To initiate
amino acid uptake, 20 pi (50 pg protein) of the vesicle
suspension was added to 20 pi of Buffer A supplemented with
10 mM MgCl£, 120 mM of either NaSCN or KSCN, and 200 pM
radioactively-label1ed amino acid. These two solutions will
be referred to as Na + and K+-uptake buffers, respective
ly. Where indicated in the figure legends, 60 mM Na2S04
or K^SO^ was used to replace the corresponding thiocyan
ate salts. Uptake was terminated by the addition of 1 ml of
ice-cold Buffer A containing 100 mM NaCl (stop-buffer). The
mixture was vortexed immediately and passed over a 0.45 pm
nitrocellulose filter. The filter was washed with another 3
ml of ice-cold stop-buffer and then analyzed for trapped
radioactivity in 5 ml of Bray's scintillation cocktail
(Bray, 1960). Unless otherwise indicated in the figure
legends, the results were from a single membrane preparation
and the S.D. of triplicate assays was less than 10% of the
mean.
Enzyme Marker Assays
The activities of 5'-nuc1eotidase (Morre, 1971), glu-
cose-6-phosphatase (Swanson, 1955), succinate:cytochrome c
reductase (K i 1 berg and Christensen, 1979 ), and cytochrome ox
idase (Kilberg and Christensen, 1979) were assayed by


pmol AIBmg-1 protein
57
0


136
Modified Lowry Protein Assay
Materials:
1. 10%(w/v) sodium dodecyl sulfate (SDS) 10 g/100 ml
2. 24%(w/v) TCA 48 g/200 ml
3. Lowry copper reagent
0.58 mM EDTA (copper disodium salt) 0.25 g/1
189 mM NaC0- 20 g/1
100 mM NaOH J 4 g/1
l%(w/v) SDS 10 g/1
4. Folin-Ciocalteu reagent (2 N) Sigma
5. 0.2%(w/v) SDS/0.2 N NaOH
Procedure:
1. Use the desired amount of protein sample or bovine serum
albumin standard (5-50 ug) and bring the final volume of
the sample to 1 ml in a 15 ml conical centrifuge tube.
2. Add 10 pi of 10% SDS to all of the tubes, vortex, and
incubate the tubes at room temperature for 15 min.
3. Add 750 pi of ice-cold 24% TCA to all of the tubes and
centrifuge the tubes for 20 min at 10,000 RPM in a
Sor val 1 SM-24 rotor.
4. Pour off the supernatant and shake the tube dry.
5. Add 100 pi of 0.2% SDS/0.2 N NaOH to all of the tubes
and vortex.
6. Add 600 pi of Lowry copper reagent to all of the tubes,
vortex, and incubate the samples for 10 min at room
temperature.
7. Add 60 pi of Folin-Ciocalteu reagent, which has been
diluted with water to 1 N phenol concentration (dilute
phenol reagent 1:1 just before adding), to all of the
tubes.
8. Incubate the samples for an additional 30 min at room
temperature and then record the absorbance of each sample
at 750 nm.. For protein concentrations higher than 50
pg, the absorbance at 500 nm can be used instead.
Reference: Bensadoun, A., and Weinstein, D. (1976) Anal.
Biochem. 7j0, 241-250.


58
The System A activity in primary cultures of rat hepato
cytes is also induced if the cells are incubated in an amino
acid-free medium (Kelley and Potter, 1978). Like glucagon-
dependent stimulation, this process, referred to as adaptive
regulation, is thought to result from increased synthesis of
a System A-associated glycoprotein located in the plasma mem
brane (Barber et al., 1983). To test this hypothesis, cul
tured hepatocytes were incubated for 6 h in amino acid-free
medium (NaKRB) or NaKRB supplemented with 20 mM asparagine
and then membrane vesicles were prepared from those cells.
The System A activity was enhanced nearly 6-fold in the vesi
cles from the starved cells. The rate of Na+-dependent
AIB uptake in the vesicles from starved (no amino acid) and
fed (20 mM asparagine) cells was 189 + 14 and 33+6
pmol per mg protein per min, respectively. The degree of
induction for System A transport activity measured in intact
cells is generally 5- to 10-fold.
After several different membrane preparations had been
tested for the level of glucagon-dependent stimulation of
Na+-dependent AIB transport, it appeared as though the
amount of hormone-induced activity for intact cells was not
reflected in the membrane vesicles. To monitor the degree
of hormone stimulation, hepatocytes from glucagon-injected
rats were assayed for System A activity and then plasma mem
brane vesicles were isolated from the same preparation of
cells. The data shown in Table 2-5 are representative of
many experiments. In this instance, the intact cells showed
a 30-fold increase in System A transport following glucagon


97
lipid: protein


155
Wray, W., Boulikas, T., Wray, V. P. and Hancock,
Anal. Biochem. 118, 197-203
Xie, X-S., Stone, D. K. and Racker, E. (1984) J.
259, 11676-11678
R. (1981)
Biol. Chem.


17
enzyme markers. The level of subcellular contamination can
also be quantitated using enzyme marker assays (Evans,
1980). Indicators of plasma membrane purity include 5'-
nucleotidase, Na+,K+-ATPase, alkaline phosphatase,
leucine ami nopeptidase, and adenylate cyclase. Endoplasmic
reticulum contamination can be quantitated through the use
of glucose-6-phosphatase or NADPH:cytochrome c reductase
assays. Mitochondrial contamination can be determined using
either succinate:cytochrome c reductase as an indicator of
the inner mitochondrial membrane or monoamine oxidase as an
indicator of the outer mitochondrial membrane. Contamina
tion by Golgi remnants can be quantitated using galactosyl
transferase and lysosomal contamination can be determined
using acid phosphatase. Nuclear contamination can be deter
mined using DNA as a marker and cytoplasmic contamination
can be quantitated using latent lactate dehydrogenase activ
ity. A qualitative determination of contamination by sub-
cellular organelles can also be performed using transmission
electron microscopy.
Enzyme markers can also be used to distinguish the func
tional domains of the hepatocyte plasma membrane. The bile-
canalicular surface contains most of the activity for leu
cine aminopepti dase and 51-nuceoti dase (Roman and Hubbard,
1983), whereas glucagon-activated adenylate cyclase is found
primarily on the blood-sinusoidal surface (Wisher and Evans,
1975) and Na+,K+-ATPase is located primarily on the
contiguous and sinusoidal surfaces of the rat liver plasma
membrane (Poupon and Evans, 1979).


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.
May, 1986 //& ^anTTolTege-of-MeWcTf^r *
; L-t^_
Dean, G r ad
>*yc
uate School


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Barber, E. F., Handloqten, M. E., Vida, T. A. and Kilberg,
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Biol. Chem. 242, 5237-5246


68
System A Transport Activity in Membrane Vesicles from Human
Hepatoma Cells
Several investigators have reported that glycolysis and
nutrient transport activities are increased in transformed
cell lines (Parns and Isselbacher, 1978). Amino acid trans
port, in particular, is increased in membrane vesicles pre
pared from SV40-transformed 3T3 cells (Lever, 1976). Our
laboratory has studied System A activity in a number of hepa
toma cell lines from both human and rat. For example, a
human hepatoma cell line (HepG2) shows an increased basal
transport rate of nearly 12-fold when compared to the normal
hepatocyte (38.6 + 6.1 versus 444 + 22 pmol AIB per
mg protein per min for the normal and hepatoma cells, respec
tively). To test whether or not the increased System A-
mediated transport activity observed in the human hepatoma
cells is retained in isolated membranes prepared from HepG2
cells, several membrane vesicle fractions were prepared as
described in the Methods section. Each of the subcellular
membrane fractions tested displayed Na+-dependent AIB
transport activity (Fig. 2-11). The PI fraction (lOOOxg
pellet) showed a sodium-dependence of 137 + 18 pmol AIB
per mg protein per min, while the P2 (45,000xg pellet) and
the P3 fractions (discontinuous sucrose gradient pellet)
showed 614 + 24 and 631 +_ 18 pmol AIB per mg protein
per min, respectively (Fig. 2-11). It appears, from these
results, that either sucrose-gradient centrifugation or a
differential centrifugation procedure at 45,000xg will
result in a similar level of specific activity for System A-
mediated transport. The data from either of these two


82
Materials and Methods
Materials
Cholic acid was obtained from Sigma Chemical Company
and was recrystallized three times in ethanol as described
by Kagawa and Racker (1971). Ultrapure urea was obtained
from Pierce. Protein concentration by ultrafiltration in a
stirred cell was performed using an Amicon model 8010 or
8050 cell fitted with a YM-30 membrane. Asolectin was
obtained from Associated Concentrates and was stored at
-20C. (U-*4C)-sucrose in 20% ethanol was obtained
from ICN. The detergent NP-40 was obtained from Particle
Data Laboratories, Inc. and octyl-glucoside was supplied by
Calbiochem. Hydroxylapatite (Bio-Gel HTP) and all reagents
for electrophoresis were obtained from Bio-Rad. All experi
ments requiring sonication were performed at 22C in a bath
sonicator supplied by Laboratory Supplies Co., Inc. All
other reagents were obtained from Sigma Chemical Company.
Preparation of EAT Cell Membrane
EAT cell membranes were prepared as described by Racker
et al ( 1984 ). 75 Swiss white mice (Charles River--19-21 g)
were injected intraperitone ally with 0.25 ml of EAT cell sus
pension which had been removed from mice and filtered
through two layers of cheesecloth. After 8-10 days, the
mice were sacrificed by cervical dislocation, the intraperi-
toneal cavity was carefully opened, and the ascites fluid
was collected and filtered through two layers of cheese-


39
structure (Christensen and Handlogten, 1977); therefore,
alanine was chosen as an additional test substrate.
Lithium-dependent alanine transport via System ASC has been
demonstrated for isolated rat hepatocytes (Ki 1 berg et al.,
1981; Kilberg et al., 1979; Edmondson et al., 1979),
although Quinlan et al. (1982) reported that the rate of
Li+-dependent alanine uptake by liver plasma membrane
vesicles was only 15% of the corresponding rate in Na+.
Alanine uptake mediated by System A, estimated by subtrac
ting the alkali-ion dependent velocity in the presence of
MeAIB from that seen in the absence of the inhibitor, was
249 and 88 pmol per mg protein per min in Na+ and Li+,
respectively (Table 2-2). In contrast, the rates of alanine
transport by System ASC, as monitored by MeAIB-insens itive
uptake, were the same in the presence of Li+ and Na+
(Table 2-2). These data are in agreement with those
obtained with intact hepatocytes and demonstrate that in iso
lated vesicles System ASC accepts Li to a greater degree
than does System A. They also provide additional evidence
for the heterogeneity of alanine transport in the isolated
plasma membrane vesicles.
One of the established tests for distinguishing between
Systems A and ASC is the greater degree of inhibition of the
former system by increased H+ concentration in the uptake
buffer (LeCam and Freychet, 1977). To further demonstrate
the heterogeneity of Na+-dependent alanine transport in
isolated liver membrane vesicles, we assayed alanine uptake
in the presence or absence of MeAIB at pH values between 5.5


84
before. The pellet was gently homogenized and centrifuged
again at 27,000xg for 30 min. The pellet was resuspended as
before and centrifuged at 27,000xg for 30 min. The final
pellet was resuspended in a small volume of sucrose-EDTA-
PMSF and frozen at -70C in 0.5 ml aliquots (total yield =
200-300 mg per 150 g of packed cells).
Oetergent-Extraction of EAT Cell Membrane
EAT cell membrane was extracted with detergent as
described in the figure legends or in the text. The details
of the protocol for optimized extraction of the EAT mem
branes using cholate and urea by the technique of McCormick
et al. (1984) is described here. Twenty mg of EAT cell mem
brane protein was mixed with 10 ml of solubilization buffer
(2.5% cholic acid, 4 M urea, 0.1 mM EDTA, 100 mM NaCl, 5 mM
Tris-HCl, pH 7.4) at a protein concentration of 2 mg/ml at
40C for 30 min. The mixture was centrifuged at 125,000xg
for 45 min. The supernatant was removed and placed in a dia
lysis bag, then the suspension was dialyzed overnight
against 100 volumes of dialysis buffer (0.2% C^Eg,
5 mM Tris-HCl, 0.1 mM MgCl2, 0.1 mM CaCl2, 100 mM
KC1, 1 pM PMSF, pH 7.45) at 4C. The dialysate could be
stored at -70C for up to one month with minimal loss of
activity. After the determination of protein content by a
modified Lowry procedure (Bensadoun and Weinstein, 1976),
the suspension was prepared for reconst itution .


142
7. The enzyme activity is expressed in terms of pmol of
P.+re]_eased per mg protein per hour.
N ,K -ATPase activity is the difference between
the activity in the presence and in the absence of
ouabain.
Reference: Kilberg, M. S., Christensen, H. N. (1979)
Biochem. J_8, 1525.


4
System ASC was also originally characterized in EAT
cells {Christensen et al., 1967). It was detected because
the System A-specific probe, MeAIS, did not completely inhi
bit Na+-dependent alanine, serine, or cysteine uptake.
System ASC prefers neutral amino acids with small, polar
sidechains with particular affinity for those containing
oxygen or sulfur atoms, such as serine, threonine, and cys
teine. In rat hepatocytes, Na+-dependent cysteine
uptake was observed to be totally insensitive to MeAIB inhi
bition and cysteine is considered, therefore, to be a selec
tive probe for hepatic System ASC activity (Ki1 berg et al.,
1979). System ASC is not as sensitive as System A to lower
ing the pH of the medium and, in liver tissue, it readily
accepts Li+-for-Na+ substitution. System ASC also
shows a high degree of stereospecificity for l-amino acids
and appears to be ubiquitous, as it has been found in all
eukaryotic cells so far tested (Shotwell et al., 1983).
System G1y is a Na+-dependent glycine-specific
transport system first described in rabbit reticulocytes
(Winter and Christensen, 1965), but is also present in
pigeon erythrocytes, rat hepatocytes, and hepatoma cells.
Sodium-dependent glycine uptake is not restricted entirely
to System Gly in rat hepatocytes because MeAIB inhibits a
portion of the transport (Christensen and Handlogten, 1981)
System 3 is a Na+-dependent transport system which
supports the uptake of 3-amino acids. Taurine has served
as a model substrate for System 3, but 3-alanine can also
be used as a selective substrate. System 3 has been well


88
for radioactivity. Visible turbidity was observed in the
void volume (fractions 16-20) for both samples. The total
dpm applied to the column was 98,000 and 77,560 for the
trapped sample and for the control sample, respectively.
The total dpm recovered in the lipid-containing fractions
(void volume) was 21,896 for the trapped sample and 2790 for
the control sample. The remaining dpm were recovered in a
broad peak beginning at fraction 35. In both cases, 99% of
the total dpm applied to column were recovered after wash
ing.
Hydroxy1apat ite Chromatography of Detergent-Extracts
Hydroxylapatite was prepared by suspending the crystals
in 6 volumes of low-salt dialysis buffer (0.2%
C12Eg, 10 mM K2HP04, pH to 7.45 using HC1).
The suspension was swirled gently and the fines were poured
off after the crystals were allowed to settle for 10 min.
The crystals were resuspended and the fines were poured off
two more times then the slurry was poured into a 0.7x10 cm
column which was half-filled with low-salt buffer at 4C.
A buffer reservoir was placed 72 cm above the head of the
column and the column was washed with 3 volumes of low-salt
dialysis buffer (flow rate = 36 ml/h). For a typical experi
ment, 10 mg of detergent-extract was charged onto the column
and 1 ml fractions were collected while washing with low-
salt dialysis buffer. The column was eluted step-wise using
buffers of increasing ionic strength up to 0.5 M K 2 H P 0 4.
Protein-containing fractions (determined by absorbance at


pmol AIB*mg~ protein
44
[sue rose] t M'
0
4
5


100
Detergent-Extraction and Reconstitution Using
129
The final stage of reconstitution of transport activity
requires detergent-extraction of membrane proteins to remove
them from their native environment. Fig. 3-5 depicts an
experiment in which EAT cell membranes (5 mg protein/ml)
were extracted for 30 min at 4C with increasing concentra
tions of the non-ionic detergent, polyoxyethylene-9-1auryl
ether (C^Eg). Longer periods of extraction resul
ted in loss of reconstituted System A activity. The
detergent-extracts were then reconstituted using the optimal
lipid to protein ratio of 20:1 and the optimal cholate to
protein ratio of 3:1. Each mixture was then diluted by 4-
fold and centrifuged to collect the proteoliposomes. In an
additional series of experiments, dilutions of up to 40-fold
did not yield any significantly higher transport activity
than the 3 to 5-fold dilution used typically. The results
of Fig. 3-5 indicate that the highest level of transport
activity was obtained when the membranes were extracted at a
detergent concentration of 0.2% C^Eg. Higher con
centrations of C12E9 resulted in lower transport
activity. In a separate experiment, protein-extracts
obtained with 0.2% C^Eg and 0.5% C^Eg were
reconstituted and then diluted to the same final concentra
tion of C^Eg (0.03%). The System A activity was
941 + 131 and 445 + 110 pmol of AIB accumulated per
mg protein per min for the 0.2% and the 0.5% extracts,


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
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 Ooctor of Philosophy.
u. /2L
Charles M. Allen
Professor of Biochemistry and
Molecular Biology
degree of Doctor of Philosophy
Biochemistry
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~-tf'gre'$ of Doctor*-pf /hi 1 osophy.
Richard P. Boyce
Professor of Bioche
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.
Vincent Chau
Assistant 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.
}u< (
Merle A. Battiste
Professor of Chemistry


38
TABLE 2-2
Alkali-Ion Specificity for Systems A and ASC
in Rat Liver Plasma Membrane Vesicles
Membrane vesicles (2.5 mg protein/ml) were diluted into
Buffer A containing 10 mM MgCl? and one of the following:
120 mM NaCl, 120 mM LiCl, or 120 mM KC1. Incubation of the
vesicles at 22C in the presence of 200 pM substrate was
for 1 min (alanine) or 5 min (AIB). All other assay condi
tions were the same as described in the text. The veloci
ties are expressed in terms of pmol amino acid accumulated
per mg protein per unit time. In parentheses, the Li-
dependent data arj_e shown as a percent of control values
determined in Na The results are reported as the aver
ages + S.D. of three determinations.
Substrate
Tested
Alkali ion
Velocity
Na+- or Li+-
dependent uptake
AIB
Na +
371 + 12
196 (100)
Li +
217 + 7
42 (21)
K+
175 + 16

A1anine
Na +
654 + 37
542 (100)
Li +
526 + 57
414 (76)
K +
112 + 10

Alanine
Na +
405 + 55
293 (100)
+ 5 mM MeAIB
Li +
438 + 42
326 (111)
K +
112 + 10


pmol mg*1 proltin
35
500
400
300
300
100
880
660
440
220
0
500
400
300
200
100
0
700
560
420
280
140
0
M inure t


55
Na+-dependent overshoot was too rapid to measure. In
order to demonstrate the peak of Na+-dependent AIB uptake,
sulfate was used to replace thiocyanate as the counter-anion
in the uptake buffers. The lower 1ipophi1icity of the
sulfate anion slowed the Na+-dependent uptake so that the
peak of the Na+-dependent overshoot could be measured
accurately.
Fig. 2-8 illustrates the time course for the Na+-
dependent uptake of AIB in normal or glucagon-treated vesi-
* +
cles It is evident that the Na -dependent AIB
uptake in the glucagon-stimulated vesicles is increased at
least 2-fold over the normal vesicles. The System A activ
ity peaks at approximately 3 min in the control membranes,
whereas the maximal uptake occurs at only 1 min in the
glucagon-treated vesicles. The higher rate of decline of
the Na+-dependence in the glucagon-treated vesicles
compared to the normal vesicles is probably due to either a
more rap id dissipat i on of the trans-membrane Na+ gradi
ent or a faster equilibration of AIB across the membrane.
All subsequent assays of AIB transport that were designed to
make comparisons between control and hormone-treated vesi
cles were performed for 15 s in sulfate-containing buffers.
These data indicate that the glucagon-induced activity is
retained in the membrane vesicles isolated from those same
cells.

For brevity, the vesicles prepared from the hepato-
cytes that were isolated from the glucagon injected rats
will be referred to as "glucagon-treated vesicles".


BIOGRAPHICAL SKETCH
Mark Schenerman was born on February 20, 1959, in
Plainfield, New Jersey. He received his Bachelor of Science
degree in 1980 from the University of Maryland School of
Medical Technology. From the spring of 1980 until the fall
of 1981, he worked as a Medical Technologist at the Univer
sity of Maryland Hospital and Kernan Hospital in Baltimore.
In the fall of 1981, he began his graduate education in the
Department of Biochemistry and Molecular Biology at the Uni
versity of Florida, working under the direction of Dr.
Michael S. Kilberg. After receiving his doctoral degree, he
will continue his training as a post-doctoral fellow in the
laboratory of Dr. Efraim Racker at Cornell University in
Ithaca, New York.
156


12
effects of inhibitors of protein and RNA synthesis on trans
port activity; therefore, many questions remain as to the
exact mechanisms that exist for the regulation of System A
transport. For example, how does a hormone like glucagon
activate transport activity in a protein synthesis-indepen-
dent manner? What other evidence besides kinetics and
cycloheximide-sensitivity can be collected to support the
notion that hormone-induction, amino acid-deprivation, or
cellular transformation causes an increased number of System
A carrier molecules to be inserted into the plasma membrane?
How does the amino acid-dependent decay process relate to
the regulation of transport activity? How is transport regu
lated by trans-effects?
The strategy we have used to begin our study of these
questions is to prepare isolated membrane vesicles which
mediate System A transport. These membrane vesicles can be
used in studies of trans-effects, hormonal, and adaptational
control to establish if these modes of regulation are associ
ated with the plasma membrane. For example, the argument
that hormones, amino acid-deprivation, or transformation
causes an increased number of System A carriers to be inser
ted into the plasma membrane would be strengthened if it
could be determined that transport activity was increased in
membrane vesicles prepared from those induced cells. Like
wise, if trans-inhibit ion could be demonstrated in isolated
membrane vesicles, it would support the notion that trans
inhibition is a membrane-associated phenomenon.


132
The recent successes of various groups in partially
purifying transporter proteins provides support for the
concept that transporter proteins can be isolated and
studied in the purified state* Unfortunately, the isolation
of the System A transporter has proven more difficult
because there are no specific inhibitors available like
phlorizin for the Na+-dependent glucose transporter or
cytochalasin B for the Na+-independent glucose trans
porter from red blood cells. Another difficulty is that the
carrier will likely be present in a very low amount; an esti
mate of the amount of carrier protein expected to be present
in the membranes can be performed if several assumptions are
made. If it is assumed that the transporter is a single
polypeptide of 50 kDa and that there are approximately
-12
10 moles of transporters per mg of membrane protein
(as is the case for the glucose carrier in adipocytes), then
there should be about 50 ng of transporter protein per mg of
membrane protein. It should, therefore, take about 20 mg of
membrane protein to isolate 1 pg of transporter protein, an
amount easily observable by SDS-PAGE and coomassie stain.
Kaback's group, who have investigated the lactose
carrier in Escherichia coli, has shown that a great deal of
information can be obtained about the functional operation
of a carrier protein once it has been reconstituted and
isolated (Newman et al., 1981). Future research in the
field of nutrient transport will focus on several
fundamental questions. What signals a cell to increase its
transporter activity in response to extracellular stimuli


120
the fractions were dialyzed against 0.2% C^Eg, 100
mM l^HPO^, 1 mM PMSF, pH 7.45. It is unclear from
these negative experiments why System A activity cannot be
recovered after ammonium sulfate treatment. Separation of
polypeptides forming a multimeric protein complex was tested
for by treating the extract with 20% ammonium sulfate for 30
min and then dialyzing it immediately without centrifuga
tion. No System A activity was measurable suggesting that
ammonium sulfate exposure may cause an irreversible inactiva
tion of the carrier.
SDS-Polyacrylamide Gel Electrophoresis of Membrane-
Extracts
To compare the protein composition of the detergent-
extracted membrane proteins and the reconstituted proteolipo
somes with native EAT cell membranes, the proteins were
resolved by SDS-po1yacrylamide gel electrophoresis (Fig.
3-10). Lane A shows the proteins present in the native EAT
cell membranes. When compared to the 100,000xg supernatant
of the cholate/urea extract (Lane B), there is no readily
apparent enrichment of any individual proteins although
several proteins appear to have been decreased in concentra
tion by the extraction process. The insoluble pellet
( 100,000xg) of the cho1 ate/urea-extract (Lane C) appears to
be poorly resolved in the high molecular weight range
although some individual proteins are visible around 50 kDa.
The poor resolution is probably due to aggregation because
the pellet was difficult to solubilize in the sample


90
albumin (66 kDa), and egg albumin (45 kDa). Gels were
silver-stained as described by Wray et al* (1981).
Results
Enzyme Marker Analysis of EAT Cell Membranes
EAT cell membranes prepared as described in the Methods
section were analyzed for enzyme markers to determine the
plasma membrane content and the degree of contamination by
microsomes and mitochondria. The membranes showed no 5'-
nucleotidase activity, which is consistent with results
reported by Rittenhouse et al. (1978) for EAT cell mem
branes. Ouabain-inhibitable Na+,K+-ATPase activity
showed a 12-fold increase over the homogenate indicating
enrichment of plasma membrane (Table 3-1). NADPH:cytochrome
c reductase showed no significant decrease in enrichment,
indicating some microsomal contamination. The succin
ate: cytochrome c reductase activity was enriched approxi
mately 3-fold in the membrane vesicles (Table 3-1) suggest
ing a significant level of mitochondrial contamination.
Optimization of Conditions for Membrane-Fragment Reconstitu
tion
Several approaches were possible to begin the optimization
of conditions for reconstitution of System A transport
activity. The approach that we chose was to begin using mem
brane fragments and vary the incubation conditions during
the freeze-thaw step until optimal activity was obtained.


126
could be increased. Hydroxylapatite chromatography, which
separates proteins primarily on the basis of hydrophobicity,
revealed that System A activity probably bound to the cry
stals but after elution with high-salt buffers by a variety
of methods no increase in specific activity was observed.
Following ammonium sulfate fractionation, System A activity
was present in the supernatant of an extract brought to 30%
of saturation and centrifuged. The extract did not show any
increase in specific activity and only 20% of the total
transport units treated were recovered.
There have been other reports of irreversible inactiva
tion of Na+-dependent transporters during the course of
purification as assayed by reconstitution. For example,
Radian and Kanner (1985) performed a partial purification of
the Na+-dependent y-aminobutyric acid (GABA) trans
porter from rat brain membranes by ammonium sulfate fraction
ation, OEAE-cel 1ulose chromatography, and hydroxylapatite
chromatography. The protein-containing fractions were
tested for activity by forming proteoliposomes in the pre
sence of excess lipid by centrifugation through a Sephadex
G-50 mini-column. These authors concluded that a resulting
60-70% loss of activity was due to the difficulties in wor
king with low protein concentrations as well as the high
detergent:protein ratio required to maintain the carrier in
the solubilized state.
Analysis by SDS-PAGE of the native EAT cell membranes
along with the detergent-extract and the reconstituted pro-
teoliposomes revealed that most of the proteins observed in


Fig. 2-6. Effect of Incubation Temperature on the
Naf-Dependent Uptake of AIB. Membrane vesicles (2.5 mg
protein/ml) were incubated at either 22C (), 15C, ()
or 4C (A) for 15 min prior to assay of System A activity
as described in the Methods section. The uptake was per
formed as usual except that the uptake buffer was pre
incubated at the indicated temperature and the uptake was
performed for the indicated period of time at the appropri
ate temperature.


ACKNOWLEDGMENTS
I would like to thank my mentor, Dr, Michael S.
Kilberg, for his patience and understanding even in the
midst of my impatience. I would also like to thank Dr.
Efraim Racker for his kind advice and hospitality during my
visits to Ithaca. Finally, I would like to thank Mary
Handlogten, Donna Bracy, and Tom Chiles for their friendship
and assistance during the course of my studies.
Hi


pmol AIB*mgl protein.min
94


50
vesicles with a Na+-selective ionophore such as gramici
din or monensin (Pressman, 1976), the rate of Na+-
dependent transport is decreased significantly (Table 2-3).
To demonstrate that the membrane vesicles retained
their native permeability with respect to anions, and to
show that the transport occurred by an electrogenic process,
Na+-dependent alanine uptake was measured in the presence
of different counter-anions. If Na+-dependent uptake of
200 pM alanine in NaSCN-containing buffer was set equal to
100% (698 pmol per mg protein per min), the Na+-dependent
transport rates in NaCl or Na^SO^ were 86% and 73%,
respectively. The ability of an anion to cross a lipid
bilayer depends on the lipophilic nature of the anion; order
ing the anions used from most lipophilic to least lipophilic
is as follows: thiocyanate > chloride > sulfate. Hence, the
order of effectiveness for these anions in increasing trans
port activity parallels their relative lipophilic properties
and illustrates the electrogenic nature of the process (more
lipophilic anions permeate the membrane more rapidly causing
an increase in membrane potential).
Trans-Inhibition of System A in Membrane Vesicles
The activity of System A is decreased considerably when
the cytoplasmic concentration of its substrates is elevated
(Kelley and Potter, 1978). This phenomenon, referred to as
"trans-inhibition", is cycloheximide-insensitive and gener
ally is thought to occur because the amino acids bind to the
carrier and "lock" it in the cytoplasmic orientation (White


42
pH


Table 1-1
Characteristics of Na-Dependent Neutral Amino Acid
Transport Systems
System
A
ASC
Gly
13
N
E xamp1e
Substrates
Ala, G1y,
Pro, AIB
Ala, Ser,
Cys
Gly, Sarc
13-Ala,
Tau
Asn, Gin
His
Spec i f i c
substrates
Me AI B
Cys, Thr
Gly
T au
Gin
Trans-ef Feets
i n h i b.
st i m.

?
m
?
*
Effect of
low pH
i n h i b .
variable
none
?

inhib.
Stereo -
specificity
moderate
high

?
*
high
Adaptive
control
yes
no
no
no
yes
Hormonal
control
yes
no
no
no
some


37
Hepatic Na+-dependent glycine transport is mediated
by Systems A, ASC, and Gly (Christensen and Handlogten,
1981). System Gly activity can be assayed selectively by
measuring Na+-dependent glycine uptake in the presence of
an amino acid that can inhibit efficiently the other two sys
tems. We have chosen threonine for this purpose because, in
cultured hepatocytes, this amino acid is transported effec
tively by both Systems A and ASC (Kilberg et al., 1985a).
When Na+-dependent glycine uptake was measured in the ves
icles, approximately 40% escaped inhibition by threonine
(Fig. 2 3 D). Although these data show that a significant
portion of glycine uptake occurs by Systems A and ASC, they
also demonstrate that System Gly activity can be measured
readily in isolated liver plasma membrane vesicles.
Further Evidence for Heterogeneity of Alanine Transport
For isolated rat hepatocytes, transport by System ASC
is partially retained when Li+ replaces Na+ as the
extracellular alkali ion, whereas little or no System A-
mediated uptake occurs in the presence of lithium (Edmondson
et al., 1979). Such system selectivity for Li+ is just
the reverse in the Ehrlich ascites tumor cell (Christensen
and Handlogten, 1977). To determine whether this cell-spe
cific property of System A was observed in isolated liver
membranes, Na+- and Li+-dependent amino acid trans
port was measured. Li+ substitution for Na+ caused
an 80% decrease in AIB uptake (Table 2-2). Lithium-
dependent transport depends to some degree on amino acid


72
membrane vesicles are useful for amino acid transport stu
dies by providing a system free of many of the restrictions
and complications imposed by intact cells. The charac
teristics that we have described for this vesicle system are
consistent with those of other amino acid transport studies
using isolated rat liver membranes (Sips et al., 1980b;
Samson and Fehlmann, 1982; Quinlan et al., 1982; Meier et
al., 1984b). The demonstration of active amino acid trans
port and its properties represents the first step toward
using isolated rat liver membranes to study the regulation
of System A by trans-effects, hormones, and adaptive con
trol .
Membrane vesicles facilitate the study of trans
inhibition of System A because, presumably, endocytosis and
recycling cannot occur due to the removal of intracellular
u1trastructures such as the microtubular network. Using
these membrane vesicles, trans-inhibition was detected when
the vesicles were loaded with AIB but not when the vesicles
were loaded with O-glutamine, an amino acid with no satur
able Na+-dependent uptake. Our results support the hypo
thesis that the System A carrier is locked into an internal
orientation by binding of AIB (or a Na+/AIB complex) at
the intracel1ular surface of the plasma membrane.
It is well established that glucagon treatment and star
vation of hepatocytes for amino acids results in a protein
synthesis-dependent increase in System A activity (Kilberg,
1982; Shotwell et al., 1983). The increase observed in both
cases is kinetically defined as an increase in the Vmax of


127
the native membranes were also present in the detergent-
extract. This implies that there is not a significant
amount of selective solubilization of proteins by treatment
with the cho1 ate/urea. The reconstituted proteoliposomes
incorporated some of the high molecular weight proteins pre
sent in the detergent-extract to a lesser extent; the most
intensely staining proteins in the proteo 1iposomes were poly
peptides of 56 kDa and 48 kDa. It is interesting to note
that Im and Spector (1980) observed that the predominant
Coomassie-staining proteins in their reconstituted pro-
teoliposomes from EAT membranes were 56 kDa, 45 kDa, and 40
kDa. Whether or not these proteins have any relationship to
System A activity will require further study.


25
previously described methods. Inorganic phosphate was
determined by the method of Fiske and Subbarow (1925).
Fluoride-stimulated (10 mM NaF) adenylate cyclase activity
was measured by a modification of the procedure described by
Wisher and Evans (1975). The cAMP produced was detected by
a protein-binding assay supplied as a kit by Amersham Corp.
Tests for contamination of the final membrane fraction by
intracellular membranes showed a similar profile to that
obtained by Van Amelsvoort et al. (1978).
Determination of the Extravesicular Volume
The extravesicular volume of pelleted membrane vesicles
was determined using ^C-inulin. Before use, the
14
C-inulin stock (1 mg inulin/ml; 7 pCi/ml) was fil
tered through a Gelman filter (0.22 pm) to remove particu
lates and the filtrate was counted for radioactivity.
Vesicles (2.48 mg/500 pi) were mixed with 400 pi of Buffer
A and 100 pi of ^C-inulin and the mixture was vor-
texed. After 10 pi was removed for determination of the
total radioactivity, the suspension was centrifuged at
100,000xg for 1 h and 100 pi of the supernatant was removed
for determination of the total radioactivity. The sides of
the centrifuge tube and the surface of the pellet were
washed 3 times with 3 ml aliquots of ice-cold Buffer A,
then the pellet was resuspended in 1 ml of 8uffer A and 200
pi of the suspension was removed for determination of the
total radioactivity. The total dpm remaining in the pellet
after the washes was divided by the total dpm present in the


pmol AIB mg'1 protein
30
0 10 20 30 40 SO 60
Minutes


APPENDIX A
ANALYTICAL ASSAYS AND PROCEDURES
Inorganic Phosphate Assay
Materials:
1. Molybdate reagent
(NH )£Mo,0OJI 4Ho0
Dissolve^in 75§fl ml ^
25 g/L
concentrated H^SO^.
of water and slowly add 139 ml
Dilute to 1 L with water.
2.Phosphate reducing agent
1-amino-2-naphthoi-4-su1fonic acid 0.25 g/100ml
sodium bisulfite 14.6 g/100ml
sodium sulfite 0.5g/100ml
Dissolve the mixture in warm water, filter, cool, and
recheck the volume. This solution must be prepared fresh
weekly and must be stored in a dark bottle.
3.Phosphate standard
10 mM K2HP04
0.0174 g/10 ml
Procedure:
1.Prepare the phosphate standards from the phosphate
standard stock solution as follows in 15 ml plastic
conical centrifuge tubes:
nmol Pi
H.O
stock Pi
0
1000 pi
200
980 (i 1
20 pi
500
950 Hi
50 pi
750
925 Hi
75 pi
1000
900 Hi
100 pi
1500
850 Hi
150 pi
2. Add 1 ml of 10% trichloroacetic acid (TCA) to all the
P ^ standard tubes and vortex.
3. Transfer 0.5 ml from each standard tube to a new conical
tube for assay.
4. Add 2.5 ml of water to all the standards and samples to
bring the final volume to 3 ml.
134


119
Unfortunately, the detergent-extract appeared to be sensi
tive to the high-salt conditions required for elution
because 80% of the initial activity in low-salt buffer was
lost. When the high-salt extract was dialyzed against a
low-salt buffer, no restoration of activity was obtained
indicating that the high-ionic strength conditions cause
irreversible inactivation of transport activity.
Ammonium Sulfate Fractionation of the Detergent-Extract
A large number of experiments were performed to deter
mine if the specific activity of the initial membrane-
extract could be enriched by selective ammonium sulfate frac
tionation. Cecchini et al. (1978) have reported that
Na+-dependent amino acid transport activity can be found
in the supernatant of a detergent-extract from EAT cell mem
branes brought to 20% of saturation with ammonium sulfate.
In our hands, treatment of the C^Eg extract with
ammonium sulfate at 20% of saturation or 40% of saturation
followed by dialysis of both supernatant and precipitate,
against the low-salt C12 Eg buffer resulted in com
plete loss of System A activity.
When the detergent-extract was fractionated in ammonium
sulfate at 30% of saturation and then both the soluble and
the precipitated fractions dialyzed against 0.2%
C^Eg* 100 mM Tris, 1 mM PMSF, pH 7.45, transport
activity was found in the supernatant. The recovery was
only 20% of the total activity and no increase in specific
activity was observed. Similar results were obtained when


87
expressed as pmol of AIB trapped per mg protein per min.
Unless otherwise indicated in the figure legends, the
results were from a single membrane preparation and the S.D.
of triplicate determinations was less than 10% of the mean.
Concentration of Detergent-Extracts Using Ultrafiltration
Detergent-extracts (1-2 mg protein/ml) were placed in
an Amicon ultrafi 1tration cell (10 ml or 50 ml size) fitted
with a YM-30 membrane. The apparatus was immersed in a tray
of ice water and the tray was placed on a magnetic stirring
plate. Nitrogen gas was introduced into the cell at 60 psi
while stirring was continued on ice. Concentration was
allowed to continue until the volume was reduced to 1-2 ml.
The concentrated extract was removed with a Pasteur pipette.
Sucrose Trapping in Reconstituted Proteoliposomes
Detergent-extract (0.5 mg protein/413 pi), 10 mg of
asolectin (250 pi) and 1.5 mg (15 pi) of K+ cholate
were combined with 5 pi (8.28 x 106 dpm) of de
suerse (0.746 pCi/pl; 1.21 mM) and then frozen in liquid
nitrogen. After thawing, the suspension was diluted with 2
ml of K+-uptake buffer and 100 pi was removed for deter
mination of radioactivity. A control sample was prepared in
14
which the C-sucrose was added after the freeze-thaw
step. Each suspension (100 pi) was passed over a Sephadex
G-50 column (1x16 cm; flow rate = 1.33 ml/min) equilibrated
with K+-uptake buffer. Fractions (0.22 ml) were col
lected in plastic scintillation mini-vials and then analyzed


2
Christensen ( 1963 ) as System A (Na + -dependent) and
System L (Na+-independent). Since that time, numerous
other amino acid transport systems have been defined and
characterized in animal cells (Christensen, 1984). The
present work will focus on Na+-dependent neutral amino
acid transport systems in isolated membranes derived from
rat hepatocytes and EAT cells.
At least five Na+-dependent neutral amino acid
transport systems have been described in animal cells. Some
of the characteristics of these systems are summarized in
Table 1-1. System A prefers amino acids having short,
polar, or linear sidechains such as alanine, serine, methio
nine, and glycine. Two non-metabo 1izable amino acids have
proven to be particularly useful for characterizing System A
activity. The Na + -dependent uptake of 2-ami no isobutyric
acid (AIB) and its N-methylated derivative, 2-(methylami no ) -
isobutyric acid (MeAIB), have proven to be highly specific
for System A activity, particularly in rat hepatocytes
(Ki 1 berg et a1 ., 1985a). System A is stereospecific for L-
amino acids and is strongly inhibited as the pH of the
medium is lowered (Kilberg et al., 1980). The alkali-ion
specificity of System A depends somewhat on the cell line
tested because Li + is not acceptable as a substitute for
Na+ in rat hepatocytes (Edmondson et al., 1979), but
Li -for-Na+ substitution is well tolerated by the
activity in EAT cells (Christensen and Handlogten, 1977).
System A activity has been observed in all nucleated cells
tested.


145
7. The results are expressed as pmol of cytochrome c
reduced per mg protein per min (extinction coefficient of
reduced cytochrome c = 18,500 M" x cm*1).
Reference: Kilberg, M. S., Christensen, H. N. (1979)
Biochem. 1J5, 1525.


103
respectively. Collectively, these data indicate that the
optimum concentration of detergent necessary for protein
extraction and reconstitution is 0.2% Cj^Eg ancl
the loss of activity at concentrations greater than 0.2%
C12^9 not simPly due to a higher final concentra
tion of detergent after dilution.
Maximization of Protein Solubilization Using Various Chao-
tropic Agents
Although the optimized conditions described above for
reconstitution resulted in maximal System A transport activ
ity, only 20-30% of the total membrane protein was solubil
ized using C^Eg alone. Other detergents were
tested in an attempt to improve the extraction of membrane
proteins and make more efficient use of the isolated mem
branes. Several detergents such as cholate, deoxycholate,
NP-40, CHAPS and octy1-g1ucoside were tried at concentra
tions as high as 2%(w/v). After a 30 min incubation of each
detergent with the membranes at 4C, the best solubiliza
tion achieved was only 23% of the total protein (2% NP-40).
Longer periods of incubation (6 h) with 2% C^Eg,
NP-40 or deoxycholate resulted in 30%, 45%, and 73% solubil
ization, respectively. Unfortunately, the conditions neces
sary to improve solubilization efficiency resulted in an
inability to recover active transport; none of the alternate
detergents tested, gave any activity upon reconstitution.
Even with the C^Eg extracts (0.2%), periods of
extraction longer than 30 min resulted in lower transport
activity following reconstitution.


27
TABLE 2-1
Enzyme Activities in the Plasma Membrane-Enriched Fraction
Membrane vesicles were tested for the presence of particular
enzyme markers for plasma membrane, endoplasmic reticulum, and
mitochondrial inner membrane. The activities of 5'- nucleotidase
and glucose-6-phosphat ase are expressed in terms of pmol Pi
formed per mg protein per h. Adenylate cyclase activity is
expressed as pmol cAMP formed per mg protein per h. Succin
ate : cytochrome c reductase and cytochrome c oxidase activities
are expressed as nmol cytochrome c reduced per mg protein per
min. Relative specific activity (R.S.A.) is determined by divi
ding the specific activity of the enzyme in the plasma membrane-
enriched fraction by the specific activity in the homogenate.
The data are the averages + S.D. of triplicate determina
tions.
Enzyme Activity
Homogenate
Ves i cl es
R.S.A.
5'-nucleotidase
4.1
+
0.2
38.9 +
1.8
9.4
Adenylate cyclase
9.1
+
3.6
94.2 +
8.9
10.4
Glucose-6-phosphatase
10.2
+
1.5
31.9 +
1.6
3.1
Succinate:cytochrome c
14.0
+
0.31
1.32 +
0.09
0.1
reductase
Cytochrome c
13.2
+
0.35
1.01 +
0.18
0.1
oxidase


Fig. 3- 7. Time Course of AIB Uptake into Proteoliposomes
in the Presence and Absence of Valinomycin. Detergent-
extracts were reconstituted as described in the Methods sec
tion (note that the liposomes were prepared in 120 mM KC1
uptake buffer). Reconstituted proteoliposomes were incuba
ted in 200 pM AIB uptake buffer in NaCI (B,A) or KC1
(,) for the indicated times at 22C either in the
presence (A,V) or the absence () of 10 pg/ml of
valinomycin in 95% ethanol. The control mixtures contained
the appropriate concentration of ethanol without valinomy
cin. Results are expressed as the mean of triplicate deter
minations of the pmol AIB accumulated per mg protein per
unit time. The standard deviations were less than 20% of
the means.


73
the carrier rather than a change in the Km for the test sub
strate (Shotwell et al., 1983). Taken together, the kinetic
effects and the protein synthesis-dependency are interpreted
to indicate that the stimulated transport activity is due to
a greater number of active carrier molecules in the plasma
membrane. We have utilized isolated membrane vesicles to
provide additional support to that proposal. Vesicles iso
lated from amino acid-starved hepatocytes displayed elevated
System A transport at a similar magnitude to that observed
in intact cells, a result that is consistent with the hypo
thesis.
A large number of experiments were performed in which
rats were treated with glucagon in vivo and then membrane
vesicles were prepared from isolated hepatocytes. Increased
transport activity was always detected in the freshly iso
lated vesicles and the level of stimulation paralleled, but
typically did not equal, the degree of induction observed in
the intact hepatocytes. Several explanations are possible
for the apparent incomplete retention of glucagon-stimulated
activity. We have eliminated some of these through experi
mentation including differences in membrane composition of
the vesicles preparations from normal and glucagon-treated
cells and differences in the time at which the maximal
Na+-dependent transport is observed. It is also clear
from our studies that irreversible inactivation of System A
activity in the membrane vesicles from hormone-treated hepa
tocytes contributes in a significant way to the loss of ele
vated transport activity in the isolated membranes.


89
280 nm) were pooled, concentrated, and reconstituted as
described above.
Ammonium Sulfate Fractionation of Detergent-Extracts
Detergent-extracts (20-30 mg protein) were brought to
the appropriate percent saturation of ammonium sulfate by
adding 6.61 ml of saturated ammonium sulfate solution, pH
7.45 containing 0.2% C^Eg and bringing the final
volume of the solution to 22 ml at 4C. The solution was
stirred at 4C for 30 min and then was centrifuged at
10,000xg for 10 min. The supernatant was removed and the
pellet was resuspended in the same volume of low-salt dia
lysis buffer. Both the supernatant and pellet suspensions
were dialyzed overnight at 4C with one change of buffer
against 200 volumes of 100 mM K^HPO^, 0.2%
C^Eg, PMSF, brought to pH 7.45 using HC1.
Occasionally, the pel 1et-fract ion would precipitate during
dialysis; when this occurred, it was centrifuged at 10,000xg
for 10 min and the resultant supernatant was used for fur
ther studies. The dialyzed fractions were concentrated and
reconstituted as described above.
SDS-Polyacrylamide Gel Electrophoresis
One-dimensional SDS-polyacrylamide gel electrophoresis
was performed using a 7.5% separating gel as described by
Laemmli ( 1970 ). Molecular weight markers used were: 13-
gal actos i dase (116 kDa), phosphory1 ase b (97.4 kDa), bovine


139
8. The enzyme activity should be expressed as pmol of
P. released per mg protein per hour.
Reference: Morre, D. J. (1971) Meth. Enzymol* 22,
130-148.


71
systems found in rat liver. Alanine, cysteine, histidine,
and glycine were used as model substrates for Systems A,
ASC, N, and Gly, respectively. The specificities of these
amino acids for their respective transport systems were simi
lar to those observed in intact hepatocytes (Kilberg, 1982).
The only notable difference was that Na+-dependent hist
idine uptake showed a greater degree of inhibition by MeAIB
in membrane vesicles than in whole cells (Kilberg et al.,
1980).
Several of the criteria that Lever (1980) has applied
to establish active transport of nutrient molecules by mem
brane vesicles were tested in this system. Uptake of alan
ine and histidine was stereospecific and AIB was actively
accumulated into an enclosed, osmoti cal 1y-sensitive space.
AIB uptake was also dependent on temperature and protein con
centration. AIB uptake was inhibited by the ionophores gram
icidin and monensin which collapse the trans-membrane
Na+ gradient by either forming channels for Na+ ions
(gramicidin) or carrying Na+ ions across the membrane
(monensin). The electrogenicity of transport was demonstra
ted by observing that the use of less permeant counter
anions in the buffer resulted in lower transport velocities.
The membrane vesicle preparation was shown to be
enriched in plasma membrane through measurements of 5'-
nucleotidase and adenylate cyclase activities. Enzyme
marker studies indicated that the vesicles had some contamin
at ion by microsomes, but little or no contamination by mito
chondria. Collectively, the results demonstrate that these


150
Doolittle, R. F., Hunkapiller, M. W., Hood, L. E., Devare,
S. G., Robbins, K. C., Aronson, S. A. and Antoniades, H.
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Edmondson, J. W. and lumeng, L. (1980) Biochem. Biophys.
Res. Commun. 9j>, 61-68
Edmondson, J. W. Lumeng, L. and Li, T. K. (1979) J. Biol.
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Evans, W. H. (1980) Biochim. Biophys. Acta 604, 27-64
Exton, J. H., Mallette, L. E., Jefferson, L. S., Wong, E. H
A., Friedmann, N., Miller, T. B. and Park, C. R. (1970)
Recent Prog. Horm. Res. ^6, 411-457
Eytan, G. D. (1982) Biochim. Biophys. Acta 694, 185-202
Eytan, G. D., Matheson, M. J. and Racker, E. (1975) FEBS
Lett. 57, 121-125
Eytan, G. 0., Matheson, M. J. and Racker, E. (1976) J. Biol
Chem. 251, 6831-6837
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375-400
Foster, D. 0. and Pardee, A. B. (1969) J. Biol. Chem.
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Friedmann, N. and Dambach, G. (1980) Biochim. Biophys. Acta
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52
and Christensen, 1983; Kilberg et al., 1985a). An alternate
explanation for trans-inhibition is possible. In response
to increased cellular levels of System A substrates, the
carrier may be rapidly internalized by a process analogous
to the response observed for the adipocyte glucose carrier
after removal of insulin from the medium (Lienhard, 1983;
Simpson and Cushman, 1985). Likewise, if the elevated amino
acid levels were decreased, the carrier would be shuttled
back to the plasma membrane location resulting in a protein
synthesis-independent increase in transport. The latter
response, referred to as "release from trans-inhibition",
has been demonstrated with intact hepatocytes by several
1aboratories. This "shuttle hypothesis" is made even more
plausible by recent evidence for a serum-dependent shuttling
of System A carriers in human fibroblasts (Gazzola et al.,
1984).
Isolated plasma membrane vesicles provide an excellent
model system to test whether trans-inhibition involves inter
nalization of endocytic vesicles, and therefore requires in
tact cells. If the phenomenon can be demonstrated in plasma
membrane vesicles, the results would argue that trans
inhibition is, indeed, a membrane-associated response and
not dependent on extensive cellular machinery or architec
ture. The results of Table 2-4 show that vesicles previ
ously loaded with MeAIB exhibited a 26% reduction in measur
able System A activity. When D-glutamine was used, an amino
acid with little or no saturable transport in hepatocytes,
no decrease in activity was observed. These data indicate


20
other chemicals were reagent grade or better and were
obtained from Sigma Chemical Company. Rats were from a col
ony maintained by the University of Florida Animal Resources
facility.
Hepatocyte Isolation and Transport Assay
Hepatocytes were isolated from male Sprague-Dawley rats
( 100-200 g) as described previously (Ki1 berg et al., 1983).
Usually, more than 90% of the cells were viable as deter
mined by the trypan blue exclusion assay. Both the control
and the hormone-treated rats were fasted overnight prior to
cell isolation. The experimental animals were injected with
1 mg of glucagon per 100 g body weight 4 h before surgery.
A small portion of both control and hormone-treated hepato
cytes was suspended in Na+-containing Kreb's-Ringer bi
carbonate buffer (NaKRB) containing 0.1 mM cyclohexi mide
(CHX) and placed in monolayer culture. Following a 2 h cul
ture period, the activity of System A was determined by
assaying the Na+-dependent transport of 50 pM AIB for 1
min at 37C as described by Ki 1 berg et al ( 1983 ). The
remaining cells were resuspended in 40 ml of ice-cold Buffer
A (0.25 M sucrose, 0.2 mM MgCK, 10 mM HEPES-K0H, pH 7.5)
for preparation of a plasma membrane-enriched subcellular
fr action .


70
fractions indicate that System A activity is greater in
membrane vesicles from human hepatoma cells when compared to
the transport observed in vesicles from normal rat
hepatocytes (Fig. 2-8). When vesicles from the HepG2 cells
were prepared by similar centrifugation methods, but using
either bath sonication or nitrogen cavitation for cellular
disruption, the results were the same. Furthermore,
membrane vesicles from a rat hepatoma, H4-II-EC3, also
retained elevated rates of System A activity (data not
shown). These data argue that the increased transport
activity observed in transformed liver cell lines is
retained in membrane vesicles prepared from those cells.
Discussion
Membrane vesicles have been prepared from either rat
liver tissue or isolated rat hepatocytes. These vesicles
actively accumulated AI3 and more than 85% of the AIB accumu
lation was inhibited by the System A-specific analog, MeAIB.
These data indicate that A13 is useful as a selective probe
for System A activity in isolated membrane vesicles. The
accumulation of AIB was energized by the imposed trans
membrane Na+ gradient. The uptake of AIB was against a
concentration gradient as indicated by the distribution
ratio which was greater than one at the maximal overshoot
point.
The vesicles also actively accumulated typical sub
strates of the other sodium-dependent amino acid transport


130
the cell can "lock" the System A carrier into an internal
orientation. The question of whether or not trans
inhibition also causes internalization of System A transport
ers could be addressed using vesicles formed during the sub-
cellular fractionation.
My studies on the reconstitution and purification of
the System A carrier in EAT cells will continue in the labor
atory of Dr. Efraim Racker. Attempts to isolate the carrier
will be made using various protein purification techniques
utilizing reconstitution to test for System A activity.
Using the reconstitution system developed for EAT cell mem
branes as a model, Donna Bracy in our laboratory has suc
ceeded in reconstituting System A activity from rat liver
plasma membrane extracts. We find that the glucagon-
stimulated transport activity seen in whole cells is
retained in the proteoliposomes. The proteo 1iposomes from
glucagon-treated cell membranes have similar levels of trans
port activity as those from EAT cell membranes. It is known
that exposure of cells to EGF stimulates System A activity
but none of the biochemical steps from the binding of the
hormone to its specific membrane-bound receptor until the
observed increase in transport activity are known. Studies
reconstituting the detergent-extract with purified EGF recep
tor, followed by EGF treatment of the proteo 1iposomes may
begin to answer some questions regarding signalling System A
to increase its activity.
Future studies in Dr. Kilbergs laboratory will be
directed toward identifying the System A-associated protein


21
Rat Liver Plasma Membrane Isolation
Plasma membrane vesicles were prepared as described by
Van Amelsvoort et al. (1978). The liver of a 24 h-fasted
male Sprague-Dawley rat (150 to 200 g) was perfused with an
iso-osmotic homogenization buffer consisting of 0.25 M
sucrose, 0.2 mM MgCl2, 10 mM HEPES-K0H, pH 7.5 (Buffer
A). All subsequent procedures were carried out at 4C.
The blanched liver was removed and homogenized with 22
strokes using a Potter-Elvehjem homogenizer with a motor-
driven, loose-fitting teflon pestle. After the addition of
E0TA to a final concentration of 1 mM, the homogenate was
forced through a nylon screen (75 pm) and then centrifuged
at 1000xg for 10 min. The supernatant and the loose upper-
layer of the pellet were saved and the remaining solid
pellet was resuspended in Buffer A containing 1 mM EDTA.
This suspension was centrifuged again at 1000xg for 10 min.
The resulting supernatant was collected as before, pooled
with the previous one, and then centrifuged at 20,000xg for
30 min. The loose upper-layer of the pellet was collected
and resuspended in Buffer A containing 1 mM EDTA by passing
the material through a 19 ga. needle six times. This mem
brane fraction was purified further by placing it on a dis
continuous sucrose gradient composed of 20%(w/v) and
39.5%(w/v) sucrose each containing 10 mM HEPES-K0H, pH 7.5.
The gradients were centrifuged at 50,000xg for 2.5 h. Vesi
cles enriched in plasma membrane were collected from the
20^/39.5% sucrose interface and diluted 1:1 with 0.2 mM
MgCl^, 10 mM HEPES-K0H, pH 7.5. These membranes were


151
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96, 217-229
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18, 1525-1530
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(1985b) J. Cell. Physiol. 122, 290-298
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(1980) J. Biol. Chem. 255, 4011-4019
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Physiol. 114, 45-52


117
14
contained the proteoliposomes When the C-sucrose was
added before the freeze-thaw step, 22% (21,896 dpm) of the
total dpm (98,000 dpm) added to the column were found in the
void volume (i.e., associated with the liposomes). When
^C-sucrose was added after the freeze-thaw step, only 4%
(2790 dpm) of the total dpm (77,560 dpm) applied to the
column were found in the void volume. In both cases, most
of the remaining dpm were eventually eluted in a single
peak. Greater than 95% of the total dpm applied to the col
umn were recovered after the elution process. These results
are in good agreement with Pick (1981) who observed, using a
similar procedure, that 19.6% of the total sucrose added was
trapped in proteo 1iposomes prepared by the freeze-thaw tech
nique.
Miscellaneous Properties of the Detergent-Extract
Several experiments were performed in which the
detergent-extract was concentrated after dialysis using an
Amicon stirred-cell ultrafiltration concentrator under nitro
gen pressure. Usually, greater than 90% of the initial pro
tein and 80% of the total transport units were recovered
after concentration of 5-fold. The concentration procedure
was performed on ice to minimize the degradation of the pro
teins within the extract. Other techniques for concentra
tion of the proteins in the extract, including Amicon Centri-
con 10 microconcentrat or tubes, proved unsatisfactory due to
aggregation of protein on the filter. The stirred-cell


ABBREVIATIONS USED
AI B
2-ami noisobutyric acid
C12 E 9
polyoxyethylene-9-1auryl ether
cAMP
adenosine 31:51 eye Tic monophosphate
CHAPS
3-[(3-cholamidopropyl)dimethyl-ammonio]-
1-propanesulfonate
CHX
cycloheximide
CMC
critical micellar concentration
EAT
Ehrlich ascites tumor
EDTA
ethylenediamine tetraacetic acid
EGTA
ethyleneglycol-bis-(6-ami no-ethyl ether)
N, N'-tetraacetic acid
FBS
fetal bovine serum
GABA
Y-aminobutyric acid
HEPES
4-(2-hydroxyethyl )-l-piperazineethane-
sulphonic acid
HepG2
human hepatoma cell line
MeAIB
2-(methylamino)-isobutyric acid
MEM
Eagle's minimal essential medium
NADPH
reduced nicotinic dinucleotide phosphate
NaKRB
sodium-containing Kreb's-Ringer bicarbonate
buffer
NP-40
non-ionic industrial detergent
NRK-49F
normal rat kidney cell line


octyl -
g1 neos i de
N-octyl-B-O-glucopyranoside
osM
osmolarity
Pi
inorganic phosphate
PMSF
phenyl methylsulfonyl fluoride
R.S.A.
relative specific activity
sarc
sarcosine
S.D.
standard deviation
SOS
sodium dodecyl sulfate
t au
taurine
TCA
trichloroacetic acid
PAGE
po1yacrylamide gel electrophoresis
X


nmol mg"' protein*2h-1
- M
0> M
o o o o o


UNIVERSITY OF FLORIDA
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3 1262 08554 7940


Fig. 2-4. Effect of pH on Alanine Uptake in Membrane
Vesicles. Na -dependent alanine uptake was assayed in the
presence () or absence (A) of 5 mM MeAIB. The pH of the
uptake buffers was varied from 5.9 to 8.1. All other assay
conditions were the same as those described in the Methods
section. Data are presented as the+averages + S.D. for
triplicate determinations of the Na -dependent transport.


19
acid transport are found in the basolateral and the canalicu
lar surfaces. Increased Na+-dependent alanine transport
has been reported in rat liver vesicles after starvation of
the animals (Quinlan et al., 1982) or after treatment of
hepatocytes with dibutyryl cAMP (Samson and Fehlmann, 1982).
In our work, we have demonstrated that membrane vesi
cles from rat liver can be prepared and these vesicles activ
ely accumulate amino acids. The vesicles were also prepared
from glucagon-treated hepatocytes as well as amino acid-
starved hepatocytes in order to show that increased System A
transport observed in the intact cells was retained in the
membrane vesicles. Vesicles were also prepared from a human
hepatoma cell line (HepG2) and those vesicles retained
increased System A activity as is observed in intact cells.
Materials and Methods
Mater i a 1s
The rad i o 1 abe 11ed compounds used were [carboxyl-
14 1
C] inulin, [methyl- H] 2-aminoisobutyric acid (AI8),
ICN Pharmaceuticals; L-[^H] cystine, Schwarz/Mann; L-[2,5-
^H] histidine, [2-^H] glycine, L-[2,3-^H] alanine, and
14
3-0-methyl-D-[U C] glucose, Amersham. Filters used for
transport assays were either Millipore type HAWP (0.45 pm)
or Gelman type GN-6 (0.45 pm). Highly purified glucagon
was a generous gift from Dr. Mary Root of Lilly Laborator
ies. Fetal bovine serum (FBS) and Eagle's Minimal Essential
Medium (MEM) were obtained from Flow Laboratories. All


LIST OF TABLES
Page
1-1 Characteristics of Na+-Dependent Neutral
Amino Acid Transport Systems 3
2-1 Enzyme Activities in the Plasma Membrane-
Enriched Fraction 27
2-2 Alkali-Ion Specificity for Systems A and ASC
in Rat Liver Plasma Membrane Vesicles 38
2-3 Effect of Gramicidin or Monensin on AIB Uptake.. 51
2-4 Trans-Inhibition in Isolated Plasma Membrane
Vesicles 53
2-5 Glucagon Stimulation of System A in Rat
Hepatocytes is Retained in Isolated Plasma
Membrane Vesicles 59
2-6 Enzyme Activities in Membranes from Control
and Glucagon-Treated Hepatocytes 61
3-1 Enzyme Marker Activities in EAT Cell Membranes.. 91
3-2 Reconstitution of System A Activity into
Proteoliposomes Following Detergent
Extraction of EAT Cell Membranes 105


54
that trans-inhibition can occur in isolated membrane
vesicles and probably does not depend on internalization of
the System A carrier as is observed for the down-regu1 at ion
of the insulin receptor. It is important to note that these
data could also be explained by competitive inhibition of
AIB uptake caused by MeAIB trapped in the extravesicular
volume. The data should be regarded with caution until the
concentration of MeAIB in the extravesicular volume is
actually determined.
Effect of Glucagon-Treatment in vivo and Amino Acid-
Starvation in vitro on System A Activity in Plasma Membrane
Vesicles
It is well documented that glucagon-treatment, either
H vivo (Handlogten and Kilberg, 1984) or j_n vitro
(LeCam and Freychet, 1976; Pariza et al., 1976), causes a
protein synthesis-dependent increase in hepatic System A
transport activity. Our laboratory has provided evidence
that the hormone-induced molecule responsible for the stim
ulation of System A activity is a glycoprotein (Barber et
al., 1983). The stimulation by glucagon is generally consid
ered to be the result of ^ie novo synthesis of carriers or
carrier-associated molecules and their subsequent insertion
into the plasma membrane. To determine whether the System
A-associated glycoprotein induced by glucagon is also
located in the plasma membrane, the System A activity in
membrane vesicles from hepatocytes taken from either normal
or glucagon-treated rats was first tested using thiocyanate
as the counter-anion in the uptake buffer, but the


106
activity in membrane-fragment reconstitution assays. All of
the remaining studies employed the cholate/urea solubiliza
tion procedure so as to benefit from the increased extrac
tion efficiency.
Temperature Stability of the Deter gent-Extract
Fig. 3-6 illustrates the results of an experiment in
which the EAT cell extract in 0.2% ^12^9 was ^ncu^a"
ted at 4C or -70C for varying periods of time and then
the extract was reconstituted using the optimized conditions
described above. During the course of incubation at 4C,
the activity of System A continued to decay at a reasonably
steady rate of about 60-70 pmol AIB per mg protein per min
every 24 h (the data at 2 h are probably the result of an
artifact in the reconstitution of those membrane proteins).
Incubation of a different membrane- extract preparation
(note the inherent difference in transport activity between
the two preparations) at -70C for 10 days did not appear
to have a significant effect on the ability to reconstitute
transport activity (Fig. 3-6). These data indicate that the
detergent-extracted membrane proteins can be stored frozen
for more than a week, but purification procedures requiring
incubation at 4C, will result in a continual loss of trans
port activity. Any estimate of carrier enrichment by speci
fic activity will have to be corrected to account for the
concomitant decay of activity.


148
3.
C12^9 dial>sls
0. c% CEq
5 mM TrTs-HCl
0.1 mM MgCl9
0.1 mM CaCl
100 mM KC1
1 pM PMSF
buffer
Adjust the pH of the solution to 7.4 using
5 ml of stock
0.394 g/500 ml
0.0102 g/500 ml
0.0074 g/500 ml
3.73 g/500 ml
50 pi of 10 mM
stock in DMS0
4 N K0H.
4. NaCl uptake buffer
120 mM NaCl
10 mM MgCl9
10 mM HEPES-KOH
Adjust the pH of the solution to 7.5 using
5. KC1 uptake buffer
120 mM KC1
1.404 g/200 ml
0.406 g/200 ml
0.4766 g/200 ml
4 N KOH.
1.789 g/200 ml
0.406 g/200 ml
0.4766 g/200 ml
Adjust the pH of the solution to 7.5 using 4 N KOH.
10 mM MgCl5
10 mM HEPES-KOH
6. Stop buffer (PBS)
154 mM NaCl 36.0 g/4 L
10 mM Na?HP0. 5.68 g/4 L
Adjust the pH or the solution to 7.5 using concentrated HC1
7. Bray's cocktail
Napthalene (scintillation grade) 240 g/4 L
Methanol 400 ml/4 L
Ethylene glycol 80 ml/4 L
Omnifluor (2a 60 ) 32 g/4 L
Bring the final volume to 4 L usinq dioxane (scintillation
grade) and stir in a ventilator hood until dissolved.
Note: Do not breath the vapors or allow the solution to
come in contact with skin.
8.10%(w/v) K cholate (recrystal 1ized ) 5 g/50 ml
Stir the cholate in 25 ml of water while heating. Add 4 N
KOH to adjust the pH of the solution to 7.5. Allow the
solution to cool and recheck the pH.
9.Sonicated asolectin ^
(Associated Concentrates, Inc.) 80 mg/2 ml
Place the asolectin and KC1 uptake buffer in a thick-walled
Pyrex test tube and flush it with nitrogen gas. Seal the
tube and vortex until all of the the particles are in
suspension. Sonicate the suspension at room temperature
in a bath sonicator (Laboratory Supplies Co.) for 15 min.


Hours
Percent of control


53
TABLE 2-4
Trans-Inhibition in Isolated Plasma Membrane Vesicles
Membrane vesicles (5 mg of protein) were incubated at 4C
for 2 h in Buffer A containing either no additions (con
trol), 25 mM O-glutamine, or 25 mM MeAIB. After the incuba
tion period, 0.5 ml of ice-cold Buffer A was added to each
condition. The samples were immediately vortexed and centri
fuged in a microcentrifuge (15,000xg) for 5 min at 4C.
The supernatants were removed and 1 ml of ice-cold Buffer A
was added back without disturbing the pellet. The tubes
were centrifuged again in the microcentrifuge at 4C for 2
min. The supernatants were removed and the pellets were
resuspended in 200 pi of ice-cold Buffer A. The uptake of
200 pM MeAIB for 1 min at 22C was immediately tested as
described in the Methods section. The velocities are
expressed as pmol MeAIB per mg protein per min and are repor
ted as the averages + S.0. of triplicate determinations.
The values in parentheses are the percent of control for
the Na -dependent velocity. Theoretical calculations
indicate that leakage of MeAIB into the extravesicular space
could not account for the degree of inhibition observed.
Actual determinations of the extraves i cu 1ar concentration of
MeAIB were not performed and the MeAIB trapped in the extra-
vesicular space may contribute to the inhibition observed.
Loading Alkali Total Na+-dependent
Condition Ion velocity velocity
Control
Na +
173
+
4
136
(100)
K +
37
+
3

25 mM D-glutamine
Na +
166
+
14
136
(100)
K +
30
+
8

25 mM MeAIB
Na +
130
+
9
101
(74)*
K +
29
+
7

*
p < 0.025


7
stimulatory effects of most of the hormones tested were
kinet i cal 1y-defined as an increase in Vmax (Shotwell et al.,
1983). Insulin- and glucagon-dependent stimulation of
System A is also blocked by cycloheximide, actinomycin, and
tunicamycin indicating that the continuous synthesis of RNA
and glycoprotein is required to express glucagon-stimulated
activity (Kilberg et al., 1985a). The increase in Vmax and
the dependence on de novo glycoprotein synthesis has led
to the proposal that hormonal induction of System A trans
port results in the insertion of a greater number of active
carrier molecules in the plasma membrane.
The first 15-30 min after exposure of freshly isolated
hepatocytes to glucagon is characterized by a stimulation of
System A activity which is independent of jde novo pro
tein synthesis. After 30 min, cycloheximide eliminates any
further increase in transport activity (Edmondson and
Lumeng, 1980). There are several possible explanations for
the protein synthesis-independent portion of the hormone-
stimulated transport activity. One possibility is that glu
cagon is causing a redistribution of the trans-membrane ion
gradients resulting in increased electro genic transport.
Friedmann and Dambach (1980) showed that glucagon-treatment
of rat liver resulted in a hyperpolarization of the cells
and an increase in the membrane potential from 39.0 mV to
47.2 mV. Bradford et al (1985) showed that the membrane
potential increased in isolated hepatocytes after treatment
with dibutyryl cAMP using a ^C1 uptake procedure to
measure the potential. The increase in potential was


Fig. 2-10. Decay of Systems A, N, and ASC in Vesicles from
Glucagon-Treated and Normal Hepatocytes. Membrane vesicles
from glucagon-treated (A) or normal control rats (B) were
incubated at 4C for the indicated times. After each period
of incubation, the vesicles were tested for amino acid
transport activity as described in Fig. 2-3. System A (),
System N (), and System ASC (A) were tested as described
in the Methods section. The results are expressed as the
mejn + S.D. of triplicate determinations of the
Na -dependent pmol of amino acid accumulated per mg pro
tein per 15 s. Where not indicated, the error bars are con
tained within the symbols.


Fig. 3-3. Titration of the Lipid to Protein Ratio for
Reconstitution of System A Activity Using EAT Cell Mem
branes. Membrane fragments (0.5 mg protein) were mixed with
sonicated asolectin so that the lipid to protein ratio (w/w)
varied from 5 to 100. The cholate to protein ratio was main
tained at 3:1. Proteoliposomes were prepared and tested for
System A activity as described in the Methods section. The
dal^a are expressed as the averages + S.D. of the
Na -dependent AIB uptake measured in triplicate.


22
then pelleted by centrifugation at 100,000xg for 40 min.
The resulting pellet was resuspended by vortexing in Buffer
A to a final concentration of approximately 10 mg pro
tein/ml. The overall yield of the procedure was about 2-3
mg protein per g liver (wet weight). Vesicles could be
stored for up to one month at -70C with minimal loss of
transport activity.
Preparation of Membrane Vesicles from Cultured Hepatocytes
When membrane vesicles were prepared from cultured
cells following substrate starvation, the freshly isolated
hepatocytes were placed in 150 mm collagen-coated dishes
(Kilberg et al., 1983) in NaKRB (amino acid-free medium) or
NaKRB containing 20 mM asparagine (amino acid-supp1emented
medium) at a density of 27 million viable cells per dish.
The cells were incubated at 37C in a humidified atmosphere
of 5% 00^/95^ air for 6 h and then each dish was rinsed
with 10 ml of phosphate buffered saline (154 mM NaCl, 10 mM
Na^HPO^, brought to pH 7.5 with HC1). The cells were
scraped into 5 ml of Buffer A and homogenized by 25 strokes
with the Potter-Elvehjem homogenizer with a tight-fitting
teflon pestle. Membrane vesicles were isolated as described
above. The total yield from 10 dishes of cultured cells was
approximat ely 3 mg of membrane protein.


40
and 8.0 (Fig. 2-4). Decreasing the pH caused an inhibition
of System A-mediated Na+-dependent alanine transport by
about 67% over the pH range tested, whereas Na + -
dependent alanine uptake via System ASC remained relatively
constant (Fig. 2-4).
Characterization of Amino Acid Uptake into Membrane Vesi
cles
An important criterion of carrier-mediated transport is
the stereospecificity of the process. When the Na + -
dependent uptake of radioactively-1abel1ed 200 pM L-alanine
was measured in the presence of 5 mM of unlabelled alanine,
the L-isomer caused a 54% inhibition of transport (control =
553 + 39, plus inhibitor 256 _+ 12 pmol per mg pro
tein per min), whereas the D-isomer caused only a 17% inhib
tion (control = 553 + 39, plus inhibitor = 457 + 36
pmol per mg protein per min). Kinetic analysis of l-alanine
transport is consistent with the observed level of inhibi
tion by the L-isomer. Likewise, experiments testing inhibi
tion of Na+-dependent L-histidine uptake by the corre
sponding L- or D-isomers showed that they inhibited trans
port by 94% and 17%, respect i ve 1y. These results demon
strate the stereospecificity of amino acid transport by the
isolated membranes.
To determine if the plasma membrane vesicles were tight
ly sealed, the i ntraves i cu 1ar space was altered by changing
the osmolarity of the incubation medium. Fig. 2-5 illus
trates that the uptake of AI3 in sodium or in potassium was


144
Succinate: and NADPH:Cytochrome c Reductase Assay
Materials:
1. 10X buffer + +
Use the same buffer as described for the Na ,K -
ATPase assay.
2. 10X substrates (prepared separately)
10 mM succinic acid
0.4 mM cytochrome c (Siama type III)
10 mM KCN
2 mM NADPH
Procedure:
1. Prepare a reference cuvette with the following contents:
100 pi buffer
100 pi KCN
100 pi cytochrome c
700 pi water
2. The non-enzymatic rate of reduction of cytochrome c is
measured by adding the following to a tube:
100 pi buffer
100 pi cytochrome c
100 pi KCN
100 pi membrane (100 pg protein)
600 pi water
3. Record the absorbance change at 550 nm for 5 min to
determine the non-enzymatic rate of reduction.
4. To determine the enzymatic rate of reduction prepare the
following tubes in triplicate:
100 pi buffer
100 pi cytochrome c
100 pi KCN
100 pi succinate (or NADPH)
500 pi water
5. To initiate the reaction, add 100 pg of membrane protein
in 100 pi to the cuvette and record the absorbance
change at 550 nm at 37C for 5 min. The rate of
reduction minus the non-enzymatic rate gives the activity
of the enzyme.
6. If these assays are repeated in the absence of KCN then
the cytochrome oxidase activity can be determined by
subtracting the rate of reduction in the absence of KCN
from the rate of reduction in the presence of KCN.
0.0118 g/10 ml
0.0495 g/10 ml
0.0070 g/10 ml
0.0091 g/5 ml


118
apparatus and the use of YM-30 membranes (rather than PM-30)
alleviated the aggregation problem.
Hydroxylapatite Chromatography of the Low-Salt Extract
Hydroxylapatite chromatography of the detergent-extract
revealed that no reproducible Na+-dependent transport
activity could be recovered in the low-salt column wash
after sample application and that the protein in those frac
tions accounted for 17% of the total protein applied. Only
45% of the total protein applied to the column was recovered
after eluting with buffers of increasing ionic strength up
to 500 mM. In several experiments, transport activity was
found after elution of the bound protein with high-salt
buffer, however, in the best experiment only 25% of the
total transport units was recovered in the eluate and no sig
nificant increase in specific activity was observed in any
of the fractions after reconstitution. To determine if the
transport activity was sensitive to the ionic condition
required for the hydroxylapatite column chromatography, the
protein extract was dialyzed against low-salt (0.2%
C12Eg, 10 mM K2HP04, 1 pM PMSF, pH 7.45) or
high-salt buffers (0.2% C12Eg, 0.5 M K2HP04,
1 pM PMSF, pH 7.45). The deter gent-extract could be dia
lyzed against the low-salt buffer with no apparent loss of
activity. This observation is an important prelude to puri
fication studies because it is necessary to apply proteins
onto certain columns under conditions of low-ionic strength
and then elute the column with a high-ionic strength buffer.


11
to amino acids. In fact, certain amino acids, such as aspar
agine, cause a protein synthesis-dependent repression of
activity. Other amino acids, such as AIB, cause a rapid
decline in transport activity which is not protein
synthesis-dependent and is probably attributable to trans
inhibition (Handlogten et al., 1985). The decay process is
blocked by cycloheximide and actinomycin (Handlogten and
Kilberg, 1984) implying that de novo protein and RNA syn
thesis are required for the decay to occur. Interestingly,
the decay process is also blocked by a-amanitin, an inhibi
tor of RNA polymerase II at the concentrations used in the
study, but not by cordycepin or ara-A, inhibitors of poly(A)
polymerase (Handlogten et al., 1985). These results suggest
that a mRNA must be synthesized de novo for the decay
process to operate and that the mRNA has the unusual feature
of lacking a poly(A) extension. The major group of mRNA
molecules known to lack poly(A) code for the histone pro
teins. The role of these proteins in controlling the decay
process is, at present, unclear. What process is actually
responsible for causing the loss in System A activity is
unknown also. Whether it is a protease, post-translational
modification, internalization, or some other process remains
to be elucidated.
The purpose of this study is to learn more about the
hormonal and adaptational regulation of System A, and to
learn about some of the molecular aspects of System A-asso-
ciated proteins. At present, we can only examine the regula
tion of transport activity in whole cells by observing the


Fig. 2-3. Alanine, Cysteine, Histidine and Glycine Trans
port by Rat Liver Plasma Membranes. Membrane vesicles were
used to measure the transport of the indicated substrate.
The transport of alanine (A), cysteine (plus 1 mM dithio-
threitol) (B), and histidine (C) was tested in the presence
or absence of 5 mM MeAIB. Glycine (D) uptake was assayed in
the presence or absence of 5 mM threonine. The substrate
concentration in each case was 200 pM and transport waj.
measured at 22C. +The difference between uptake i rj Na
() and that in K () was takej as the total Na -
dependent transport (A). The Na -dependent component in
the presence of the inhibitor (MeAIB or threonine) is also
shown (). See the text for interpretation of these data as
evidence for the specific transport systems indicated. The
results are presented as the averages + S.D. of three
determinations.


5
characterized in EAT cells (Christensen, 1964) and rat hepa-
tocytes.
System N was first described in rat hepatocytes as a
system which was specific for Na+-dependent asparagine,
glutamine, and histidine uptake (Ki1 berg et al., 1980).
System A mediates a portion of the transport of these sub
strates in rat hepatocytes, but this component can be elimin
ated by measuring uptake in the presence of an excess of the
System A-specific probe, MeAIB. System N is highly sensi
tive to decreasing the pH of the medium and also exhibits a
high degree of specificity for l-amino acids. System N has
also been shown to occur in fetal rat hepatocytes and the
rat hepatoma cell line H4-II-EC3 (Vadgama and Christensen,
1983).
Neutral amino acid transport activity is regulated by
three different processes: trans-effects, hormones, and sub
strate availability. The transport systems affected by
these factors are indicated in Table 1-1. Combined, the
characteristics of specificity and regulation of each trans
port system make it possible to uniquely identify a given
transport system operating in a particular cell line.
Regulation of transport activity by trans-effects is an "j"
acceleration or deceleration of amino acid uptake caused by
an elevated concentration of substrates inside the cell. An
acceleration of the transport rate, or trans-stimulation, is
a common characteristic of System ASC. In other words, when
cells are pre-loaded with a System ASC substrate then washed
free of external substrate and tested for System ASC uptake,



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pmol AIB
120
mg"' protein
*
O
T


113
O
1
2 3
OsM"1
4


D SOLUTIONS FOR THE PREPARATION AND
RECONSTITUTION OF EAT CELL MEMBRANE 147
BIBLIOGRAPHY 149
BIOGRAPHICAL SKETCH 156
V


81
Coomassie-staining bands. McCormick et al. (1985) reconsti
tuted Na+-dependent AIB transport from EAT cell mem
branes that had been extracted with cholate/urea as
described above and showed an apparent enrichment in a pro
tein of 125 kOa in the proteo1iposomes when compared to the
native plasma membranes. Using reconstitution as an assay,
Takahashi et al. (1985) have determined by radiation-
inactivation studies that the target size for the Na+-
dependent glucose transporter from kidney brush border mem
branes is one-million daltons and 1.2 million daltons for
the Na+-dependent alanine transporter.
The object of the present studies was to devise a
rapid, efficient system to reconstitute System A activity
from EAT cell membranes. Techniques for reconstitution of
System A described by McCormick et al. (1984) and Cecchini
et al. (1978) were attempted in our laboratory and in the
laboratory of Dr. Efraim Racker but no reproducible activity
could be obtained. In collaboration with Dr. Racker, we
developed a novel reconstitution technique for System A
activity which was rapid and reproducible. Having estab
lished the technique, future research in the laboratories of
both Dr. Kilberg and Dr. Racker will focus on the purifica
tion of the transporter using reconstitution as an assay.


95
freeze-thaw step. Fig. 3-3 illustrates the results of an
experiment in which the lipid to protein ratio (w/w) was
varied by adding more lipid to the mixture while using a
fixed amount of protein and keeping the cholate to protein
ratio constant at 3:1. The results indicate that as the
lipid to protein ratio was varied up to 20:1, an optimum
level of activity was obtained. Further increases in the
lipid to protein ratio resulted in lower transport activity.
Brief periods of sonication after a freeze-thaw step
have been reported to result in further stimulation of
transport activity (Kasahara and Hinkle, 1976). Fig. 3-4
shows that periods of sonication less than 30 s resulted in
slight increases in System A activity, but the first three
time points are not statistically different from each other
because of the variations in the means. Longer periods of
sonication resulted in significantly lower transport activ
ity. These results indicate that, for our particular car
rier, sonication does not cause a significant enhancement in
transport activity and it will probably not be useful for
application to reconstitution of EAT cell membrane
detergent-extracts.
For the purposes of further reconstitution studies
using detergent-extracts, the optima obtained in these exper
iments for the lipid to protein ratio and the cholate to pro
tein ratio were used. It is important to note that the
optima obtained through these experiments are considered to
be a range of values and may vary between different reconsti
tutions and different membrane preparations .


pmol AIB-mg-1 protein *30 tec'l
33
[MeAlB] mM


153
Owen, A. J. Ill, Geyer, R. P. and Antoniades, H. N. (1982)
Proc. Natl. Acad. Sci. ^9, 3203-3207
Oxender, D. L. and Christensen, H. N. (1963) J. Biol. Chem.
238, 3686-3699
Pariza, M. W., Butcher, F. R., Kletzien, R. F., Becker, J.
E. and Potter, V. R. (1976) Proc. Natl. Acad. Sci.
73, 4511-4515
Parns, J. R. and Isselbacher, K. J. (1978) Prog. Exp. Tumor
Res. 22, 79-122
Pick, U. (1981) Arch. Biochem. Biophys. 212, 186-194
Poupon, R. and Evans, W. H. (1979) FEBS Lett. 108,
374-378
Pressman, B. C. (1976) Ann. Rev. Biochem. 4jj, 501-530
Prpic, V., Green, K. C., Blackmore, P. F., Exton, J. H.
(1984) J. Biol. Chem. 259, 1382-1385
Quinlan, D. C., Todderud, C. G., Kelley, D. S. and Kletzien,
R. F. (1982) Biochem J. 208, 685-693
Racker, E. (1972) J. Memb. Biol. 10, 221-235
Racker, E. (1976) "A New Look at Mechanisms in
Bioenergetics". Academic Press, New York
Racker, E. ( 1979 ) Meth. Enzymol 55, 699-711
Racker, E., Abdel-Ghany, M., Sherrill, K., Riegler, C. and
Blair, E. A. (1984 ) Proc. Natl. Acad. Sci. 81,
4250-4254
Racker, E., Chien, T-F. and Kandrach, A. (1975) FEBS Lett.
57, 14-18
Racker, E., Resnick, R. J. and Feldman, R. (1985) Proc.
Natl. Acad. Sci. 82, 3535-3538
Radian, R. and Kanner, B. I. (1985) J. Biol. Chem. 260,
11859-11865
Ray, T. K. (1970) Biochim. Biophys. Acta 196, 1-9
Rittenhouse, H. G., Rittenhouse, J. W. and Takemoto, L.
(1978) Biochem. V, 829-837
Roman, L. M. and Hubbard, A. L. (1983) J. Cell Biol. 96,
1548-1558
Sachs, G., Jackson, R. J. and Rabn, E. C. (1980) Am. J.
Physiol. 238, G151-G164


TABLE 2-5
Glucagon Stimulation of System A in Rat Hepatocytes is
Retained in Isolated Plasma Membrane Vesicles
Rats were injected with 1 mg of glucagon per 100 g body weight 4 h prior to
hepatocyte isolation. Hepatocytes and the corresponding membrane vesicles
from the same preparation of cells were isolated and analyzed for System A
activity as described+in the section on Materials and Methods. The veloci
ties given are for Na -dependent AIB uptake and are the averages +
S.O. of three individual assays. Note that the length of the assays in the
whole cells was one minute, whereas the assays employing membrane vesicles
were for 15 s. Values shown in the last column indicate the degree of the
glucagon-dependent stimulation determined by calculating the ratio of the
velocities (glucagon-treated/control ).
Preparation Control Glucagon-treated Glucagon-dependent
stimulation
(pmol AIB per mq protein per unit time)
Intact 28.9 + 4.9 872 + 64 30.2
Cel Is
45.4 + 22.0
Membrane
Vesicles
571 + 5.4
12.6


CHAPTER I
AMINO ACID TRANSPORT IN ANIMAL CELLS
Amino acids are pivotal contributors to many metabolic
pathways in cells including gluconeogenesis, glycolysis, and
the Kreb's cycle. For example, amino acids released from
muscle provide the carbon skeletons for de novo glucose
synthesis in the liver through the glucose-alanine cycle
(Felig, 1973). The supply of amino acid precursors for glu-
coneogenesis has long been considered to be a control point
for the entire pathway (Exton et al., 1970). It has been
shown more recently that the rate-limiting step in alanine
metabolism in rat hepatocytes is the transport of alanine
into the cells (Sips et al., 1980a). In this way, the intra
cellular concentration of amino acids is tightly regulated
according to the nutritional needs of the cell.
Amino acids can be transported into cells through two
types of carrier-mediated pathways. Eukaryotic cell trans
port can either be by active accumulation, that is, coupled
to a trans-membrane Na+ gradient (Na+-dependent), or
by facilitated transport not driven by ion fluxes (N a + -
independent). Neutral amino acid transport was first
observed as two distinct pathways in Ehrlich ascites tumor
(EAT) cells and the pathways were designated by Oxender and
1


pmol AIB (6 min)
49
0 40 80 120 160 200
ug vesicle protein


91
TABLE 3-1
Enzyme Marker Activities in EAT Cell Membranes
Membrane vesicles were tested for the presence of particular
enzyme markers for plasma membrane, endoplasmic reticulum,
an< mjtochondr i al inner membrane. The activity of
Na ,K -ATPase is expressed in terms of pmol Pi
formed per mg protein per h. NADPH:cytochrome c reductase
and succinate:cytochrome c reductase activities are
expressed as nmol cytochrome c reduced per mg protein per
min. Relative specific activity (R.S.A.) is determined by
dividing the specific activity of the enzyme in the plasma
membrane-enriched fraction by the specific activity in the
homogenate. The data are the averages + S.D. of tripli
cate determinations.
Enzyme
Tested
Homogenate
Membranes
R.S.A.
Na+,K+-
ATP ase
0.46 + 0.54
5.55 + 1.21
12.1
NA DPH:cyto
chrome c reductase
13.3 + 0.50
12.2 + 0.26
0.9
Succinate:cyto-
chrome c reductase
5.85 + 0.11
15.2 + 0.31
2.6


APPENDIX B
ENZYME ASSAYS
5 1-Nuceoti dase Assay
Materials:
Substrate mix:
5.5 mM Mg Cl
0.056
g/50
ml
55 mM Tris Dase
0.333
g/50
ml
11 mM 51tAMP (Sigma Type II)
0.191
g/50
ml
10 mM Na -K tartrate
0.141
9/50
ml
This mixture is prepared just prior to use.
2. 10%(w/v) TCA
Procedure:
1. Place the substrate mix in a 37C water bath and allow
the temperature to equilibrate. Place 10% TCA on ice.
2. Dilute the membrane fractions to be tested to 1 mg
protein/ml and warm to 37C.
3. Add 0.9 ml of the substrate mix to conical tubes (15 ml)
in a water bath at 37C. Prepare a membrane blank (0.9
ml water + 0.1 ml membrane) for each membrane preparation
to be tested and a substrate blank (0.9 ml substrate mix
+ 0.1 ml water).
4. To initiate the assay, add 100 pi of membranes (100 pg)
to the substrate mix at 37C. Vortex and continue
adding the samples at 30 s intervals.
5. Incubate the mixtures at 37C for 15 min and then
terminate the reaction by adding 1 ml of ice-cold 10%
TCA.
6. After vortexing the mixture, centrifuge the samples at
10,000 RPM for 20 min in a Sorvall SM-24 rotor.
7. Remove 0.5 ml of supernatant for P determination by
the procedure described in Appendix A.
138


Fig, 3-5. Determination of the Optimal Concentration of
C,Eq for Extraction of EAT Cell Membrane Proteins
Prior to Reconstitution. EAT cell membranes (5 mg pro
tein/ml) were extracted in various concentrations of deter
gent for 30 min at 4C. Following the extraction period,
the suspension was centrifuged at 100,000xg for 1 h and the
supernatant was saved (deter gent-extract). The detergent-
extract (0.5 mg protein) was mixed with sonicated asolectin
(10 mg) and cholate (1.5 mg) frozen in liquid nitrogen, and
then thawed at room temperature. The reconstituted proteo-
liposomes were collected by centrifugation and tested for
System A activity as described in the Methods section. The
velocities are expressed as the averages + S.D. of the
Na -dependent AIB uptake measured in triplicate.


123
preparation buffer. The reconstituted proteoliposomes (Lane
D) appear to have incorporated most of the proteins observed
in the supernatant from the extraction (Lane B) but a 56 kDa
and a 48 kDa polypeptide appear to be more prevalent in the
proteoliposomes than in the supernatant of the cholate/urea
extract. Further experiments will be necessary before one
can speculate as to the importance of any such enrichment.
Discussion
Reconstitution of System A transport activity has been
achieved using EAT cell membranes and a freeze-thaw tech
nique. Optima determined for the lipid to protein ratio and
the cholate to protein ratio during membrane-fragment recon
stitution are similar to previously published values for the
Na+,K+-ATP ase (Karlish and Pick, 1981). The
Na+-dependent uptake of AIB into the proteoliposomes was
also completely eliminated in the presence of 5 mM MeAIB
(data not shown) indicating that the reconstituted amino
acid transport activity shows similar sensitivity to MeAIB
as previously reported in intact EAT cells and isolated rat
hepatocytes (Shotwell et al., 1983). The choice of
C^Eg as the detergent for the initial extraction
studies was made on the basis of its successful use for the
reconstitution and partial purification of the H+-ATPase
from cl at hrin-coated vesicles (Xie et al., 1984).
Preliminary results using reconstituted membrane pro
teins from the human hepatoma HepG2 cell either by a


69
HOMOGENATE (ND)
*
lOOOXG CENTRIFUGATION
sir N
SI (ND) PI (137 + 18)
45,OOOXG CENTRIFUGATION
Ml.
S2 (0) P2 (614 + 24)
P 3 (631 + 18)
s/
SUCROSE-STEP
GRADIENT
50,OOOXG
CENTRIFUGATION
Fig. 2-11. Flowchart of HepG2 Membrane Preparation. HepG2
membranes were prepared as shown above and described in the
Methods section. The supernatant fractions (S) and the
pellet fractions (P) are indicated for each step. Fractions
which were tested for System A+transport activity are indi
cated in parentheses as the Na -dependent pmol of AIB
accumulated per mg protein per min + S.O. for triplicate
determinations. Fractions which were not tested for activity
are indicated by ND.


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vi 1
ABBREVIATIONS USED ix
ABSTRACT xi
CHAPTERS
I AMINO ACID TRANSPORT IN ANIMAL CELLS 1
II CHARACTERIZATION OF SYSTEM A AMINO ACIO
TRANSPORT ACTIVITY IN MEMBRANE VESICLES.... 14
Introduction 14
Materials and Methods 19
Results 26
Discussion 70
III RECONSTITUTION OF SYSTEM A TRANSPORT
ACTIVITY INTO ARTIFICIAL PROTEOLIPOSOMES... 76
Introduction 76
Materials and Methods 82
Results 90
Discussion 123
IV FURTHER DISCUSSION ON THE USE OF MEMBRANE
VESICLES AND RECONSTITUTION 128
APPENDICES
A ANALYTICAL ASSAYS AND PROCEDURES 134
8 ENZYME ASSAYS 138
C SOLUTIONS FOR THE PREPARATION OF PLASMA
MEMBRANES AND TRANSPORT OF VESICLES 146


This dissertation is dedicated to my parents, whose constant
love and support made this work possible.


Fig. 2-7. Relation Between Membrane Protein Concentration
and JIB Uptake. Membrane vesicles were diluted into Na -
or K -uptake buffer containing 200 pM AIB. The amount
of membrane protein in the assay was varied from 10 to 200
pg. Uptak^ of the amino acid was measured £or 6 min at
22C in Na containing buffer(B). The Na -dependent
AIB uptake is also shown (). The data are the averages
+ S.O. of triplicate determinations. Where not shown,
the standard deviation bars are within the symbol.


26
initial suspension before centrifugation. This value was
divided by the total mg of protein present in the suspension
to give 23.2 pi of extravesicular volume per mg protein for
the 100,000xg pellet.
Protein Determination
Vesicle protein was determined by a modification of the
method of Bensadoun and Weinstein (1976). Approximately
10-50 pg of membrane protein was suspended in 1 ml of 0.1%
sodium dodecyl sulfate (SDS). Following a 10 min incubation
at 22C, the protein was precipitated by adding 750 pi of
ice-cold 24%(w/v) trichloroacetic acid (TCA) and then
pelleted by centrifugation at 12,000xg for 20 min. The pro
tein content of the pellet was measured by a modification of
the Lowry technique as described previously (Kilberg et al.,
1983). Bovine serum albumin (5 to 100 pg) was used as the
standard.
Results
Enzyme Marker Activities in Rat Liver Membrane Vesicles
Enzyme marker analysis of the membrane vesicles pre
pared from rat liver revealed that the vesicles are enriched
approximately 10-fold in the plasma membrane marker enzymes
5'-nucleoti dase and adenylate cyclase (Table 2-1). A marker
enzyme for microsomal contamination, glucose-6-phosphatase,
showed 3-fold enrichment. There was a 10-fold reduction in
the level of the mitochondrial enzyme marker activities,


79
easily fractured, exposing hydrophobic regions which can
fuse to form large liposomes.
Another technique used successfully to achieve recon-
+?
stitution of transport processes is the Ca "-fusion pro
cedure. This alternate procedure capitalizes on the observa
tion that liposomes which contain phosphatidylethanol amine
and phosphatidylserine or cardiolipin rapidly fuse in the
+ 2
presence of Ca (Miller and Racker, 1976). This tech
nique has proven useful for reconstitution of cytochrome
oxidase activity from mitochondria and has also found wide
use in drug delivery systems (Szoka and Papahadjopoulos,
1980).
Reconstitution of Na+-dependent amino acid trans
port activity has been reported in kidney brush border mem
branes (Kinne and Faust, 1977; Takahashi et al., 1985),
SV-40-transformed 3T3 fibroblasts (Nishino et al., 1978),
and EAT cell membranes (McCormick et al., 1984; Cecchini et
al., 1977). Reconstitution of amino acid transport activity
from kidney brush border membranes has been achieved,
although the level of Na+-dependence was low. Kinne and
Faust (1977) extracted membranes with Triton X-100, then
removed excess Triton by passing the detergent-extract over
Bio-Bead SM-2 columns. The detergent-extract was reconsti
tuted by combining it with lipid and sonicating the mixture.
Sodium-dependent alanine uptake was quantitated, but less
than a 2-fold difference was reported for uptake in Na+
versus K+-containing buffers. The authors did not dis
cuss the margin of error present in the assays so the data


15
similar specificities in membrane vesicles when compared to
intact cells as has been demonstrated for the Na + -
dependent glucose transporter in intestinal brush border
vesicles (Kessler and Semenza, 1983).
Several criteria have been applied to membrane vesicles
to determine decisively if solute is accumulated in the
i ntravesi cu 1ar space via a carrier-mediated process: 1)
exchange diffusion of the solute with an internal solute, 2)
temperature dependence of influx and efflux, 3) osmotic sen
sitivity of the vesicles for accumulation of solute, 4) sat
uration kinetics of the substrate and, 5) selective effects
on ion-dependent transport caused by ionophores.
Advantages of Studying Transport in Membrane Vesicles
There are several advantages to studying transport phe
nomena in isolated membrane vesicles versus intact cells.
Membrane vesicles offer the possibility of examining trans
port processes in the absence of i ntracel1ular metabolism.
Many nutrients, such as amino acids or glucose, are trans
ported into a cell and then rapidly metabolized, complica
ting transport studies considerably. Membrane vesicles also
allow one to introduce defined media to either side of the
membrane, thus facilitating carrier function studies from
the cis or trans side. Various electrochemical driving
forces can be introduced and the effects on the carrier
readily observed. Vesicles are, additionally, ideal systems
for monitoring electrochemical potenti a 1-sens itive and pH-
sensitive dyes. Membrane vesicles which show transport


CHAPTER II
CHARACTERIZATION OF SYSTEM A AMINO ACID TRANSPORT
ACTIVITY IN MEMBRANE VESICLES
I ntroduction
Use of Membrane Vesicles for Study of Nutrient Transport
Membrane vesicles from various cell types and tissues
have proven to be extremely useful for studying nutrient
transport processes. The earliest studies of active trans
port systems in membrane vesicles were reported using bacter
ial membranes (Kaback, 1960). Transport systems for various
sugars and amino acids were characterized by using bacterial
vesicles prepared by osmotic lysis and testing uptake using
a rapid filtration system (Kaback, 1974). The development
of methods for preparation of vesicles from bacterial cells
led other groups to develop similar techniques for eukaryo-
tic cells.
Nutrient transport in membrane vesicles from eukaryotic
cells and tissues has been reviewed recently in several ex
cellent articles (Lever, 1980; Murer and Kinne, 1980; Sachs
et al., 1980). Sodium-dependent transport systems for D-
hexoses, L-ami no acids, bile acids, and various ions have
been demonstrated in vesicles from brush border as well as
basolateral membranes. Most transport systems tested show
14


Regulation of System A-mediated transport activity was
studied by stimulating transport activity using various
effectors, then isolating membrane vesicles from those
cells. When plasma membrane vesicles were isolated from
either glucagon-treated or normal hepatocytes, it was dis
covered that the membrane vesicles from cells exposed to the
hormone partially retained the stimulated transport activ
ity. The lack of retention of all of the enhanced activity
in the membrane vesicles is explained, in part, by a rapid
loss of activity during storage at 4C (half-life = 13 h).
Neither protease inhibitors nor dithiothreitol blocked this
decay.
A procedure for reconstitution of transport activity
was developed using Ehrlich ascites tumor cell membranes.
The membrane proteins were solubilized in cholate and urea
which were exchanged for the non-ionic detergent polyoxy-
ethy 1 ene-9-1 aury 1 ether (C^Eg) by dialysis. After
mixing with sonicated asolectin and cholate, the detergent-
extract was reconstituted by a freeze-thaw procedure. The
proteoliposomes were collected by centrifugation and tested
for System A activity by a rapid filtration assay. Reconsti
tuted transport activity was determined to be optimal when
the cholate to protein ratio (w/w) was 3:1 and the lipid to
protein ratio (w/w) was 20:1. The development of this pro
cedure for reconstitution will facilitate further studies
toward purifying the protein(s) associated with System A
because reconstitution can be used to monitor the increase
in specific activity of the carrier during purification.


SYSTEM A AMINO ACID TRANSPORT
VESICLES AND RECONSTITUTED
ACTIVITY IN MEMBRANE
PROTEOLIPOSOMES
By
MARK ALLEN SCHENERMAN
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
1986


104
After a recent report of successful reconstitution of
amino acid transport activity from EAT cell membranes by
McCormick et al. (1984), we attempted to repeat their stu
dies using the same system of membrane solubilization. Mem
branes were stirred with 2.5516 cholate and 4 M urea at 4C,
centrifuged at 125,000xg, and then the supernatant was
dialyzed against 100 volumes of 0.25% cholate buffer. The
dialysate was reconstituted exactly as described by
McCormick et al. (1984) by mixing with asolectin, passing
through a Sephadex G-50 column, and then performing a
freeze-thaw step on the proteoliposome fractions. Despite
several attempts, by workers in both Gainesville and
Cornell, no measurable transport activity could be detected.
Table 3-2 shows the results of an experiment in which the
cholate/urea extract prepared by the method of McCormick et
al. (1984) was dialyzed against 0*25% cholate, as they
suggest, or was dialyzed against 0.2% C^Eg and then
reconstituted by our procedure. The results indicate that
transport activity could not be recovered after dialysis
against cholate, but after exchanging the cholate for
C12^9 3 S19ni^icdnt amount of System A activity was
obtained (Table 3-2).
Using the cholate/urea system for solubilization of EAT
cell membrane, it was possible to consistently extract grea
ter than 85% of the total membrane protein. After exchang
ing cholate and urea for C1?Eg by dialysis over
night, System A activity was always observed in protein-
extracts prepared from membranes which showed transport


Fig. 3-10. SDS-Polyacry1 amide Gel Electrophoresis of EAT
Cell Membranes and Reconstituted Proteoliposomes. Native
EAT cell membranes (Lane A), the supernatant (100,000xg) of
the cholate/urea extraction (Lane B), the pellet of the
cholate/urea extraction (Lane C), and the reconstituted pro-
teoliposomes (Lane D) were resolved on a 7.5% SDS-polyacryl-
amide gel as described in the Methods section. Each sample
(25 pg of protein) was diluted into 100 pi of sample dilu
tion buffer and the mixture was placed in a boiling water
bath for 2 min. After cooling to room temperature, the sam
ples were applied to the gel and constant current was
applied at 15 mA for 2 h to allow stacking. After stacking,
the current was increased to 20 mA for 3 h until the dye
front was 1 cm from the bottom edge of the gel. The gel was
fixed in TCA/methanol/acetic acid (5:30:10) for 2 h, and
then soaked overnight in 50% methanol (several changes).
The gel was silver-stained as described in the Methods
section. The positions of molecular weight standards are
indicated.


114
leakage which in our case represents approximately 30% of
the total uptake in isotonic buffer.
The intraves i cu 1ar volume of the proteoliposomes was
measured using the 3-0-methyl-glucose method of Kletzien et
al. (1975). Fig. 3-9 indicates that increasing the
3-0-methyl-glucose concentration resulted in a linear
increase in 3-0-methyl-glucose uptake. The slope of the
line, which is an estimate of the average intravesicular
volume, was approximately 29 pl/mg protein in both the NaCl
and the KC1 buffers. This value is considerably larger than
the intravesi cu 1ar volume of approximately 2 pl/mg protein
determined by McCormick et al. (1985). This difference
could be explained by variation in the amount of protein
incorporated per liposome formed because the results from
both studies are expressed per mg protein. Calculation of
the distribution ratios for the time course of AIB uptake
indicates that values greater than 1.4 were obtained at incu
bation times longer than 30 min. These data demonstrate
that the reconstituted proteo 1iposomes are capable of accumu
lating solutes against a concentration gradient.
Proteoliposomes were prepared in the presence of
14
C-sucrose, a relatively impermeant molecule, to deter
mine if sucrose could be trapped during the freeze-thaw
step. As a control, proteoliposomes were prepared first and
then the ^C-sucrose was added after the freeze-thaw
step. Both mixtures were passed over a Sephadex G-50 column
and fractions were collected. The void volume for both pre
parations showed visible turbidity, presumably because it


143
Adenylate Cyclase Assay
Materials:
1* Substrate mix (1.1X)
3.52 mM ATP (disodium salt)
5.5 mM Mg Cl9
27.5 mM Tris-HC1
0.11% bovine serum albumin
22 mM creatine phosphate (200 mM stock)
1.1 mM EDTA
0.55 mg creatine phosphokinase
(135 U/mg)
Adjust the pH of the solution to 7.6 using 3 N NaOH.
11 mg/5 ml
6 mg/5 ml
22 mg/5 ml
6 mg/5 ml
0.55 ml stock
2 mg/5 ml
3 mg/5 ml
Procedure:
1. Add 180 pi of substrate mix to as many 1.5 ml microfuge
tubes as are necessary for the assay. Place the tubes in
a 30C water bath and allow 10 min for temperature
equilibration to occur.
2. Prepare a substrate blank which contains 20 pi of water
and 180 pi of substrate mix. To determine the level of
endogenous cAMP present in the membranes, add 180 pi of
water to 20 pi of membrane (200 pg protein).
3. Initiate the assay by adding 200 pg of membrane protein
in 20 pi to each tube in the presence or absence of 10
mM NaF. Vortex and incubate each tube for 10 min at
30 C.
4. To stop the reaction, transfer the tubes to a boiling
water bath for 3 min.
5. Remove the precipitated proteins by centrifuging each
tube in a microfuge (15,000xg) for 2 min.
6. Remove 150 pi of the supernatant for cAMP determination
using the Amersham protein binding kit. Samples can be
stored frozen until the cAMP assay can be performed.
7. The results are expressed as the difference between the
nmol of cAMP formed per mg protein per hour in the
presence and absence of 10 mM NaF.
Reference: Wisher, M. H. and Evans, W. H. (1975) Biochem. J.
146, 375-388


13
Having perfected this artificial system for assaying
the transport of amino acids, the next part of the strategy
is to reconstitute System A activity after extracting it
from its native membrane environment and replacing it into
artificial lipid bilayers. If this assay system could be
developed, it would then be possible to perform several puri
fication steps on the membrane-extract and assay purifica
tion by reconstitution. Purification and identification of
System A-associated proteins would be an important prelude
to the elucidation of the mechanism of amino acid transport
because studies could be done on the carrier in an isolated
state in an artificial membrane. The consequences of reveal
ing the regulation and mechanism of System A transport activ
ity would be medically relevant because its activity is
increased not only in tumor cells but also in hepatic cells
from diabetic animals.


pmol AIB*mg~l protein* min-
102
0
0.1 0.2 0.3 0.4
c12e9 (%)


Fig. 3-6. Temperature Stability of the Membrane Protein
Extract. Detergent-extracts (cho1 ate/urea) were dialyzed
against 0,2% C1?Eq and then incubated at 4C ()
or -70C () rorythe indicated time. At the end of the
incubation period, an aliquot (1.0 mg protein) was removed
reconstituted, and tested for System A transport activity
described in the Methods section. Extracts incubated at
-70C were frozen in aliquots and were thawed only once
prior to the ass^y. The results are expressed as the mean
+ S.D. of the Na -dependent pmol of AIB accumulated
per mg protein per min (N = 5).


51
TABLE 2-3
Effect of Gramicidin or Monensin on AIB Uptake
Membrane vesicles (2.5 mg protein/ml) were incubated at
22C for 15 min with either 2% ethanol, 20 pg/ml gramici
din in 2% ethanol, or 20 pg/ml monensin in 2% ethatjol The
suspensions were then diluted (1:1) into either Na or
K -uptake buffer containing 200 pM of radioacti ve 1y-
labelled AIB. The transport was measured in the usual
manner as described in the text. The velocities are
expressed as pmol AIB per mg protein per 6 min and are the
averages + S.O. of three individual assays.
Add itions
Alkali ion
Velocity
Na+-dependence
Ethanol
Na +
232 + 8
118
Ethanol
K +
114 + 16

Gramicidin
Na +
146 + 3
29
Gramicidin
K +
117 + 13

Monensin
Na +
130 + 5
8
Monensin
K +
122 + 6


140
G1ucose-6-Phosphatase Assay
Mater 1als:
1. Buffer
100 mM maleic acid 1.16 g/100 ml
Adjust the pH of the solution to 6.5 using 4 N NaOH.
2. Substrate
0.1 mM D-glucose-6-phosphate 0.0282 g/ml
(monosodium salt)
3. 10*(w/v) TCA
Procedure:
1. Warm the buffer and substrate to 37C in a water bath.
2. Add 300 pi of buffer and 100 pi of substrate to the
sample tubes (15 ml conical centrifuge tubes) in the
water bath. Prepare a sample blank for each different
membrane preparation tested (300 pi of buffer + 100 pi
of water) and a substrate blank (300 pi of buffer + 100
pi of substrate + 100 pi of water).
3. Dilute the membrane to be tested to 1 mg protein per ml
and warm to 37C.
4. To initiate the assay, add 100 pi of membranes to the
appropriate tubes, vortex, and incubate the mixtures at
37C for 15 min.
5. Terminate the assay by adding 1 ml of ice-cold TCA. Add
0.5 ml of water to all the samples to bring the final
volume to 1 ml. Vortex and then centrifuge the tubes at
10,000 RPM for 20 min in a Sorvall SM-24 rotor.
6. Remove 0.5 ml from each tube for P. determination as
described in Appendix A.
7. The enzyme activity is expressed as pmol of P.
released per mg protein per hour.
Reference: Swanson, M. A. (1955) Meth. Enzymol. 2,
541-543.


45
decreased when the concentration of sucrose in the incuba
tion medium was increased. The inversely linear relation
between the sucrose concentration and the steady state amino
acid accumulation indicates that AIB is being transported
into an osmoti cal 1y-sensitive compartment, presumably the
intravesicular space. Extrapolation of the data to infinite
sucrose concentration suggests that some leakage of sucrose
or some non-specific binding to the vesicles does occur
(Fig. 2-5).
The Na+-dependent uptake of AIB into vesicles was
dependent on temperature changes and protein concentration.
Sodium-dependent uptake of AIB after a 6 min incubation at
15C was only 78% of that at 22C, whereas the uptake at
4C was only 11% of the uptake observed at 22C (Fig.
2-6). To provide evidence that the uptake of amino acids
was mediated by a membrane-bound protein or prote in-comp1 ex,
the effect of increasing the concentration of protein con
tent in each assay was measured. Fig. 2-7 shows that,
between 10 and 200 pg of protein, the rate of AIB uptake
varied linearly with the amount of vesicle protein included
in the assay. This relation was observed for both total and
sodium-dependent transport.
Two lines of evidence support the conclusion that the
Na+-dependent uptake of amino acids by the membranes is
the result of an imposed trans-membrane Na+ gradient.
First, when the extravesicular Na+ concentration is
increased the rate of transport also increases. Secondly,
if the sodium gradient is dissipated by incubation of the


92
This approach is often referred to as "membrane-fragment
reconstitution" because large fragments of membrane are
fused with sonicated lipid vesicles. The resulting proteo-
liposomes can be tested for activity, and optimization of
that activity should be a good starting point for detergent-
extraction techniques.
Fig. 3-2 indicates that the membrane vesicles prepared
from EAT cells show minimal Na+-dependent AIB uptake
(approximately 50 pmol AIB accumulated per mg protein per
min). When sonicated asolectin was added to the membrane
vesicles and the mixture was freeze-thawed in liquid
prior to assay, a 6-fold increase in transport activity was
observed (Fig. 3-2). Karlish and Pick (1981) observed that
the addition of cholate to membrane vesicles prior to such a
freeze-thaw step resulted in even greater activity for the
reconstituted Na+,K+-ATPase. For our preparation,
the addition of cholate to the mixture before freeze-thaw
resulted in further increases in System A activity until an
optimum was attained at a cholate to protein ratio of 3:1
(w/w). Higher cholate concentrations resulted in loss of
activity. These data indicate that asolectin and cholate
addition before the freeze-thaw step result in significantly
greater transport activity than shown by the membrane vesi
cles alone, and indicate that these two components may be
useful for further reconstitution studies.
Another parameter that can be varied in order to opti
mize reconstituted transport activity is the ratio of lipid
added to the protein present in the mixture prior to the


9
hypotonic conditions with cAMP-dependent protein kinase, an
inhibition of AIB uptake was observed. As expected, the
P incorporation was increased in many proteins after
the addition of protein kinase and no conclusion could be
reached correlating specific protein phosphorylation and the
System A carrier protein.
Amino acid and hexose transport have frequently been
observed to be increased in tumorigenic cells (Parns and
Isselbacher, 1978). In particular, it has been reported
that the Na+-dependent uptake of AIB, a specific probe
of System A activity in most cells, is increased in trans
formed cells (Isselbacher, 1972; Foster and Pardee, 1969).
Increased rates of glycolysis and System A transport activ
ity were detected when "normal cells" were transformed
either by transfection with specific oncogenes or by expo
sure of the cells to the low molecular weight polypeptides
called "transforming growth factors" (TGF). Exposure of rat
fibroblasts (rat-1) or myc-transformed rat-1 cells to the
23 kDa polypeptide called TGF-6 greatly increased the rate
of System A transport (Racker et al., 1985).
Stimulation of amino acid transport and glycolysis was also
observed when a normal rat kidney cell line (NRK-49F) was
exposed to TGF-B (Boerner et al., 1985). The increased
activity reported in the experiments where TGF-(5 was used
to transform the cells was blocked by cyclohexi mide. The
cycloheximide sensitivity of the transformat i on-sti mu 1 ated
System A activity appeared to be analogous to the cyclohexi-
mide sensitivity detected during the hormone- and


124
cholate-dialysis technique (Kagawa and Racker, 1971) or by
freeze-thaw followed by dilution (Alfonso et al., 1981) indi
cate that there is a significant amount of sodium-dependent
alanine and AIB uptake present in the proteoliposomes (124
+ 15 and 606 + 64 pmol per mg protein per min,
respectively). AIB transport into reconstituted proteo-
liposomes prepared from EAT cell membrane proteins was
Na+-dependent, but it did not show a rapid overshoot as
was observed in rat liver membrane vesicles. As mentioned
above, this could be explained by a slower dissipation of
the trans-membrane Na+ gradient in the proteoliposomes.
Another possibility is that the proteo 1iposomes are not main
taining a membrane potential similar in magnitude to that
observed in whole cells or in membrane vesicles. In support
of this hypothesis, valinomycin did not cause a large
increase in AIB transport. It is important to note, how
ever, that no other test was performed to determine if the
valinomycin caused a hyperpolarization of the membrane poten
tial in the proteoliposomes.
The reconstituted proteo 1iposomes transported AIB into
an osmotically-sensitive space and retained other small mole
cules such as sucrose inside that space. These character
istics, along with the fact that AIB was accumulated against
the concentration gradient, indicate that the reconstituted
proteoliposomes are tightly sealed and do not allow System A
substrates to exit at a rate equal to or greater than the
uptake rate.


CHAPTER IV
FURTHER DISCUSSION ON THE USE OF MEMBRANE VESICLES
AND RECONSTITUTION
It is evident that System A transport activity is
increased during the course of several disease states. In
the diabetic patient, for example, blood levels of insulin
are low but blood levels of glucagon are higher than normal
(Unger, 1978). It is likely that the increased blood levels
of glucagon are responsible for stimulating the hepatic
System A transport activity reported in diabetic liver cells
(Barber et al., 1982). Since many System A substrates are
glucogenic amino acids, such as alanine, it is also possible
that the increased rate of transport of glucose precursors
into liver cells is responsible, in part, for higher levels
of blood glucose. Further studies of the mechanism and regu
lation of System A using membrane vesicles and reconstituted
proteoliposomes will provide a better understanding of how
amino acid transport relates to the overall metabolism of
the cell and may aid in the treatment for such incurable
diseases as diabetes mellitus.
The establishment of a procedure in our laboratory for
the isolation of rat liver membrane vesicles either from
intact liver tissue or isolated hepatocytes will allow us to
gain valuable information on the regulation and activity of
128


60
treatment, whereas the membrane vesicles showed a 13-fold
change.
The apparent incomplete retention of stimulated System
A activity in the membrane vesicles compared to the intact
cells may be due to a difference in the composition of the
control and glucagon-treated vesicles with respect to the
three domains known to exist on the hepatocyte surface
(Wisher and Evans, 1975; Hubbard et al., 1983). It has been
reported that Na+-dependent alanine transport activity is
about equally distributed on the cell surface (Van
Amelsvoort et al., 1980; Meier et al., 1984b), but these
studies were not performed on hormone-induced membranes.
We assayed plasma membrane marker enzymes to determine
if the amount of canalicular and basolateral membrane in the
control and glucagon-treated vesicles were similar. The
control vesicles showed an enrichment of 6-fold for 5-
nucleotidase (canalicular) and 2-fold for fluoride-stimula
ted adenylate cyclase (basolateral) when compared to the
homogenate, whereas the vesicles from glucagon-treated rats
showed a 9-fold enrichment of 5'-nuc1eoti dase activity and a
5-fold enrichment of f1uoride-stimulated adenylate cyclase
(Table 2-6). These data indicate that there are differences
in the absolute content of canalicular and basolateral mem
brane between the two vesicle populations, but the glucagon-
treated vesicles actually show a higher degree of enrichment
for both markers. The ratio of the 5'-nuc1eoti dase activity
to the fluoride-stimulated adenylate cyclase activity
appears to be similar for both preparations (control = 2.5,