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Protein modification of the system A carrier and amino acid- dependent gene regulation in hepatic tissue

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Protein modification of the system A carrier and amino acid- dependent gene regulation in hepatic tissue
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Chiles, Thomas Crane, 1960-
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English
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xii, leaves : ill. ; 29 cm.

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Amino acids ( jstor )
Cell membranes ( jstor )
Cells ( jstor )
Cellulose nitrate ( jstor )
Gels ( jstor )
Hepatocytes ( jstor )
Liver ( jstor )
Membrane proteins ( jstor )
Rats ( jstor )
Reagents ( jstor )
Biochemistry and Molecular Biology thesis Ph.D ( mesh )
Carrier Proteins ( mesh )
Dissertations, Academic -- Biochemistry and Molecular Biology -- UF ( mesh )
Gene Expression Regulation ( mesh )
Membrane Proteins ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1988.
Bibliography:
Bibliography: leaves 254-270.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Thomas Crane Chiles.

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Full Text
PROTEIN MODIFICATI(ON OF THE SYSTEM A CARRIER AND AMINO
ACID-DEPENDENT GMNE RBEGUIATION IN HEPATIC TISSUE
By
1HOMAS CRANE CHILES
A DISSERTATION PRESENTED TO THE
GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUIFILIME~T
OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHIIDSOPHY
UNIVERSITY OF FIDRIDA
1988




ACUILEDGEMENTS
I would like to thank all the members of the laboratory for their assistance throughout these studies. I would also like to extend a deep appreciation to Mary Handlogten for her help with the amino acid transport and the protein electrophoresis studies, Elizabeth Dudenhausen for her assistance with the reconstitution assays, and Kathleen Dudeck-Collart for the membrane vesicles studies. In addition, I would like to acknowledge Neil Shay, William Wong, and Catherine Ketchem for their assistance in the molecular biology phase of this research. Finally, I would like to acknowledge especially my mentor Dr. Michael S. Kilberg for his time, training and guidance throughout all of these studies.
ii




TABLE OF CONTENTS
ACLISK OF FIEDGEMES ......................................... ii
LIST OF TABIES. .........................................viii
ABBREVIATIONS............................................. ix
ABSTRACr ................................................xi
CAPTER I AMINO ACID TRANSPORT IN MA24AL9AN CEIS...... 1 CHAPTER II MEMBRANE TRANSPORT) MEIHODS .................. 10
CiAPIER III DIFFERENTIAL SENSITIVITY OF AMINO ACID
TRANSPORT SYSTEMS TO SULF11YDRYL-MJDIFYING REAGENTS..23 Introduction........................................23
Results............................................25
Discussion. ..........................................49
CHAPTER IV EVIDENCE FOR THE DIRECT CIE2M4ICAL
MODIFICATION BY PCMBS OF THE SYSTEM A CARRIER
IN RAT H4 HEPATCMA CEILS............................57
Introduction........................................57
Results.............................................60
Discussion..........................................96
CHAPTER V MATERIAIS AND METHODS FOR PROTEIN CQEMISTRY
AND MOLIECULAR BIOIOGY..............................102
CHAPTER VI IDENTIFICATION AND CHARACTERIZATION
OF AMINO ACID STARVATION INJUCED HEPATIC
ME4RANE PROTEINS................................. 122
Introduction.......................................122
Results............................................129
Discussion. .........................................219
CHAPIERVII SUMMARY. ....................................226
APPENDIX I TISSUE CXURE AND TRANSPORT MEDIUM ........ 230 APPENDIX II SOIDTIONS FOR GEL ELECTROPHORESIS .......... 233 APPENDIX III GENERAL METHODS AND ENZYME ASSAYS .......... 236 APPENDIX IV SOIITYIONS FOR MOLECUIAR CIDNING ............ 246
iii




APPENDIX V GENERAL METHODS FOR MDIECULAR CIDNING ..... 249 APPENDIX VI REAGENTS FOR THE CHEMICAL MlDDIFICATION
OF PROTEINS................................253
BIBLIOGRAPHY............................................254
BIOGRAPHICAL SKETCH .....................................271
iv




LIST OF FIGURES
1. Concentration Dependence of the Inhibition of System A
Activity in Normal Rat Hepatocytes and Several Hepatama
Cell Lines by NEM or P BS..............................31
2. MeAIB Inhibition of Nat-Dependent AIB Transport in
Several Hepatama Cell Lines.............................36
3. Concentration Dependence of NEM Inhibition of System A
Activity in Fao Hepatcaa Cells Cultured in Amino Acid
Rich Medium............................................39
4. Concentration Dependence of the Inhibition of System A
Activity in Rat Hepatocytes or Rat H4 Hepatama Membrane
Vesicles by NEM........................................43
5. Reversal of PCXBS Inactivation of System A by
Dithiothreitol...........................................62
6. Concentration Dependence of the Inhibition of System A
Activity in Rat Hepatocytes or H4 Hepatama Membrane
Vesicles by PCBS......................................66
7. Kinetics of AIB Transport Following PCMBS Treatment of H4
Hepatama Cells...........................................69
8. Time-Course of AIB Uptake into H4 Hepatama Cells Following
PEMBS Treatment........................................72
9. Effect of PCHBS on the Intracellular Water Volume of H4
Hepatama Cells...........................................74
10. Time-Course of AIB Efflux from H4 Hepatama Cells Following
P MBS Treatment..................................... .....77
11. Dixon Plot of the Kinetics of Ir-Norleucine Inhibition of
AIB Uptake.............................................81
12. Inactivation of System A Transport Activity by PCBS:
Kinetics of I-Norleucine, Ir-Serine, and I-Alanine
Protection.............................................84
13. Time-Course for 3-O-Methyl-D-Glucose Exodus from H4
Hepatama Cells Following PHBS Treatment................. 92
V




14. Electrphoretic Protein Pattern of a Coamassie Blue Stained
Two-Dimensional Polyacrylamide Gel...................... 131
15. Fluorograms of the Synthesis of Individual Hepatic
Membrane Proteins in Response to Amino Acid Starvation..134
16. Quantitation of the Rates of Incorporation of RadiolabeledIr-leucine into Membrane Proteins........................138
17. Antibody Production Scheme for Preparing Monospecific
Polyclonal Antibodies.................................141
18. Detection of MP-73 by Immunablotting of Rat Liver Membrane
Proteins................................................144
19. Tine-Course of Antibody Production Against MP-73........ 147
20. Serum Antibody Titer Against MP-73.....................149
21. Immunioblot Detection of MP-73 Following Two-Dimensional
Polyacrylamide Gel Electrophoresis...................... 152
22. Immunoblot of Immune or Noninmune Serum Against MP-66
Following One-Dimensional Polyacrylamide
Gel Electrophoresis....................................155
23. Immundblot of MP-66 Following Two-Dimensional
Polyacrylamide Gel Electrophoresis...................... 157
24. Estimation of the Molecular Weight of MP-73 Following OneDimensional Sodium-Dodecyl-Sulfate Polyacrylamide
Gel Electrophoresis.....................................160
25. Immunoblot of MP-73 Following One-Dimensional
Nonreducing Sodium-Dodecyl-Sulfate Polyacrylamide
Gel Electrophoresis.................................162
26. Estimation of the Molecular Weight of MP-73 Following OneDimensional Nonreducing Sodium-Dodecyl-Sulfate
Polyacrylamide Gel Electrophoresis.......................164
27. Comassie Blue Stain of Proteins frcm the Subcellular
Fractionation of Rat Liver...........................167
28. Silver Stain of Proteins from the Subcellular
Fractionation of Rat Liver...............................169
29. Suboellular lIcalization of MP-73 by Imiunablot Analysis.171
30. Coamassie Blue Stain of Proteins from the Fractionation of
Rat Liver Mitochondria.................................175
31. Silver Stain of Proteins from the Fractionation of
Rat Liver Mitochondria..................................177
Vi




32. Inmunmoblot Analysis of Fractionated Rat
Liver Mitochondria.......................................179
33. MP-73 Inminoreactivity Following Triton X-114 Phase
Separation of Rat Liver Mitoplasts ....................... 183
34. Fast Green Stain of the Proteins Following Triton X-114
Phase Separation of Rat Liver Mitoplasts................. 185
35. Inmunoprecipitation of MP-73 from [3H]-Ir-leucine
Labeled Cells............................................188
36. Biosynthesis of MP-73 During Amino Acid Deprivation...... 190 37. Adaptive Regulation of System A During Amino Acid
Starvation of Rat Hepatocytes............................193
38. Map of the Lambda gt11 Gename Shwing Major Restriction
Sites....................................................197
39. Imminoscreening of a Human Fetal Liver cENA Expression
Library with Antiserum Against MP-73..................... 200
40. Molecular Size of the Human Fetal Liver cEDA Insert
from Bacteriophage lambda gt11 and pUC19................. 203
41. Molecular Size of the Adult Rat Liver cA Insert
fram Bacteriophage lambda gt11 and pUC19.................205
42. Restriction Map of pUC19 Gencne..........................209
43. Restriction Endonuclease Analysis of the Bacteriophage
Lambda gt11 and the Subcloned Human Fetal Liver
cENA Insert..............................................211
44. Hybridization Analysis of Rat and Human Liver cENA' s..... 213 45. Northern Analysis of Polyadenylated RNA Complementary
to the cENA Isolated from the Adult Rat Liver
cENA Expression Library..................................216
46. Northern Analysis of Polyadenylated RNA Camplementary
to the cD[A Isolated from the Human Fetal Liver
cMA Expression Library..................................218
Vii




LIST OF TABLES
1. Sensitivity of System A Activity in Normal Rat Hepatocytes
and H4 Hepatana Cells to Protein Modifying Reagents ....... 26
2. Sensitivity of System A Activity in Normal Rat Liver
and H4 Hepatama Vesicles to Phenyl Isothiocyanate ......... 28
3. Reconstitution of System A Activity fran Rat Hepatocytes
or H4 Hepatana Cells Following NEM-reatment of
Solubilized Plasma Membrane Proteins......................45
4. Amino Acid-Dependent Protection fran Inactivation by PCMBS
of System A-Mediated Transport............................47
5. Sensitivity to NEM or PCXBS of Several Amino Acid Transport
Systems in Rat Normal Hepatocytes or H4 Hepatama Cells.... 48
6. Ir-Norleucine-Dependent Protection fran PHEBS Inactivation
of System A Transport in H4 Hepatama or Rat Liver
Membrane Vesicles.........................................64
7. Sensitivity of System A Activity in H4 Hepatana Cells to
PCMBS in the Presence and Absence of Na+-ions. ............ 78
8. Protection of System A Transport Activity
by Amino Acids........................................86
9. Ir-Norleucine Protection from PCMBS-Dependent Inactivation
of Several Amino Acid Transport Systems
in H4 Hepatama Cells........... .......................88
10. Effect of PCMBS on Lactate Dehydrogenase Release fran H4
Hepatama Cells............... ...................... 94
11. Effect of PC4BS on the Nat-Dependent Uptake of
I-Pyruvate, Uridine, and AIB in H4 Hepatama Cells..... ...95
12. Enzyme Marker Analysis of Isolated Rat Liver
Mitochondria....................................... 173
13. Enzyme Marker Analysis of Rat Liver Submitochondrial
Fractions..........................................181
Viii




ABBREVIATIONS
ACI actinmiycin D AIB 2-aminoiscbutyric acid AMP ampicillin ASN Irasparagine BME 2-mercaptoethanol BSA bovine serum albumin Cho1KRB Nat-free Krebs-Ringers bicarbonate buffer CholKRP Nat-free Krebs-Ringers phosphate buffer DIT dithiothreitol EDI'A ethylenediamine tetraacetic acid FBS fetal bovine serum HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid h hour IPIG isapropyl-B-D-thiogalactopyranoside kDa kilodalton Kl/2 concentration of inhibitor that yields half-maximal
inactivation of transport activity
Ep concentration of amino acid that yields half-maximal
protection of transport activity MeAIB 2- (methylamino) -isobutyric acid 3MG 3-0-Methyl-D-Glucose MEM Eagles Minimal Essential Medium g microgram 1 microliter
ix




M micramelar IM milinolar mmol milimole min minute MW molecular weight NaCl sodium chloride NaERB Nat-containity Krebs-Ringer bicarbonate buffer NaERP Nat-containing Krebs-Rixger phosphate buffer nmol nanamole NaOH sodium hydroxide NEM N-ethylmaleimide PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCMBS p-chloramercuribenzene sulfonate pmol picamole S.D. standard deviation SDS sodium-dodecyl-sulfate Tris tris (hydroxymethyl) amincmethane X-gal 5-brcmo-4-chloro-3-indolyl-B-galactoside




Abstract of Dissertation Presented to the University of Florida
in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
PROTEIN MODIFICATION OF THE SYSTEM A CARRIER AND AMINO ACID-DEPENDENT GENE REGULATION IN HEPATIC TISSUE
BY
THOMAS CRANE CHILES
April, 1988
Chairman: Michael S. Kilberg
Major Department: Biochemistry and Molecular Biology
The transport of amino acids by System A in normal rat hepatocytes and several hepatama cell lines has been shown to be inhibited by modification of free sulfhydryl group(s). System A-mediated uptake in all of the hepatic cell types tested was inhibited by the organic mercurial p-chloramercuribenzene sulfonate (PCMBS). In contrast, the sensitivity of the System A carrier in hepatama cells to inactivation by N-ethylmaleimide (NEM) varied from moderate to no inhibition. The inactivation of System A in the H4 hepatama cells by PC4BS was rapidly reversed by treatment of the cells with dithiothreitol. The PCMBS inhibited the initial rate of AIB uptake, whereas AIB efflux was stimulated. The net effect was a decreased steady state distribution ratio for AIB accumulation (AIBi/AIBout) from 24 to 1. Kinetic analysis revealed that the effect of PC4BS was a reduction in the maximal translocation rate as well as a decrease in the affinity for System A substrates. Substrate level protection fra inhibition by xi




PCMBS was observed in H4 hepatoma cells, whereas none of the System A substrates tested protected transport activity in normal hepatocytes. Amino acid-dependent protection was stereospecific and largely restricted to System A. Similar results were obtained using plasma membrane vesicles isolated from intact hepatcma cells and rat liver.
The biosynthesis of several rat liver membrane proteins was
demonstrated to be enhanced by amino acid deprivation of hepatocytes in primary culture. One of these proteins, MP-73, has an estimated molecular weight of 73 kDa and an isoelectric point of 7.0. Monospecific polyclonal antiserum prepared against MP-73 was used to localize the protein to a rat liver subcellular fraction highly enriched for inner mitochondrial membranes. Based on Triton X-114 phase separation, the protein appears to be a hydrophobic integral membrane protein. The antiserum was used to screen both an adult rat liver and a human fetal liver lambda gt11 cENA expression library. Of five rat and four human independent clones that were plaque-purified, two rat (2000 and 1000 bp) and one human (1100 bp) were subcloned into the bacterial plasmid pUC19. cDNA probes prepared from either the rat or human inserts identified two transcripts in rat and human tissue of approximately 1900 nucleotides and 2400 nucleotides in length.
Xii




CIAPFER I
AMINO ACID TRANSPORt IN 1MA9ALIAN CELLS
All known cellular organisms utilize L-amino acids to conduct many essential processes (Meister, 1965). The liver is a major site for amino acid metabolism in the body with most of the metabolism occurring in the hepatocyte. These metabolic processes include protein synthesis, gluconeogenesis, utilization by both the urea and tricarboxylic acid cycles, as well as conversion into a wide variety of metabolites (lehninger, 1982). In order to carry out these processes, however, cells must maintain intracellular levels of both essential and nonessential amino acids. Non-essential amino acid levels are maintained by a variety of mechanisms which include transport from the extracellular medium, de novo biosynthesis, interconversion of A-keto acids, and intracellular protein degradation. On the other hand, the essential amino acids are supplied largely by intracellular protein degradation or by transport into the cell; however, the plasma membrane acts as a barrier to polar and/or charged molecules. Consequently, in order to accumulate amino acids from the extracellular medium, most eukaryotic cells possess a series of plasma membrane carriers capable of selectively transporting amino acids.
Movement of organic solutes, such as amino acids, from the extrato the intracellular environment necessitates their passage through the plasma membrane. Transport of amino acids across the plasma membrane
1




2
occurs by two distinct routes, either passive diffusion or facilitated uptake (protein-mediated). Passive diffusion is a nonprotein mediated process governed largely by the molecular size and net charge of the solute (Collander, 1949). Flux across the membrane is governed largely by the direction of the solute's concentration gradient and does not saturate at high substrate concentrations. In contrast, proteinmediated transport of amino acids occurs via two modes, designated as either facilitated transport or active transport. Facilitated transport is an energy-independent processes by which solutes are transported in the direction of their concentration gradient. Net flux ceases at electrochemical equilibrium, resulting in a distribution ratio of 1:1 for the amino acid with respect to the inside and outside of the cell (Houslay and Stanley, 1982). Facilitated carrier proteins show both stereo-specificity and saturability with respect to their substrates. Many energy-independent amino acid transport systems demonstrate the property of trans-stimulation, i.e., the stimulation of a cis-to-trans unidirectional flux of labeled substrate brought about by the presence of unlabeled substrate at the trans-face.
Cells possess two forms of active transport designated as either primary or secondary. Primary active transport by definition requires the hydrolysis of ATP; examples of this form of transport include the ion-dependent ATPases, such as the Na+/K+-ATPase and the Ca+-ATPase. Secondary active transport is an energy-dependent processes which allows transport of organic solutes against their concentration gradient. Energy is usually supplied in the form of an ion-driven gradient. In




3
the case of hepatic amino acid transport, solute flow is coupled with that of Nat-ion flux, resulting in the simultaneous transfer of one or more Na -ions with an amino acid molecule (Christensen, 1975; Crane, 1977). Although cotransport systems can operate in opposite directions (antiport), such as the H /Ca" transporter of intestinal brush border membranes, the Na--dependent amino acid carriers appear to transport both Nations and amino acids in the same direction (symport). The Na*ion gradient is provided by the plasma membrane Na /K1-ATPase which employs the hydrolysis of ATP to provide the energy needed to maintain the Nat-gradient. The Nat-ion electrochemical gradient in turn allows a distribution ratio ([amino acid]irj/[amino acid]Out) to exceed unity. The Na-dependent amino acid transport systems can also be subjected to either trans-stimulation or trans-inhibition. Trans-inhibition is a phenomenon of the cotransport systems which demonstrate a particular order for binding of substrates. The binding of unlabeled amino acid on the trans-face decreases the influx of labeled substrate at the cis-face (Christensen, 1975).
Characterization of amino acid transport systems began with the investigations by Christensen and his coworkers. Initial studies centered on the Ehrlich ascites tumor cell. These observations revealed that the neutral amino acids were transported mainly by three agencies designated as Systems A, ASC, and L. System A was originally described by Oxender and Christensen (1963). The carrier was noted to serve best for L-glycine and Ir-alanine entry. It discriminated against amino acids with either five- or six-carbon atoms, especially when the side-chains




4
were branched. N-methylation of most of the neutral amino acids restricted their uptake solely to System A (Christensen et al., 1965). Hepatic System A activity is monitored by the model amino acid substrate 2-aminoiscbutyric acid (AIB). Both AIB and its N-methylated analog, 2(methylamino)-isabutyric acid (MeAIB), are not metabolized to a significant extent due primary to the lack of an alpha-hydrogen on the alpha-carbon (Christensen and Jones, 1962; Noall et al., 1957). The carrier is also strongly dependent on Nat-ions; however, the hepatic System A carrier can accept Li+-ions as a replacement for Na+t-ions (Kilberg, 1982). System A is subjected to trans-inhibition by substrates, inhibited at pH values below 7.0, and is controlled by both hormones and substrate availability. The later process is known as adaptive regulation (Kilberg et al., 1982).
The adaptive regulation process was originally described in chick embryo heart cells (Gazzola et al., 1972) and diaphragm muscle (Riggs and Pan, 1972), and more recently in hepatocytes (Kelly and Potter, 1978), as well as the H4 hepatcma cell line (Kilberg et al., 1985). In hepatic tissue, the substrate-dependent regulation of System A consists of two distinct phases. The first phase (termed derepression) occurs when hepatocytes are starved for extracellular amino acids and results in a stimulation of transport activity. The second phase (termed repression) results from the addition of System A substrates to hepatocytes that have been previously cultured in the absence of amino acids; this results in a rapid decrease in System A activity.
The stimulation of transport activity which occurs during amino




5
acid starvation is accompanied by an increase in the substrate translocation rate (Vmag) by the carrier, without a change in the apparent affinity of the carrier for substrates (Kelly e 4., 1982). The initial increase in transport activity occurs independent of protein synthesis and has been ascribed to a release of the carrier from transinhibition. Although the basis for trans-inhibition remains unresolved, it is thought to arise primarily from the accumulation of active System A carriers in a cytosolic orientation due to increased intracellular levels of amino acids. Upon amino acid starvation, the intracellular amino acid concentration is lowered, thereby allowing more active System A carriers to be oriented on the extracellular surface (i.e., the carriers are "released" from trans-inhibition). Release from transinhibition accounts for only a small increase in transport activity following amino acid deprivation; the majority of the enhanced transport activity, however, requires the synthesis of both RNA and protein (Kilberg et al., 1985; Kelly and Potter, 1978). More recently, tunicamycin, an inhibitor of asparagine-linked glycoprotein biosynthesis, was shown to inhibit the induction of System A activity upon amino acid deprivation of hepatocytes (Barber et 4., 1983), suggesting the involvement of a glycoprotein in adaptive regulation.
The second phase of adaptive regulation is the reversal of the
stimulation of transport activity. Simply, inhibition of transport occurs when cells that have been previously starved for amino acids are placed in a medium containing System A substrates. This decay of transport activity can be induced by the addition of a single System A




6
substrate (Handlogten and Kilberg, 1984; Handlogten et al., 1985; Bracy et al., 1986). For example, within 6 h after the addition of Lasparagine to primary cultures of rat bepatocytes, System A-mediated transport is lowered to basal rates. The decay is characterized by a half-time of 1.5 h. Although the initial decrease in transport activity is protein synthesis-independent and ascribed to trans-inhibition, the majority of the decay requires the synthesis of both RNA and protein and is considered to be the result of gene repression.
System ASC was originally described in the Ehrlich ascites cell (Christensen, 1967). Its activity was defined as that portion of the Na-dependent neutral amino acid uptake which is insensitive to the System A specific substrate MeAIB. A significant amount of Ir-alanine, Ir-serine, and I-cysteine uptake by the Ehrlich cell was found to be mediated via System ASC. The carrier prefers neutral amino acids with polar side-chains containing oxygen or sulfur. System ASC is not inhibited at pH values below 7, yet is subject to trans-stimulation by substrates. Although many of the characteristics of System ASC from the Ehrlich cell are retained in the rat hepatocyte, Kilberg et Al. (1979) found that the Nat-dependent uptake of I-cysteine into rat hepatocytes was totally insensitive to MeAIB, suggesting that all of the Natdependent uptake of Ircysteine was mediated by System ASC. This is not the case, however, for the H4 (Kilberg et al., 1985) and the HTC (Hardlogten et Al., 1981) hepatcma cell lines. Ir-threonine appears to represent a more selective substrate for System ASC in these transformed liver cells. In contrast to the Ehrlich cell, however, the Nat-




dependent uptake of AIB is not solely mediated by System A. In both freshly isolated hepatocytes (LeCam and Freychet, 1977) and same hepatama cells (Kilberg et al., 1985) the hepatic Nat-dependent uptake of AIB has a small System ASC component.
System L, a Na*-independent carrier, was demonstrated by Oxender and Christensen (1963) to prefer apolar amino acids, such as those with branched and aromatic side-chains, as well as L-histidine and Imethionine. A decrease in affinity for amino acid substrates was found to occur sharply as the number of carbon atams fell below five. System L is characterized by its high capacity for trans-stimulation and can be assayed selectively with the modeled substrate BCH, 2-aminabicyclo(2,2,1) -heptane-2-carboxylic acid (Christensen et al., 1969). The activity of System L has also been characterized in isolated hepatocytes (McGivan and Bradford, 1977). Although the hepatic System L retains most of the characteristics first described in the Ehrlich cell, recent studies have demonstrated the presence of a previously undetected Natindependent system (Weissbach et al., 1982). The identification of this second component system was based largely on kinetic analysis. In these studies, the initial rate of uptake of L-leucine and BCH was found to be biphasic in nature as a function of time in primary culture. The first ccmponent (Ll) has a high affinity for System L substrates and a low capacity for transport, whereas the second component (L2) has a low affinity for substrates, yet a high capacity for transport. Furthermore, when adult rat hepatocytes were placed into primary culture the activity of the LI component increased during tim in culture,




8
whereas the activity of the 12 component actually decreased. Interestingly, fetal hepatocytes, HTC and H4 hepatama cells contain only the Li component (Kilberg et al., 1983).
Other more selective neutral amino acid carriers include System
Gly, a Nat-dependent L-glycine specific carrier. This transport system was originally described in rabbit reticulocytes by Winter and Christensen (1965) and mediates the uptake of Ir-glycine and the selective substrate, L-sarcosine (N-methyl glycine). In hepatic tissue, the carrier is insensitive to changes in the extracellular pH and is assayed by measuring the Nat-dependent uptake of L-sarcosine or Lglycine in the presence of MeAIB. System Gly is unique among the amino acid transport systems in that it requires two Nat-ions for every Lglycine molecule transported.
System N was first described (Kiberg al., 1980) in isolated rat hepatocytes by the demonstration that the Nat-dependent uptake of Iglutamine was insensitive to both System A substrates (MeAIB) and System ASC substrates (Ircysteine). System N has a high affinity for nitrogencontaining neutral amino acids, is highly stereospecific and is inhibited by lowering the extracellular pH. System N exhibits the ability, although somewhat weak (2- to 3-fold) to undergo adaptive derepression by substrate amino acids.
Most animal cells also possess a carrier, System y*, which
transports cationic amino acids. Recently, White and Christensen (1982) characterized System y+ in a variety of rat hepatama cell lines as well as isolated rat hepatocytes. The transport agency was found to be pH-




9
insensitive, was subject to trans-stinulation by substrate amino acids, and was inhibited by neutral amino acids when incubated in the presence of Nat-ions. Transport system specific for the anionic amino acids have also been identified in the Ehrlich cell (Gazzola et al., 1981) and rat hepatocyte (Ballatori et al., 1986). In isolated rat hepatocytes, anionic amino acid uptake occurs largely via a Na-dependent agency which is inhibitable by the model substrates Lr-cysteate and Lcysteinesulfinate. The transport system has been designated as System X"A,G and is subjected to trans-stimulation by substrate amino acids, possesses a low K. (0.016 rM) and is induced by dexamethasone (Gebhardt and Mecke, 1983). In hepatic tissue, this carrier appears to be accamanied by a lower affinity system, specific for Irglutamate (Km
3.24 EM) .
A system for the transport of B-alanine and taurine has been
described for the Ehrlich cell (Christensen, 1964). Hardison and Weiner (1980) have described B-alanine transport in freshly isolated hepatocytes. In these studies, the uptake of 8-alanine was shown to be strongly dependent on Nat-ions, subjected to trans-stimulation, and cmetitively inhibited by taurine and hypotaurine.




CHAPIER II
MEMBRANE TRANSPr METHODS
Male Sprague-Dawley rats weighing 100-200 g were obtained from a colony maintained by the University of Florida, Division of Animal Resources. The [methyl-3H]-2-aminoisabutyric acid, L[G-3H]-threonine, 8-[3-3H(N) ]-alanine and [methyl-14C]-3-O-methyl-D-glucose were from ICN Pharmaceuticals (Irvine, Ca). The [1-14C]-pyruvate, L[G-3H]-glutamine, L[5(n)-3H]-arginine, L[2-3H]-glycine, [5,6-3H]-uridine and L[4,5-3H]leucine was purchased from Amersham Corp (Arlington Heights, IL). The unlabeled amino acids, protein-modifying reagents, Type I collagenase, and antibiotics were obtained from Sigma Chemical Co (St. LIuis, Mo). The Eagles minimal essential medium (MEM) and fetal bovine serum (FBS) were purchased from Flow Laboratories (Mclean, Va) and the scintillation cocktail (No. 3a70B) was obtained from Research Products International (Mt. Prospect, IL). Filters for the vesicle uptake studies were Gelman type GN-6 (0.45 micron). Tissue culture dishes and the 24 well cluster trays were from Fisher Science (Orlando, Fl). All other tissue culture supplies were purchased from Corning. Highly purified glucagon was a gift from Dr. Ronald E. Chance of Eli Lilly Laboratories. Hepatcma Cell Culture
The rat hepatama cells H4-II-EC3 (H4), Fao, HTC, and the human
hepatama HepG2 were grown in 75 cm2 culture flasks (Falcon 3023, tissue culture flasks) at 370C under a humidified atmosphere of 5% 002/95% air. The hepatama tissue culture cell lines were maintained in 75 anm2 culture
10




flasks in MEM, pH 7.4, supplemnnted with 25 nM NaHOD3, 2.5 nM glutamine, 10 g/ml penicillin, 5 g/ml streptamycin, 28.5 g/ml gentamicin, 0.2% bovine serum albumin (BSA) and 5% FBS. Two to three days prior to the transport assays, the hepatama cells were rinsed with (PBS) and removed from the culture flasks by trypsinization (0.5 ml of a solution containing 0.05% trypsin and 0.02% EDIA in PBS [10 nM sodium phosphate, 150 nM NaCl, pH 7.4]). The cells were then diluted with 100 to 150 ml of MEM containing 5% FBS and transferred to 24-well cluster trays (Costar #3524, 24 well/16 m well dia.) at a ratio of 4 to 6 trays containing approximately 1 ml cells/well. Sterile Nat-containing KrebsRinger bicarbonate (NaKRB) buffer supplemented with antibiotics was used as the amino acid-free (AAF) medium for the substrate starvation experiments. The composition of the Krebs-Ringer bicarbonate is listed in Appendix I.
The origin and characterization of the hepatcaa cell lines used in these studies is described below. The rat H4 hepatama cell line, H4II-EC3 (Pitot e al., 1964), was derived from the original Reuber H4 or Reuber H35 hepatama (Reuber, 1961). The Reuber H4 hepatcma was obtained by feeding rats a diet supplemented with N-2-fluorenyldiacetamide, which resulted in a bile secreting transplantable hepatocellular carcinoma. The Fao hepatama is a well differentiated cloned cell line obtained after exposure of H4 hepatcma cells to 8-azaguanine (Deschatrette and Weiss, 1974). The Fao cell line expresses a number of liver specific proteins as well as the production and secretion of serum albumin, and hormonal induction of both tyrosine aminotransferase and alanine




aminotransferase. Interestingly, the Fao hepatama cell line does not contain readily detectable levels of alcohol dehydrogenase, glucose-6phosphate dehydrogenase or aldolase. The HTC hepatma cell line was derived originally frcm feeding male buffalo rats a diet containing N,N'-2,7-fluorenylenebis-2,2,2-trifluoroacetamide (Thampson et al., 1966). The human hepatcma, HepG2, was obtained from liver biopsies of a male exhibiting primary hepatoblastoma and hepatocellular carcinoma (Aden get al., 1979). The HepG2 is histologically a well differentiated parenchymal cell, capable of biosynthetically producing many liver specific proteins, including ceruloplasmin, albumin and transferrin (Knowles et al., 1980).
Hepatocyte Isolation and Culture
Hepatocytes were isolated from male Sprague-Dawley rats (100-200 g) as described by Kilberg (1988). Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital (65 ng/kg body wt.). The right renal vein and inferior vena cava were then rapidly cannulated and retrograde perfusion was began at 3 ml/min. The perfusion solution was maintained at 370C and consisted of 25 mM sodium phosphate, pH 7.4, 3.1 mM potassium chloride, 119 mM sodium chloride, 5.5 mM glucose, and 5 mg/l phenol red. After perfusion was initiated, the hepatic artery and portal vein were severed, the superior vena cava was clamped with the aid of a hemostat and the pump speed was then increased to 10 ml/min. When liver was perfused with approximately 100 ml of the perfusion solution, 75 units/ml of collagenase (Type I from Sigma, C-01301) dissolved in 10 ml of perfusion buffer was added to the remaining 100 ml




13
of perfusion buffer. Following perfusion, the liver was removed and placed in ice-cold NaERB (30 ml). The liver was dispersed by gentle agitation and the cells separated by filtration through a 75 m nylon cloth. The mixture was then centrifuged at 100 g for 2 min. The supernatant was discarded and the pellet containing hepatocytes was resuspended in 40 ml NaKRB and centrifuged at 100 g for 2 min (4-C). The washing and centrifugation procedure was repeated an additional three times. Hepatocyte viability was determined by trypan blue exclusion and was typically 85% to 90%. The pellet containing hepatocytes was then diluted to 8 x 105 cells/ml with warm culture medium (MEM or NaRB) and the cells were placed (0.33 ml/well) into the bottom of 24-well cluster trays which had been previously coated with collagen. Collagen coating of the cluster trays was performed by adding
0.5 ml of a stock solution consisting of 20 g/ml sterile acid-soluble collagen (Sigma Type III, C-3511) to each well. After 12 h, the solution was removed and the trays stored at room temperature. The stock solution of collagen was made by dissolving the collagen in 0.5% acetic acid (1 mg collager/ml). The hepatocytes were maintained in an incubator at 37-C with a humidified atmosphere of 5% 002 and 95% air. Determination of Intracellular Water Volume
Intracellular water was measured by the 3-0-methyl-D-glucose method of Kletzien et al. (1975). For example, H4 hepatama cells were placed in 24-well cluster trays containing MEM and 5% FBS for two days as described above. The medium was removed by rinsing the cells with NaKRP buffer (Nat-containing Krebs-Ringer phosphate buffer, Appendix I) and




14
then placed in 1 ml of NaKRP buffer (37-C) containing varying aMounts of unlabeled 3-0-methyl-D-glucose (3MG) at concentrations of 1 nM, 5 nM, and 10 inM. These 31G solutions also contained a trace amount (3.5 nmole) of [methyl-14C]-34 (S.A. = 40 Ci/xole). The 314 is used to monitor the glucose carrier, because it is not metabolized to a significant degree by cells (Stein, 1986). The cells were then incubated with the 3E solution for 1.5 h. This incubation period was intended to allow the 3M4 to equilibrate across the plasma membrane; the cells were then washed quickly four times with 2 ml of ice-cold CholERP (choline-containing Krebs-Ringer phosphate, Appendix I) solution containing 1 nM phloretin and the amount of 3M retained in the cells was determined as described below for the amino acid analysis. Phloretin is an inhibitor of glucose transport and is very effective at blocking 3MG efflux from cells previously loaded to equilibrium with the sugar (Kletzien et al., 1975). Since the glucose carrier in hepatic tissue is a facilitated transport system, the inside 314 concentration is equivalent to the extracellular concentration at equilibrium. Therefore, by determining the number of moles of 314 intracellularly per mg protein at a given extracellular concentration the intracellular water content per n protein can be ascertained. Whole Cell Transport Assay
Transport of whole cells was measured by a modification (Kilberg et a., 1988) of the method originally described by Gazzola et al. (1981). Briefly, cells plated in 24-well cluster trays were incubated in CholERP for 15 to 30 min at 37- C. This depletion period was intended to




15
minimize possible trans-effects due to intracellular Nat-ions and substrate amino acids. To initiate transport, the CholERP buffer was removed and the appropriate radioactive amino acid solution in NaERP or CholERP buffer was added. The composition of the radiolabeled uptake buffers is described in Appendix I. Exposure of the cells to the uptake solution was accomplished simultaneously for all wells by using the camplentary fitting lid to the 24-well cluster trays. The lids were modified so that they contained Beem embeddinq capsules (#00). Within 30-60 sec the radioactive solution was discarded by inverting the cluster tray and the cells washed four tines with 2 ml/wash of ice-cold Cho1RP buffer (5 seq/wash). This was also accamplished simultaneously for all wells by modifying the complementary fitting lids with 12 x 75 mm plastic test tubes. After completion of the transport assay, the hepatocytes were solubilized by the additional of 0.2 ml of a solution containing 0.2 N NaOH and 0.2 % SDS. Within 30 min, the amount of radiolabeled amino acid was determined by removing 0.1 ml of this solution and adding it to 3 ml of scintillation fluid containing 0.1 ml of 0.2 N HC1. To estimate the amount of protein in each well a modification of the lowry method was employed (Kilberg et al., 1983). For the hepatocytes, 0.6 ml of a copper reagent solution containing 0.58 mM EDUA (copper-disodium), 189 nM Na2CO3, 100 AM NaOH, and 1% SDS was added to the remaining 0.1 ml solubilized material. After 10 min, 60 1 of a Folin-Ciocalteau reagent (diluted 1:1) was added to each well. This mixture was then incubated at room temperature (30 min) and the




16
absorbance along with a bovine serum albumin standard curve was measured at 750 nm.
Upon copletion of transport assays with the hepatama cell lines,
0.22 ml of a solution containing 10 % trichloroacetic acid (TCA) was added to each well. The trays were then incubated for 1 h at 4-C. To estimate the amount of radiolabeled amino acid trapped within the cells, approximately 0.2 ml of this solution was removed and added to 3 ml of scintillation fluid. Radioactivity for all cell types was determined by scintillation spectrophotcmetry. To estimate the amount of protein in the wells following the removal of the 'ICA lysate, 0.1 ml of the 0.2 N NaOH and 0.2% SDS solution was added. After 15 min, the protein concentration was assayed as described for the hepatocytes. Data Analysis
The Nat-dependent transport was taken as the difference in the uptake rate of labeled amino acid observed in the presence of Na*containing Krebs-Ringer phosphate buffer (NaKRP) and in the absence of Nat (CholKRP). The saturable Na-independent transport was taken as the difference in the uptake rate of labeled amino acid observed in CholKRP buffer and in CholERP buffer containing 10 :WM of the appropriate unlabeled amino acid. The transport data were calculated and analyzed with the aid of a microcomputer utilizing computer programs that incorporated standard statistical analyses. The transport kinetic paramneters were calculated with FORIRAN programs that estimated and subtracted the basal activity. The corrected data were then analyzed by a non-linear least squares method (Cleland, 1979). Unless otherwise




17
indicated, the results for all whole cell experiments are reported as the averages (+ the standard deviations, S.D.) of 3 to 4 determinations for a single population of cells. Nearly all experiments were repeated using different preparations of cells. Chemical Modification of Plasma Membrane Proteins in Whole Cells
Cells were plated and cultured in 24-well cluster trays containing MEM and 5% FBS as described above. The cells were then rinsed twice with NaERP and incubated in NaERB for the indicated period of time. Preincubation of the cells in NaERB was intended to deplete the intracellular concentration of amino acids, thereby minimizing possible trans-effects and when assayed to increase the amount of measurable System A activity by adaptive regulation. The cells were then washed quickly with NaERP and exposed to NaERP buffer containing the indicated protein modifying reagent at 37*C. The protein modification reagents and their chemical reactivity is listed in Appendix VI. After 10 min, the cells were rinsed twice with 2 ml of CholKRP (370C) and then the individual transport systems were assayed. All transport systems were assayed for 30 sec at 370C, except for System A which was assayed for 1 min. To quantitate the amount of amino acid-dependent protection of transport activity during exposure of cells to protein modifying reagents, the cells were incubated for 10 min in NaERP (to yield the velocity, V), NaKRP containing the protein modifying reagent (to yield the velocity, Vi), NaERP containing amino acid (to yield the velocity, V ), or NaERP containing amino acid and the protein modifying reagent (to yield the velocity, Vaa+.) and then the rate of transport was




18
measured (Chiles and Kilberg, 1986). For those experiments that employed organic mercurials as protein modification reagents, the buffer solution was supplemented with an equal-molar concentration of EDIA. This precaution was intended to chelate any free mercury. The amount of transport activity protected by an amino acid during exposure of the cells to protein reagent was calculated by substituting the appropriate transport velocity into the following equations:
% Inactivation = [ (V-Vi) / (V) ] x 100
% Protection = [ (Vaa+i-Vi) / (Vaa-Vi) ] x 100
Plasma Membrane Vesicle Isolation from Cultured Cells
Plasma membrane-enriched vesicles were prepared from H4 hepatanma cells by culturing the cells in 150 um x 25 m Falcon 3025 tissue culture dishes in MEM and 5% FBS (Dudeck et al., 1987). About 6 to 8 h prior to the membrane isolation, the cells were incubated in NaKRB to increase the amount of measurable System A activity by adaptive regulation (Kilberg et al., 1985). The cells were then rinsed twice with Buffer A (0.25 M sucrose, 0.2 mM MgC12, 10 mM HEPES-EDH, pH 7.5), scraped from the culture dishes with a rubber policeman into 15 ml of Buffer A, and then collected by centrifugation at 3,000 g for 5 min (4-C). The cell pellets from 16-20 dishes were combined, resuspended in approximately 20 ml Buffer A containing 1 EM EDIA, 1 mM phenylmethanesulfonyl floride (PMSF) and 5 aM benzamidine (4*C) and disrupted with a Potter-Elvehjem hamogenizer using a tight-fitting pestle (100 to 125 motor-driven strokes at 930 rpm). The haomagenate was




19
then centrifuged for 10 min at 500 g to removed unbroken cells and nuclei and the resulting supernatant was centrifuged (30 min at 39,000 g) to obtain a pellet containing plasma membrane-enriched vesicles. The final vesicle preparation was stored in 50 1 aliquots at a protein concentration of approximately 10 mg/ml in Buffer A (-700C). To minimize the loss of transport activity, each aliquot was thawed only once.
Plasma Membrane Vesicle Isolation from Nonnal Rat Liver
Normal hepatocyte plasma membrane vesicles were isolated from
intact rat liver tissue as described by Prpic' et al. (1984). In order to increase the measurable System A transport activity in the resulting rat liver vesicles, the rats were injected with 1 mg of glucagon per 100 gm body weight 5 h prior to membrane isolation (Handlogten and Kilberg, 1984). In brief, male Sprague-Dawley rats, weighing 100-200 g were anesthetized and the liver perfused as described above with the exception that the liver was simply blanched free of blood with ice-cold PBS. Following perfusion, the liver was removed, weighed, and placed in an equal volume (w/v) of ice-cold Buffer B (0.25 M sucrose, 10 EM Trisbase, pH 7.5) containing 1 mM EIA. The liver was then minced and hamagenized by hand using a glass Dounce hcmxgenizer (10 strokes with a loose-fitting pestle followed by an additional 4 strokes with a tightfitting pestle). The hamgenate was diluted to 6% (w/v) with Buffer B containing 1 nI4 EDTA, the weight (grams) was based on the original amount of tissue. The mixture was then centrifuged at 120 g for 2 min (4*C) to remove unbroken cells and nuclei. The supernatant was




20
centrifuged at 1500 g for 10 min (4*C) to obtain a pellet enriched for plasma membrane. This pellet was resuspended in a final volume of 30 ml (Buffer B/1 mM EDIA), filtered through cheese-cloth, diluted with Buffer B/1 mM EDIA to 31.2 ml and then added to Percoll (Sigma, P-1644) to give a final volume of 35.4 ml. The solution was mixed with the aid of a glass rod and 11.8 ml was transferred into each of three 15 ml Corex tubes. The membranes were centrifuged at 34,500 g for 30 min (4*C). After carefully removirxng the tcp lipid-containing layer, the plasma membranes were removed (first band of membranes under the lipid layer), diluted 1:6 (v/v) with Buffer B and centrifuged at 34,500 g for 30 min (4-C). The final plasma membrane-enriched pellet was resuspended in Buffer B at a concentration of approximately 10 mg/ml and stored in 50
1 aliquots at -700C. To minimize the loss of transport activity, each aliquot was thawed only once.
Transport Assay for Vesicles
System A-mediated transport by either membrane vesicles or
reconstituted proteoliposmes was assayed as described by Bracy et al. (1987). Briefly, 20 1 (50 g) of membrane at 40C was added to an equal volume of a 2X uptake buffer (200 mM of either KC1 or NaCl, 10 mM MgC12, and 10 mM HEPES-EDH, pH 7.5, 370C) containing 0.4 mM of AIB and [3H]-AIB (1.0 Ci/ml). Therefore, the final composition of the uptake mixture was 0.125 M sucrose, 5.1 TM MgC12, 10 uM HEPES-IDH, pH 7.5, 100 mM NaCl or KCl, and 0.2 mM [3H]-AIB. The vesicles or proteoliposames were incubated with the uptake mixture for 1 min in a water bath at 220C. Transport was terminated by the addition of 1 ml of ice-cold stop-buffer




21
(125 mM NaCI, 0.2 mM MgC12, 10 MM HEPES-NRI, pH 7.5). The mixture was rapidly vortexed and filtered over a 0.45 m nitrocellulose filter. The filter was rinsed twice with 4 ml of ice-cold stcp-buffer and then the trapped radioactivity measured by scintillation spectrometry. Chemical Modification of Membrane Vesicles
Treatment of isolated membrane vesicles with protein-modifying reagents was performed at 220C for 10 min. Typically, 400 g of membrane protein was resuspended in Buffer A and incubated with the protein-modifying reagent at the indicated concentration (10 min). The final volume and protein concentration during treatment was 0.15 ml and
2.7 mg protein/ml, respectively. The membranes were centrifuged in a Beckman airfuge at 150,000 g for 5 min. The supernatant was decanted
and the membrane pellet was rinsed with 0.1 ml of Buffer A without resuspension. Following another centrifugation, the membrane pellet was resuspended in Buffer A at a protein concentration of 2.5 ng/ml. System A-mediated uptake was immediately assayed as described above. Modification and Reconstitution of Solubilized Membrane Proteins
Plasma membrane proteins from either rat liver or H4 hepatama
cells, at a concentration of 1.5 my/ml in Buffer A, were solubilized by diluting the membrane to 0.5 mg/ml (1:2) with solubilization-buffer (0.1 mM EUDA, 100 mM KCl, 1 M IMSF, 2.5% cholate/4 M urea [Pierce, sequanal grade] and 5 mM HEPES-EDH, pH 7.5). The insoluble membrane material was removed by centrifugation at 100,000 g for 30 min. The solubilized membrane proteins were incubated in the presence of 1 nM NEM for 10 min
at roam temperature and then the reaction was stopped by the addition of




22
2 i4 D-cysteine. For the control incubations, 1 TM KC1 replaced the NEM but the 2 M D-cysteine was added as usual. The solubilized proteins were precipitated by the addition of polyethylene glycol (M.W. = 8,000) as described by Gal e a. (1983).
The precipitated proteins were reconstituted into artificial
proteoliposames by the method of Bracy et al. (1987). A stock solution (40 nJ/ml) of asolectin (Associated Concentrates, BA #1267 soybean phospholipid) was prepared by suspending the lipid in (-containing uptake-buffer and sonicating the mixture in a bath-type sonicator until a clear solution was obtained (approximately 10-15 min). Reconstitution of System A activity was performed by mixing 1 mg of solubilized membrane proteins, 20 mg of sonicated asolectin, and 1 ny of potassium
colate (the stock solution of twice-recrystallized cholate was 10% w/v). The mixture, approximately 1 ml total volume, was frozen in liquid nitrogen, thawed at roam taperature,and then diluted with 4 ml of 2X e-containing uptake-buffer. The mixture was then sonicated for 20 sec in a bath-type sonicator. The proteoliposames were pelleted by centrifugation at 125,000 g for 45 min at 40C and then resuspended in
200 1 of Buffer A with gentle vortexing.




CIAPIER III
DIFFERENTIAL SENSITIVITY OF AMINO ACID TRANSPORT SYSTEMS 'IO SULFHYDRYIjroDIFYING RFEAGNTS
Introduction
Amino acid uptake by animal cells is mediated by several distinct transport systems with overlapping specificities. To date, the information concerning the twelve or more carriers described for amino acid transport has been largely descriptive. The basic characterization of transport systems includes information on the ion-dependency, substrate specificity, pH-sensitivity, trans-effects and regulation, if any, by hormones or substrate availability (Kilberg, 1982) At the molecular level far less is known about individual amino acid transport systems. None of the mmbrane proteins responsible for the hepatic uptake of amino acids has been identified or isolated. In general, proteins that catalyze ion-coupled transport of organic solutes have remained refractory to molecular characterization. This is due mainly to the lack of specific affinity probes and the necessity to employ detergents during isolation and purification. Detergents sometimes irreversibly inactivate carrier function to an extent that the only functional assay, reconstitution, is nonfunctional.
Recent studies by Hayes and McGivan (1983) have provided scme evidence for a protein involved in a*-dependent Ir-alanine transport into isolated rat hepatic plasma membranes vesicles. These authors
23




24
demonstrated that approximately 50% of the total Na+-dependent uptake of L-alanine into hepatic membrane vesicles could be inactivated by 1 rM NEM, suggesting the necessity of free sulfhydryl(s) groups in the transport of L-alanine across the plasma membrane. Preincubation of the vesicle population with 2 4M rL-alanine prior to and during the NE4 treatment reduced the inactivation by 21%, leading the authors to speculate that NEM was inactivating an rL-alanine carrier protein. When vesicles were incubated with radiolabeled NEM in the presence or absence of L-alanine and the labelled proteins analyzed by sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), six major proteins were found to be covalently modified by radiolabeled NEM. In contrast, when the same vesicle population was treated with unlabeled NEtM and L-alanine, washed to remove the excess NEM and alanine, and then incubated with radiolabeled NEM, one major protein of molecular weight 20,000 daltons was labeled. Unfortunately, the authors employed Lalanine as the substrate amino acid in these protection studies, because L-alanine is transported in rat liver by two different Na-dependent agencies, System A and ASC, as well as the Na+-independent System L, it is unclear which carrier protein was labeled.
Recently the Na+-dependent L-proline carrier (imino carrier) of
intestinal brush border membranes was identified and characterized with respect to possible amino acids involved in substrate binding using methods similar to that of Hayes and McGivan (1983). Wright and Peerce (1984) were able to take advantage of two related primary amino grop modifying reagents, fluorescein isothiocyanate (FITC) and phenyl




25
isothiocyanate (PITC) They found that the Nat-dependent uptake of Lproline was equally sensitive to both FITC and PITC and in the presence of sodium, L-proline protected the carrier from inactivation. Specific labeling by FIT was detected after first modifying unrelated proteins with PIC in the presence of sodium and L-proline, the membranes were then washed free of unbound PriC and then incubated with FTC. The proteins were analyzed by SDS-PAGE, scanned with a fluorescence detector, and a FTIC labeled polypeptide of 100,000 daltons was detected. The authors further characterized the carrier with respect to its Na -binding site (s) In this respect, the authors detected specific conformational changes upon alkali ion binding, which were blocked by Nacetylimidazole modification of the carrier.
This phase of my research was directed towards addressing questions concerning the chemical properties of the hepatic System A protein. The experiments discussed below employed the use of protein modifying reagents to determine which chemical reactive groups present on the carrier, resulted in a loss of transport activity when modified. The properties of System A with respect to protein modifying reagents was also compared between normal and transformed liver tissue.
Materials and Methods
See the transport method procedures outlined in Chapter II.
Results
In preliminary studies the sensitivity of System A-mediated
transport, as monitored by assaying the Nat-dependent uptake of AIB, toward a host of group-specific protein-modifying reagents was tested.




26
Table 1. Sensitivity of System A Activity in Normal Rat Hepatocytes and
H4 Hepatoma Cells to Protein-Modifying Reagents
Inhibitor Hepatocytes H4 Hepatana cells
Percent of Control
None 100 100 Acetic anhydride 100 85 Succinic anhydride 89 109 Chloroacetate 116 121 Iodoacetate 66 134 Iodoacetamide 86 96 N-ethylzaleimide 0 99 P-chlormercribenzene sulfonate 31 30 P-chlormercuribenzoate 30 17 Fluorescein isothiocyanate 124 106 Phenyl isothiocyanate 101 85 N-acetylimidazole 106 78 N-bramesucinimide 109 0 Trinitrobenzane sulfonate 75 112 IRenylglyaxal 157 130 Ethoxyformic anhydride 77 ND HgM2 ND 7 Hepatocytes or H4 hepatcmia cells were cultured in an amino acid-free medium (Na1RB) for 4 h. All cells were then incubated for an additional 10 min at 370C in the presence or absence of NaERP containing 1 mM of the indicated reagent. The cells were then rinsed in CholKRP for 5 min and the Na*-dependent uptake of 50 M (3H)-AIB was measured at 370C for 1 min. The total Na-dependent uptake was determined subtracting the velocity in absence of Na* (CholERP) from that in the presence of Na (NaKRP). The data are the averages of four determinations and the standard deviation were less tlpn 10%. The control velocities were 120 8.0 and 368 + 12 pmol mg protein min for the hepatocytes and H4 hepatona calls, respectively.




27
A variety of protein-modifying reagents were examined so as to probe for several functional amino acid side-chains present in proteins (Table 1). A list of all the protein modifying reagents use in these studies and their chemical reactivity is listed in Appendix VI. The cell lines used for these studies were normal rat liver tissue (freshly isolated hepatocytes) and a rat liver-derived tumor cell line, H4-II-EC3 (H4). Normal hepatocytes and the H4 hepatcma cell line were chosen in order to compare the sensitivity of System A between normal and transformed liver tissue. Many laboratories have reported distinct changes which occur in neutral amino acid transport following transformation (Handlogten e a., 1981; Kelly and Potter, 1979). Thus, it was of interest to determine if similar changes occurred with respect to chemical modification.
Reagents that show preferential reactivity with amino groups including fluorescein isothiocyanate (FITC), phenylisothiocyanate pitch) (Edman, 1950), and trinitrobenzene sulfonate, 'INBS (Kotaki et 4., 1964), produced only modest changes relative to control cells not exposed to the reagent. These initial studies were performed in the presence of Nat-ions (i.e., NaxRP buffer), so the lack of inhibition by FTITC and PITC may have resulted either from protection of essential lysyl-amino group(s) by Nat-ions or fra the low pH employed. Wright and Peerce (1984) observed that inactivation by PITC of the imino carrier was blocked by the presence of Nat-ions. In reference to the pH effects, these protein-modifying reagents are thought to react with primary amino groups at an alkaline pH. I was reluctant to test their




28
Table 2. Sensitivity of System A Activity in Normal Rat Liver and H4
Hepatoma Vehicles to Phenyl Isothiocyanate
Corxition Hepatocytes H4 Hepatca
Control (1% DF) 1151 52 954 36
10 aM Irnrlemcine 1080 192 885 123
1 rM PITC 45 4b 209 + 8b
10 M L-norlecine
+ 1 aM PI 7 4b 215 13b
Membrane vesicles were incubated with 0.25 M sucrose, 10 nM Tris, pH 9.2, 100 UM NaC1 containing 1 NM PTC in the presence or absence of 10 imM I-norleucine. The PITC was dissolved in dimethylformamide (DMF) such that the final percentage of DMF exposes to the membrane vesicles was 1%. After 20 min the membranes were centrifuged at 150,000 g for 5 min, the supernatant was discarded, and the membrane pellet washed and resuspended in Buffer A. Sv A activity was then measured by monitoring the Na*dependent uptake of 200 M [H]-B for 1 min at 370C. The data are expressed as the velocity of AIB uptake (pnol mg protein min ) and represent the averages + standard deviations of four determinations. This experiment was performed with the assistance of Kathleen Dudeck- bStatistically different fram the control values at the P<0.005 level.




29
effects at pH values above 8, because damage to the integrity of the plasma membrane or cell viability might occur, thereby resulting in considerable non-specific inhibition of Na-dependent System A transport. Mhen the System A activity in rat liver or H4 hepatama plasma membranes vesicles was assayed following treatment with 1 mM PITC (pH 9.2), transport was inhibited approximately 96% and 78%, respectively (Table 2).
N-acetylimidazole is an acylating reagent that reacts with both
amino and tyrosyl groups; however, the reaction with tyrosine has been reported to be more selective (Riodan and Vallee, 1963; Riodan et al., 1965). Inactivation of the Na-dependent L-proline carrier has been reported to occur upon treatment of brush border membranes with Nacetylimidazole (Wright and Peerce, 1984). Although this reagent decreased System A activity by 22% in the H4 hepatana cell line, both cell types were largely resistent (Table 1). Phenylglyoxalic acid, a specific reagent for guanidine and terminal amino groups (Takahashi, 1968), actually caused an increase in the observed System A activity in both cell types. Ethoxyformic anhydride (diethylpyrocarbonate), an imidazole specific reagent (Miles, 1977) produced a small decrease in System A-mediated transport (23%) in the normal hepatocytes (Table 1).
The activity of both normal hepatocytes and H4 hepatama cells denxronstrated a similar degree of sensitivity to the sulfhydrylpreferring reagents p-chloranercuribenzoate (PC4B) and pchlorcaercuribenzene sulfonate (PO4BS). Inhibition ranged from 70% to 83% for the Na+-dependent uptake of AIB. In contrast, the sulfhydryl-




Figure 1. Concentration Dependence of the Inhibition of System A Activity in Nonrmal Rat Hepatocytes and Several Hepatema Cell Lines by NEM or PCMBS. The cells were incubated in an amino acid-free medium (NaKRB) for 5 h to enhance System A transport activity by adaptive regulation and were then transferred to NaKRP containing PCMBS ( ) or NEM ( ) for 10 min at 370C. The inhibitor concentration was varied between 0.025 and 1.0 mM. After rinsing the cells with CholKRP, System A activity was assayed by measuring the Na-depedent uptake of 50 M [3H]-AIB for 1 min at 37-C. The results are reported as the percent of the rate of transport in the non-treated cells; those velocities (averages + S.D. of 3 deleminaticnsilwere 133+2.0, 766+27, 828+48, 531+15, 922+23 and pmol tg protein min for the normal rat hepatocyte, rat H4, Fao, HTC hepatamas, and human HepG2 hepatama, respectively. The KI values were calculated by computer analysis as described by Cleland




31
100 Hepatocyte HTC
* PCMBS
A NEM
75
KI;445t 15 uM
50 Ki,= 55t26uM
KV2=4619UM
0
.25 Ki,=l l 9t28uM A2
Ce
0
A H4 FAO Hep G2 c 100
K 1=271%32uM
* 72
75 Ki, 253*20uM
50
25 Ki,2=72tl2UM K, =40t4uM K1=78:27uM
2 2 '
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
[Inhibitor] IUM (.102)




32
preferring reagent NEM produced complete inhibition of System A in the hepatocytes, while the correspoxing transport activity in H4 hepatama cells was resistant to the inhibitor (Table 1). The strong inactivation of System A in hepatocytes by NEM, but not by icdoacetate, is in agreement with observations by others (Kilberg gt a., 1980; Hayes and McGivan, 1983; Sips and van Dam, 1981). Further discrimination of the System A activity present between these two cell types was achieved through the use of N-bramosuccinimide (NBS); this reagent abolished AIB uptake in the H4 hepatena cells, but was largely ineffective as an inhibitor of the activity in normal hepatocytes. Although NBS is known to cleave peptide bonds at tryptophan, tyrosine, and histidine residues, oxidation of sulfhydryl residues is reported to occur more rapidly (Fontana, 1972). Methionine and cystine are also subject to oxidation by NBS (Means and Feeney, 1971). Regardless of the mechanisms involved, there is a clear difference in the reactivity of the System A carrier in hepatocytes aid H4 hepatama cells treated with either NEM or NBS.
Additional experiments were performed to ascertain whether the
resistance to NEM by the H4 hepatama cells was a unique property of that hepatcma cell line or a general characteristic shared by other liverderived tumor cells. In this study a variety of hepatama cell lines including both human (HepG2) and rat (Fao, HIC, and H4) were examined for sensitivity of System A to inactivation by NEM or PCMBS (Chiles and Kilberg, 1986). The concentration-dependence of inactivation was monitored using concentrations of reagent ranging from 0.025 bM to 1.0 itM. PCMBS treatment was found to decrease System A-mediated transport




33
by 70% to 80% in all cell types examined; concentrations that produced half-maximal inhibition (for ease of discussion this value will be referred to as '1/2) ranged from 40 to 119 M (Fig. 1) MEN-dependent inactivation of System A in the hepatocytes was nearly complete. Greater than 95% of the total Nat-dependent AIB uptake was inhibited relative to non-treated cells with K1/2 of 46 + 19 M. In comparison, the kinetics and extent of inactivation in the hepatama cell lines was strikingly different. For example, the IC-associated System A activity was inhibited by NIPI, yet required concentrations of 1 xlM before the degree of inhibition was comparable to that observed in normal hepatocytes. Although the level of NEM required to produce halfmaximal inhibition in the rat Fao and human HepG2 hepatamas was relatively low ( 1/2 = 53 + 20 M and 271 + 32 M, respectively), less than 50% of the total Nat-dependent transport was inactivated at the most effective concentrations of inhibitor. Consistently, the System A activity in the H4 hepatama cells was largely unaffected by NEM at concentrations exceeding 1 mM (Fig. 1).
The saturable inhibition of only 40% to 50% of the total Na
dependent AIB uptake by NEM in the Fao and HepG2 hepatana cells raised the possibility that additional routes for Na -dependent AIB uptake might exist in these transformed liver cell lines. Saturable inactivation of only 40% to 50% of carrier activity has been reported for a limited number of transport systems. For example, the sensitivity of nucleaside transport in rat erythrocytes has been investigated using PCMBS (Jarvis and Young, 1986). PCMBS produced a concentration-




34
dependent inhibition of uridine uptake, yet inactivation of transport reached a plateau of 50% at 0.1 M PCMBS. No further inhibition of transport was observed using concentrations of PCMBS in excess of 1.0 nM. Detailed analysis later revealed that uridine transport into the rat erythrocytes occurred via two distinct routes. One ccuponent was highly sensitive to nitrobenzy1thioinosine (NBMFR)), an active site inhibitor of uridine transport. This ccpanent exhibited a K for uridine uptake of 163 + 18 M, whereas the other component was NBMPRinsensitive possessing a Km for uridine of 50 + 18 M. The authors observed that the NBMP-insensitive portion of uridine uptake was inactivated by PCMBS, whereas the NBMPR-sensitive component was unaffected.
A somewhat similar observation has been seen for Na -independent uptake of Ir-leucine. L-leucine transport into Chang liver cells can be resolved into two components. One component possess a low Km (44 M) for Ir-leucine uptake, whereas the other camponent has a Km of 8.0 EM. Takadera and Mahri (1982; 1983) reported that NEM actually stimulated Lleucine uptake (2-fold) via the high-affinity system. In contrast, transport-mediated by the low-affinity component was actually decreased 2-fold.
Based on these observations and the results of Fig. 1, AB uptake was tested for inhibition by the System A-specific substrate, MeAIB (Christensen et a., 1965). Operationally, the portion of the Na*dependent AB uptake that is inhibited by 2- (methylamino) -isobutyric acid (MeAIB) is assumed to be mediated by System A; any remaining Na -




Figure 2. MeAIB Inhibition of Na+-Dependent AIB Transport in Several Hepatcma Cells Lines. Hepatcnma cells were incubated in an amino acidfree medium (NaKRB) for 5 h to stimulate System A activity by adaptive regulation and were then rinsed with CholKRP for 5 min. System A activity was assayed by measuring the Na m-d uptake of 50 M [3H]-AIB for 1 min in the presence of increasing cncentrations of unlabeled MeAIB (0.01 to 20.0 nm. The results are reported as the percent of the transport rate in the absence of MeAIB. The control velocities (averages + S.. of 3 detejnitions) were 434+12, 673+15, 514+31 and 409+17 pmol mg protein min for the rat H4 ( ), HITC ( ), Fao ( ), and human HepG2 ( ) hepatama cells, respectively. The Ki values were calculated by computer analysis as described by Cleland (1979).




36
100
A Hop G2 Ki=0.4t0.07 mM FAO Ki=0.4t0.04 mM o 80
80 E HTC Ki=0.1tO.01 mM C V H4 Ki=0.2*0.04 mM
0
0
40
LE
0
0 4 8 12 16 20 [M eAI B], mM




37
dependent AIB transport is mediated by System ASC. Although AIB uptake is largely restricted to System A, it has been reported that System ASC can contribute significantly to the uptake of AIB in hepatocytes (LeCam and Freychet, 1977) and H4 hepatama cells (Kilberg et al., 1985) when the activity of System A is fully repressed by substrates. In contrast, when the activity of System A is stimulated by substrate starvation i.e., adaptive regulation, the relative contribution of System ASC diminishes rapidly. Therefore, the hepatama cells were incubated in amino-acid free medium for 5 h to enhance both the mount of measurable System A activity and to diminish any contribution by System ASC; these incubation conditions correspond to those used to obtain the data shown in Fig. 1. As seen in Fig. 2, nearly all of the Na-dependent AIB uptake was inhibited by an excess of MeAIB in the Fao and HTC hepatcmas with Ki values of 400 + 40 M and 110 + 10 M, respectively. Greater than 85% of the Na dependent uptake of AIB in the HepG2 and the H4 hepatama cells was inhibited by MeAIB (Ki = 400 + 70 M and 200 + 40 M, respectively) The strong inhibition by MeAIB demonstrates that System A mediates nearly all of the Nadependent AIB uptake in all four hepatama cell lines. The results indicate that the saturable but incanplete inhibition by NEM in both the Fao and HepG2 cells does not appear to represent selective inactivation of one of multiple routes for AIB transport. Rather, the data suggest two possibilities: First, only a subset of the System A carrier proteins in these cells is sensitive to NEM (i.e., the sensitivity may be restricted to those carriers which were synthesized during adaptive regulation, whereas the basal carriers




Figure 3. Concentration Dependence of NE4 Inhibition of System A Activity in Fao Hepatema Cells Cultured in Amino Acid-Rich Medium. The Fao cells were incubated in an amino acid-rich medium (MEM) for 24 h to decrease the measurable amount of System A transport activity. The cells were then transferred to NaKRP containing NEM for 10 min at 37*C. The inhibitor concentration was varied between 0.01 and 1.0 nM. After rinsing the cells twice with CholKRP (37 *C), System A activity was assayed by measuring the N*-dependent uptake of 50 M [3H]-AIB for 1 min at 370 C. The results are reported as the percent of the rate o transport in1the non-treated cells havir a velocity of 39+6.4 pool mg protein min (averages S.D. of 4 determinations) The K value was calculated by computer analysis as described by Cleland (197.




39
100
FAO
80 Kg= 0.05 mM 60
40 20 0
0I I I I
0 0.2 0.4 0.6 0.8 1.0
[NEM], mM




4o
are unaffected or vice-versa). Second, all of the carriers are modified, but the activity of the transporter is slowed rather than ccmpletely prevented.
In reference to the later suggestion, the basal or fully repressed System A activity in the Fao hepatama cells, which demonstrated a saturable inhibition of 40% to 50% of total activity upon amino acid starvation, was tested for sensitivity to NM. The System A activity was repressed by culturing the cells for 24 h in a medium rich in amino acids (MEM). The basal activity was then assayed for sensitivity to NEM using conditions corresponding to those used to obtain the data for the amino acid starvation-induced activity. As seen in Fig. 3, only 40% to 50% of the total Na*-dependent AIB uptake was inhibited by NEM. Although the measurable K1/2 value was somewhat higher (50 + 0.1 M), the results are qualitatively similar to the data obtained when System A activity was stimulated by amino acid deprivation. These results suggest that at least for the Fao hepatama cell line all of the carriers are modified, yet the activity is slowed rather than completely prevented.
It is interesting to note, however, that studies of System A
transport during amino acid deprivation have suggested that the newly synthesized carriers may be distinct frcm the basal carriers in same tissue types. Klip e; al. (1982) noted that I-proline uptake following amino acid starvation of I6 myoblast cells was inhibited by 1 mM NEM (50%), whereas uptake by System A in amino acid-suplemented cells (basal System A activity) was unaffected. Presumably, the System A




41
carriers that are synthesized during adaptive regulation contain sulfhydryl group(s) essential for activity that are not displayed by the carriers present prior to substrate starvation. It was also noted that the basal System A carrier possessed a higher Egm for substrates relative to the newly synthesized carrier. The pH cptimm for transport activity was shifted fra 7.8 to 7.5 for the amino acid-fed and amino acidstarved cells, respectively. The authors concluded that the carriers synthesized during adaptive regulation were chemically distinct from the basal System A carrier.
In reference to the sensitivity of System A transport to NEM between the normal hepatocytes and the hepatama cell lines, these differences could be attributed to: 1) selective modulation of the carrier by NEM-sensitive cytoplasmic elements present at varying degrees in the individual cell types, 2) structural differences in the carrier protein itself, or 3) differences in the membrane environment that may exist between the cell types (i.e., lipid composition ard/or organization). To test for cell-specific differences in sensitivity to NEI modification in the absence of cellular integrity, System A activity was assayed in isolated plasma membrane-enriched vesicles prepared fran either normal liver tissue or H4 hepatama cells (Dudeck et al., 1987). Fig. 4 shows the results of a series of experiments designed to examine the effects of varying concentrations of NIZ on System A-mediated uptake. When the Nat-deperdent uptake of AIB was assayed following treatment of the membrane vesicles with NEI, the activity of System A in the normal liver tissue was inactivated effectively (K112 = 370 1), but




Figure 4. Oncntration Dependence of the Inhibition of System A Activity in Rat Hepatocyte or H4 Hepatama Membrane Vesicles by NEM. The mbrane vesicles were prepared as described in Chapter II and then were treated with NEM for 10 min at 22*C. The inhibitor concentration was varied between 0.5 and 5.0 nM. After rinsing the membranes with Buffer A, System A activity was assayed by measuring the Na-dependmt uptake of 200 M [3H]-AIB for 1 min at 22-C. The results are reported as the percent of the rate of transport in the non-treated vesicles; those velocities (averages + S.D of 3 determinations) were 1518+6.7, and 984+41 pmol mg protein min for the normal hepatocyte and H4 hepatama membrane vesicles, respectively. This experiment was performed with assistance from Kathleen Dudeck-Collart.




43
100
FAO
80 K /= 0.05 mM 60 & 40 w 20
0
0 0.2 0.4 0.6 0.8 1.0
[NE M]. mM




44
transport by the H4 hepatcma-derived vesicles was unaffected at concentrations up to 5 nM (Fig. 4). Although the concentration of NEM required to produce half-maximal inhibition in the membrane vesicles is somewhat higher than that measured for intact hepatocytes (370 M verses 46 M), the results are consistent qualitatively with those reported for the intact cells (Figure 1).
The selective inactivation by NEM was further substantiated by treating detergent-solubilized plasma membrane proteins with NEM. Following solubilization of either normal or H4 hepatama plasma membranes, the protein mixture was exposed to 1 mM NEM for 10 min at roam temperature. The excess NEM was then quenched by the addition of 2 :mM D-cysteine and the proteins reconstituted into artificial proteoliposames (Bracy, 1987). As shown in Table 3, NEM was effective in partially inactivating the System A activity solubilized fra normal liver tissue, although the inhibition was not as strong as that observed for intact membrane vesicles or whole cells. In contrast, NEM had little or no effect on the activity of the solubilized carrier from H4 hepatcma cells.
Additional evidence for the marked heterogeneity of System A
between the normal rat hepatocytes and the transformed liver tissue was obtained by examining the ability of neutral amino acid substrates to protect System A activity from PCMBS-depexent inactivation (Chiles et al., 1988). In these studies PCMBS was chosen as an inhibitor, because this protein modifying reagent inactivated System A-mediated transport by aoroximately 80% to 90% in both cell types. L-serine, L-proline,




45
Table 3. Reconstitution of System A Activity from Rat Hepatocytes or H4
Hepatoma Cells Following NEM-Treatment of Solubilized
Plasma Membrane Proteins
Membrane Preparation Assay System control + NEM Reconstituted
Normal Liver 22*C:EC1/NaCl 933 46 696 l9b
H4 Hepatma Call 22'C:EC1/NaCl 1042 202 1056 100
Normal Liver 37'C:ESCN/NaSCN 2649 251 1677 + 188c
H4 Hepatca Call 37'C:ESCN/NaSCN 2673 114 2293 216
Plasma membranes were isolated and the proteins solubilized in diolate/urea as described in Chapter II. Solubilized membrane proteins were incubated with Buffer A in the presence or absence of 1 uM NIM for 10 min at roon tanperature and then subjected to reconstitution. Proteoliposanes fran two different membrane preparations were assayed for System A by measuring the uptake of 200 M [3H)-AIB for 1 min at either 220C with the transport buffers listed in Chapter II (KC1 or NaCl containing buffer) or 37'C with ESCN or NaSCN containing buffers. The data are expressed as the averages + standard deviations of three determinations. This experiment was performed by Elizabeth E. Dudenhausen of this laboratory. bStatistically different fran the value for normal liver at the P<0.01 level.
CStatistically different fru the value for normal liver at the P<0.025 level.




46
ard L-norleucine were chosen as protector substrates because a large percentage of their uptake by liver tissue is mediated by System A (Kilberg et al., 1985). As seen in Table 4, when H4 cells were incubated with 0.2 mM PCMBS in the presence of the neutral amino acids, the inactivation of System A activity was blocked significantly. Depending on the amino acid tested, the amount of transport activity protected ranged from 41% to 72%. In each case the corresponding Discmer yielded lower transport activity. In contrast, the PCMBSinactivation of System A from the normal liver cells (freshly isolated hepatocytes) was not blocked by the presence of any of the neutral amino acids tested.
The remaining major amino acid transport system in normal and H4 hepatcma cells were also assayed for sensitivity to NEM and PCMBS. The results of these inactivation studies are shown in Table 5. In the H4 hepatama cells, the Nat-dependent amino acid transport Systems ASC, N, and the Na -independent System L were inhibited effectively by PaMBS, whereas the Na*-independent System y+ activity was decreased by only 28%. In contrast, the reagent decreased transport via Systems ASC, N, and y+ in normal liver cells, however amino acid uptake by System L was slightly elevated in these cells. These data indicate that all of the Na -dependent transport systems tested are sensitive to PCMBS in both cell types, whereas the Na*-independent agencies for amino acid uptake were differentially inactivated in the normal and transformed cells.
Heterogeneity between these transport systems in the normal and hepatcma tissues was also noted through the use of NEM. All of the




47
Table 4. Amino Acid-Dependent Protection from Inactivation by PCMBS
of System A-Mediated Transport
Amino Acid Control PCMBS-treated cells %Protection Amino Acid Amino Acid Amino Acid Hepatocytes present absent present D-proline 78 13 36 5 15 5 0 L-proline 93 7 20 9 13 3 0 D-serine 222 4 53 4 58 + 4 3 L-serine 226 25 59 7 12 + 4 0 L-norleucine 106 4 25 5 26 4 1 Hepatcma Calls
D-prolirn 889 58 219 19 267 11 7b L-proline 357 12 152 6 236 11 41c D-serine 333 24 112 14 191 5 35c
-Irserine 634 4 162 10 502 9 72c L-norleucine 372 + 15 92 15 237 27 52c
Hepatocytes or H4 hepatona cells were cultured in amino acid-free medium (NaxRB) for 5 h to enhance System A activity and were then transferred to NaKRP containing 0.2 tmM NEM or PCMBS in the presence or absence of the indicated amino acid (5 rM) for 10 min at 370C. Cells were washed extensively with CholFRP and then the System A activity was measured by assaying the Na+-depne upte of 50 M [3i]-AIB for 1 min at 370C. The velocity data are expressed as pmol mg protein min- and are the averages + S.D. of four determinations. The percent protection was calculated by the following equation: [ (V +-Vi) / (V -Vi)] 100, where aa and i indicate the presence of amino acid and iz i ip ively.
bese values are significantly different to p values < 0.025. lhese values are significantly different to p values < 0.005.




48
Table 5. Sensitivity to NEM or PCMBS of Several Amino Acid Transport
Systems in Rat Normal Hepatocytes and H4 Hepatoma Cells
System Control NEM %of Control Control PCM4BS %of Tested Control
H4 Hepatcma
A 652 60 767 77 118 372 15 92 3 250 ASC 3188 109 3183 263 100 2988 292 294 19 10b N 362 11 589 34 163c 468 10 41 7 9 L 1672 100 1689 111 101 6900 510 25 4 c10 y+ 262 18 277 15 106 174 15 125 4 740
Hepatocytes
A 140 5 22 4 160 100 1 27 3 27c ASC 50 1 17 2 340 40 2 13 2 32c N 375 7 373 + 12 99 282 27 81 9 290
L 111 11 67 10 60 108 2 131 10 121b y+ 38 11 33 11 87 47 1 9 11 19c
Individual amino acid transport systems were tested for activity after incubating the cells for 10 min with Na1RP containing 0.2 M NEM or PCMBS. The specific systems ware measured as follows: System A, Na-dependent AIB uptake; System ASC, Na+-dependent Lthreonine uptake in the presence of 5 mM MeAIB; System N, Na+-dependent L-glutamine uptake in the presence of 5 mM MeAIB; System y+, saturable Na+-independent L-arginine uptake; and System L, saturable Na+-independent L-leucine uptake. In each case, the cells used to assay Systens ASC, N, L, and y+ were incubated in NaKRB buffer for 1 h prior to addition of the inhibitor, while those cells used to assay System A were incubated for 4 h. The substrate concentrations were 50 M and all transport assays were performed for 30 sec at 370C except for Sy stf A, which was rasured for 1 min. iThe velocity data are expressed as pmol mg protein unit time and are the averages standard deviations of four determinations. blhese values are significantly different to p values < 0.025. olhese values are significantly different to p values < 0.005.




49
amino acid transport systems tested in the hepatana cell line were resistant to inhibition by NEM (Table 5); System N was actually increased to a significant degree. Even after increasing the NEM concentration above 1 IrM, no significant inhibition of these carriers was observed (Table 5). The results contrast observations by others who have reported partial inactivation of Systems A, ASC, and N in an alternate strain of H4 hepatcma calls (Vadgama and Christensen, 1983). Tests for NEM-dependent inactivation of Systems ASC and L in the normal hepatocytes showed transport rates decreased by 66% and 40%, respectively, whereas Systems N and y* were relatively resistant to the inhibitor.
Discussion
The aim of this phase of my research was to determine which amino acids are important for System A carrier function. Investigations centered around testiM a wide variety of protein modifying reagents so as to individually modify specific amino acid side-chain groups which are present on the carrier protein. Following a brief exposure (10 min) to the protein-modifying reagent, whole cells or plasma membrane vesicles were assayed for System A activity by monitoring the Na dependent uptake of AIB. The results of Table 1 clearly demonstrate that System A transport activity is largely resistant to covalent modification by both alkylating reagents, including iodoacetate, iodoacetamide and chloroacetate, and to acylating reagents, such as acetic anhydride and succinic anhydride. The reagents specific for amin.-grup, fluorescein isothiocyanate and pbenyl isothiocyanate, were




50
ineffective in whole cells, presumably due to the low pH at which these reagents were tested (i.e., pH 7.4). Inactivation of System A transport activity was, however, observed in plasma menbrane-enriched vesicles isolated from both normal rat hepatocytes and H4 hepatcama cells when the pH was raised from 7.4 to 9.2 (Table 2). Unfortunately, no System A substrate tested was effective at blocking this inactivation. These reagents may be modifying plasma memrbrane proteins other than System A or may be modifying the carrier protein at a location removed from the amino acid binding site and thus not protected by substrates.
System A-mediated uptake of AIB was highly sensitive to the
noncovalently interacting mercurials, HgC12, RMB and PB. Transport activity was completely abolished in the H4 hepatana cells by HgC12, indeed, this mercurial was the most potent inhibitor of transport activity. These results complement the studies of Stirling (1975) who also demonstrated that HgC12 was 10 to 20 times more potent than PClBS in blocking galactose and amino acid uptake into rabbit brush border plasma membrane vesicles. The organic mercurial compounds PCMB and PCMBS were equally effective as inhibitors of transport in both the normal hepatocytes and H4 hepatana cells. Mercurial compounds have long been known to perturb membrane structure and function (Rothstein, 1970). For example, same of the membrane systems susceptible to sulfhydryl agents include: alkali metal permeability (Rega et Al., 1967); a variety of active transport processes for sugars (Van Steveninck et al., 1965), amino acids (Schaeffer, et al., 1973), and nucleosides (Hare, 1975);




51
hormone binding to receptors including insulin (Dixit and Lazarow, 1967) and acetylcholine (Karlin and Bartels, 1966).
It was also demonstrated that the System A carrier from normal and transformed liver tissue has undergone significant changes with respect to its sensitivity to chemical modification by protein-modifying reagents (Table 1). The inherent changes in the carrier are dmnstrated by the differential inactivation by NEM and NBS. NBS completely abolished System A-mediated transport in the H4 hepatama cell, whereas this reagent was ineffective as an inhibitor of transport in the normal hepatocytes. Particularly striking is the differential sensitivity of System A to the covalent alkylating agent NEM. All of the transformed liver cell lines tested were either weakly inhibited or unaffected by NEM. In contrast, the System A activity in the normal hepatocytes was completely inactivated (Fig. 1).
Several possibilities can be postulated to account for such
differences in chemical reactivity. There may be an alteration in the primary sequence of the carrier protein that eliminates or masks a highly reactive cysteine group. In this case, a second less reactive cysteine may be responsible for the partial inhibition that is observed in the HTC, FAO, and HepG2 cell lines. Alternatively, the carrier protein in the normal and transformed tissues may assume different conformation states as a result of differences in the primary sequence of the protein that do not eliminate the cysteine residue but do alter its reactivity. It was possible that either cytosolic factors or the membrane lipid environment itself could account for the differential




52
sensitivity observed with NEM. Tob eliminate any secondary effects by the cytosol, NEM inactivation of System A transport was assayed in plasma membrane-enriched vesicles isolated from both normal hepatocytes and H4 hepatama cells (Fig. 4). Clearly, the inability of NEM to
inhibit System A transport was retained in the H4 hepatama membrane vesicle population, whereas the membrane vesicles derived from normal hepatocytes demonstrated sensitivity.
In an effort to reduce the possible influence of the plasma
membrane lipid environment, total plasma membrane proteins from both normal hepatocytes and H4 hepatama cells were detergent-solubilized, treated with NEM, and then reconstituted into artificial liposomes prepared from soybean phospholipid (Table 3). Analysis of AIB uptake revealed that the solubilized System A activity had retained the differential sensitivity to NEM. It is important to note that while the reconstitution methodology was designed to remove much of the bulk lipid, tightly bound annular lipid may be present and conferring the differential sensitivity on the carrier. Finally, we needed to eliminate the possibility that AIB was transported via an agency other than System A in the hepatama cell lines. In this case, the new route for AIB uptake would be largely resistent to NEM. To test if additional routes of entry existed in the hepatama cells, the effects of MeAIB on the uptake of AIB was ascertained (Fig. 2) and MeAIB was shown to inhibit greater than 90% of the AIB uptake in all hepatama cells, except the human cell line HepG2 (80%). If either the transmembrane Natgradient or the membrane potential had been differentially affected in




53
the normal hepatocyte by NEM, System A would have been inhibited. This is unlikely because both System N and System y were not affected by NEM in the hepatocyte. These observations argue strongly against the proposal that cytosolic factors and membrane environment alter the sensitivity of the carrier protein and indicates that the disparate inactivation between normal and transformed liver tissue is due to inherent structural differences in the System A carrier protein itself.
Recently, Lea t al. (1987) demonstrated striking differences
between rat liver and a variety of rat hepatama cells with respect to the uptake and incorporation into protein and EA of Ir-leucine and thymidine, respectively. In their study, the action of the carbamaylating agents 2-chloroethylisocyanate, ethylisocyanate and sodium cyanate was compared in normal rat hepatocytes and the Morris hepatama cells 7288CTC, 7777, 5123C, and 8999. In all of the hepatcmia cell lines tested, the carbamoylating agents strorly inhibited the uptake of L-leucine and thymidine, whereas there was little or no effect with normal hepatic tissue. Similar observations were noted for the incorporation of L-leucine into protein and the incorporation of thymidine into IIM.
Additional evidence for the inherent changes in the System A carrier was afforded by amino acid-dependent protection from inactivation (PCMBS) studies. Protection of transport activity by amino acids was observed only in the H4 hepatama cells and membrane vesicles. Although it is unclear why System A substrates provide no observable protection of transport activity in either normal hepatocytes or




54
membrane vesicles isolated from rat liver tissue, there are a number of possible explanations. 1) PCMBS may be modifying the carrier protein at a sulfhydryl group distant from the amino acid binding site; in this case, binding of amino acid substrate must have little or no effect on the interaction of PCMBS with the carrier protein. 2) PCMBS may react with an amino acid residue in or near the substrate binding site, yet, structural features within this area do not allow the bound amino acid to block the reaction. 3) In contrast to its action in the transformed cells, PCMBS may inactivate transport activity in the normal hepatocytes by means other than direct chemical modification of the carrier protein. For example, any interaction of PGBS with plasma membrane components which would perturb the trans-membrane sodium electrochemical gradient, such as inactivation of the Na+/K1 ATPase, would result in inhibition of active transport. Indeed, other laboratories have sugested that the action of PCMBS on many Nat-dependent transport processes is due to a general inrrease in the plasma membrane permeability to small inorganic cations (Will and Hcpfer, 1979), rather than direct transporter modification.
These results extend and compliment the work of several laboratories that have reported inherent differences in the characteristics of amino acid transport between normal rat (freshly isolated hepatocytes) and transformed liver epithelial cells. These changes include the disappearance of System y* during maturation of fetal hepatocytes to adult and its reappearance upon transformation to a hepatcma cell line (White and Christensen, 1982). Irglutamine uptake in




55
mature hepatocytes is mediated largely by the Na+-dependent System N carrier (Kilberg et al., 1980); however, greater than 80% of glutamine uptake in HTC hepatama cells is sensitive to the System A-specific substrate MeAIB (Vadgama and Christensen, 1983). In normal hepatocytes, Na-dependent Ir-cysteine uptake is considered a selective test for System ASC (Kilberg et al., 1981), whereas in the HIC hepatmia cell line threonine represents a better selective substrate for the ASC carrier (Handlogten et al., 1981). In the mature rat hepatocyte, the Natdependent anionic carrier, System X-AG, transports LI-aspartate and Lglutamate equally well (Gazzola et al., 1981). However, in both fetal hepatocytes and HTC hepatma cells, the substrate specificity of this system has been altered such that only the shorter (i.e., Tr-aspartate or L-cysteate) anionic amino acids are acceptable, there appears to be little or no uptake of either Li-glutamate or hamocysteate (Makowske and Christensen, 1982).
Irrespective of the mechanisms involved, the observations described above provide additional evidence that distinct structural changes in amino acid transport systems result from transformation of normal rat hepatocytes. Although only four hepatama cell lines have been tested to date (Chiles and Kilberg, 1986), the fact that amino acid carrier proteins in all of them show the same changes in chemical reactivity when ccpared to normal hepatocytes suggests that same fundamental alteration in transport systems occurs following development of a chemically-induced transformed state. The differential sensitivity of System A to inactivation by NEM has been examined in a stable SV40-




56
transformed liver cell line. The laboratory of Ciou developed this cell line by infecting fetal rat hepatocytes with a SV40 mutant that is temperature-sensitive with respect to growth and to the transfonmed phenotype (Chou and Schlegel-Haueter, 1981; Chou and Ito, 1983; Chou, 1985). These cells are referred to as RIA209-15 and exhibit properties characteristic of transformed cells at 330C, but at the restricted temperature of 400C the cells behave like nontransformed cells. To determine if the RIA209-15 hepatocytes retained characteristics similar to the normal rat and transformed hepatma cells, their sensitivity to either NEM or PCMBS was ascertained following growth at permissive or restricted temperatures. Handlogten and Kilberg (1988) noted that System A activity was inhibited by PCMBS regardless of the incubation temperature; however, treatment with NEM resulted in no noticeable inactivation of transport activity at the restricted temperature, suggesting that the SV4-transformed cells behave similar to that of chemically-induced transformed cells.




CHAP ~ER IV
EVIDENCE FOR THE DIRECT CHEMICAL MODIFICATION BY PCMBS
OF THE SYSTEM A CARRIER IN RAT H4 HEPAT4MA CELLS
Introduction
The results presented in Chapter III demonstrated that the hepatic System A carrier protein contains a sulfhydryl group(s) which appears to be essential for transport activity. Although the carrier shows sensitivity to chemical modification by PCMES in all liver-derived cells tested, substrate level protection was observed in the H4 hepatama cell line but not in normal hepatocytes. These results suggest that the inactivation of the H4 hepatoma System A activity is a result of direct chemical modification. The goal of this phase of my research was to a) characterize the mode of PCMBS inactivation with respect to active transport by System A in the H4 hepatoma cell and b) to determine to what extent the inactivation was due to direct carrier modification as opposed to general membrane perturbation.
Cultured cells and tissues have proven extremely useful in the study of organic solute transport; however, a primary draw back with whole cells is intracellular metabolism of the transported solute. This also applies to the use of protein modifying reagents, especially when these campounds are used to study plasma membrane phenomena. Typically cells are exposed to a protein modifying reagent for a given period of time and then the carrier activity ascertained. Although the specific
57




58
carrier function under study may be significantly altered due to direct chemical modification, secondary effects which are the result of the modifying reagent interaction with intracellular components on which carrier function is dependent can also occur. Such secondary effects can lead to false conclusions attributing the change in a particular carrier property to direct modification. An example is the inactivation of glucose transport in yeast cells (Van Steveninck et al., 1965). Iodoacetate inhibits the active transport of glucose into yeast cells by 90%. The inhibition was initially attributed to direct chemical modification of the glucose carrier by iodoacetic acid. Subsequent analysis revealed, however, that iodoacetate Was not inhibiting transport, but was instead inhibiting glycolysis via modification of triose phosphate dehydrogenase. This resulted in a rapid depletion of cellular ATP production and ultimately lead to inhibition of active transport. It has been suggested that the inactivation of the mammalian kidney tissue Nat/K1-ATPase activity (Taylor, 1963) by iodoacetate may also be due to secondary interactions of the chemical reagent with intracellular proteins. (Rothstein, 1970).
One means of distinguishing between menbrane verses intracellular effects is to assay internal metabolic markers. For example, following exposure of cells with a sulfhydryl modifying reagent, one may choose to monitor cellular respiration or assay for the presence of oxidized glutathione. The most direct method for avoiding secondary effects, however, is to employ either plasma membrane vesicles or reconstituted proteoliposames derived fran the tissue under study. Given that plasma




59
membrane vesicles are essentially devoid of cytosolic elements, their use eliminates the potential for secondary effects. Unfortunately, they do not allow the differentiation between nonspecific inhibition due to interactions of the modifying reagent with unrelated ocuponents within the plasma membrane. For example, active transport by System A is strongly dependent on the trans-nembrane sodium electrochemical gradient (Kilberg and Christensen, 1980; Kristensen, 1980). Any interaction of a protein modifying reagent which would allow rapid dissipation of the artificially-imposed sodium electrochemical gradient would result in inhibition of System A-mediated uptake.
Sulfhydryl modifying reagents have been known to perturb the sodium electrochemical gradient across the plasma membrane (Van Steninck et al., 1965; Aledort et al., 1968 Khauf and Rothstein, 1971). PCMBS inactivates the erythrocyte plasma membrane Na /K'-ATPase, resulting in an increase in sodium influx and a general inhibition of all ion-coupled transport processes (Aledort et al., 1968). Will and Hopfer (1979) demonstrated recently that PCMS increased the sodium penmability of isolated rat brush border membrane vesicles. These authors concluded that the increased Nat-ion permeability was sufficient to account for the observed inhibition of both valine and glucose active transport.
Same information concerning PCMBS-dependent inactivation of System A transport has been reported in the literature. Chemical modification of System A activity has been served by Wock et al. (1976). PCMBS at concentrations below 10-5 M was shown to stimulate AIB transport into thymocytes, however, inhibition of uptake was observed with PCIMBS




60
concentrations in excesses of 10-5 M. Klip et al. (1980) demonstrated in L6 myoblasts that PCM4BS inhibited adaptive regulation induced System A transport activity in a concentration dependent manner. Unfortunately, no information concerning the actual mode of inactivation by PCMBS was reported in these studies. It remains unclear if the
inhibition was due to direct carrier modification or modification of unrelated plasma membrane components.
Perhaps the most extensive study concerning mercurial perturbation of neutral amino acid uptake was reported by Stirling (1975). This investigator used Hg+ and PCMBS to inhibit uptake of L-alanine (80% to 90%) in brush border membrane vesicles. Elemental mercury was about 10fold more effective than PCMBS in abolishing transport. Dithiothreitol (DIT) reversed Hg+ and PCMBS inhibition by 40% and 100%, respectively. There appeared to be no noticeable changes in Na+-permeability across the membrane, suggesting that a significant amount of the inhibition was due to direct carrier modification.
Materials and Methods
See the methods and experimental procedures outlined in Chapter II.
Results
The results of Table 1 indicate a general refractoriness of the System A protein in H4 hepatcma cells to inhibition by covalent modification, yet, the noncovalently interacting mercurials inhibited System A activity by 85% to 90%. To characterize further the inactivation of System A, a time-course of PCMBS inhibition was measured. As seen in Fig. 5, greater than 85% of the total Na+-




Figure 5. Reversal of PCMBS Inactivation of System A by Dithiothreitol. H4 hepatcma cells were incubated in NaKRB for 5 h to allow induction of System A activity by adaptive regulation. The cells were then incubated in NaKRP containing 0.2 rM PCMBS ( ) for the indicated times. The H4 cells were rinsed twice in NaKRP for 2 min to remove the inhibitor and then incubated in NaKRP containing 1 mM ( ) or 5 mM ( ) dithiothreitol (DIT). At the indicated times, the cells were rinsed in CholKRP for 2 min and System A activity tested by measuring the Na-dependent uptake of 50 M [3H]-AIB for 1 min at 37C. The results are reported as the percent of the transport rate in te absence PCMBS treatment; the control velocity was 386+15 pml n prtein min .




62
100
0 200.0 uM PCMBS
O I.OmM DTT
80 A 5.0mM DTT
C
0
c
60
w 60
O
0
c 40
a. 20
I IIII
0 5 10 15 20 25
Minutes




63
dependent AIB uptake was inactivated within 8 min after exposing the H4 hepatcmna cells to an external concentration of 0.2 mM PCMBS; the tl/2 for inhibition was 3 min. The inhibition of System A was not reversed by extensive washing with NaKRP buffer, however, a significant amount of the total activity was recovered when the cells ware exposed to 5 nmM dithiothreitol (DIT). The rapidity of inhibition by PCMBS and the reversibility by DIT suggested a direct interaction of the chemical reagent with plasma membrane components. Although all of the experiments involving PCMBS-dependent inactivation were performed in the presence of 170 mM sodium, the inactivation process does not appear to require sodium during the treatment period (Table 6). Given the considerable difference in affinity of PCMBS for sulfhydryl groups versus other ligands (Means and Feeney, 1971), the data presented above point to the presence of an essential cysteine residue(s) contained within the System A carrier. Furthermore, the negatively charged nature of PCMBS does not allow this compound to cross the plasma membrane readily (Aledort et al., 1968); hence, the PCMBS-sensitive group on System A is likely located on the extracellular surface of the plasma membrane.
In order to confirm that the PCM4BS-sensitive sulfhydryl group(s)
was located within the plasma membrane, the effect of PCMBS on System A activity was tested using H4 hepatcma-derived plasma membrane vesicles (Dudeck et al., 1987). Membrane fractions enriched for plasma membrane vesicles were isolated and then exposed to increasing concentrations of PCMBS using conditions corresponding to those used to obtained the data




64
Table 6. Sensitivity of System A Activity in H4 Hepatoma Cells to PCMBS in
the Presence or Absence of Na -ions
condition Velocity %ontrol % Protection
NaKRP 648 52 100 NaERP + I-norleucine 505 30 78 NaIN + PCMBS 138 8 21b NaKRP + Irnorleuine
+ PCMBS 337 16 52b 54
CholERP 712 40 100 CholERP + -norleucine 615 34 86 OolERP + PoBS 185 + 8 26 CGolEP + Irnorleucine
+ PCMBS 348 40 49b 38
H4 hepatama cells were cultured in an amino acid free medium (NaKRP) for 3 h to enhance System A activity by adaptive regulation. Cells were incubated an additional 10 min in the above conditions. The concentration of L-norleucine and PCM S was 5 mM and 0.2 rM, respectively. Following this incubation period, the cells were washed twice with CholKRP and then the total Na-dependent uptake of 50 M [3H]-AIB was assayed as described in Chapter II.
b~hese values are significantly different from the control values to p values < 0.005.




Figure 6. Concentration Depexxence of the Inhibition of System A Activity in Rat Hepatocyte or H4 Hepatcma Membrane Vesicles by PClBS. Plasm membrane vesicles were prepared as described in Chapter II and were treated with PIMBS for 10 min at 22*C. The inhibitor concentration was varied between 0.1 and 1.0 nm. After rinsing the membranes with Buffer A, System A activity was assayed by measuring the Na-dependent uptake of 200 M [3H]-AIB for 1 min at 22-C. The results are reported as the percent of the rate of transport in the non-treated vesicles; those velocities (avages + S.D.1 of 3 determinations) were 2291+106, and 590+75 pol m g protein min for the normal hepatocyte and H4 hepatcama membrane vesicles, respectively. This experiment was performed by Kathleen Dudeck-Collart of this laboratory.




100
o 80
A H-4
C
0 n Hepatocyte v 60
0
c 40
20
0 0.2 0.4 0.6 0.8 1.0
[PCMBS], mM




67
shown in Fig. 5 for intact cells. As shown in Fig. 6, PC4BS treatment of H4 membrane vesicles, as well as vesicles derived from normal rat liver, decreased the total amount of measurable Na+-dependent AIB uptake by 80%-90%. The inactivation of transport was concentration-dependent and the external concentration of PC4BS required to produce half-maximal inhibition was approximately 200 M. Collectively, these results suggest that the action of PC4BS is due to direct modification of plasma membrane components.
Additional information about the inactivation of System A-mediated transport was gained by studying the initial-rate kinetics of Na+dependent AIB uptake before and immediately following PC 4BS treatment. In this experiment, H4 cells were treated with 200 M PCMBS (10 min) at room temperature, the cells were washed rapidly with CholKRP buffer, and the transport of AIB was monitored over a concentration range of 0.02 to 50.0 rM. The kinetic data was then represented by the method of Eadie (1942) and Hofstee (1959). The results in Fig. 7 are consistent with the proposal that transport over the concentrations of AIB employed occurs via a single Na -dependent agency exhibiting a Km for AIB of 1.2 1l -1
+ 0.1 mM and a Vm of 6.4 + 0.1 nmol mg- protein 15 sec -. Kinetic
- max
analysis of Na-dependent AIB uptake after treatment of cells with PCMBS also revealed linear kinetics, but the Km for the Na+-dependent AIB uptake was increased about three-fold (3.3 + 0.2 AM) and the Vx was max
decreased by nearly 40% (4.1 + 0.3 nmol mg-lprotein 15 sec-1 ) .
The following series of experiments were designed to investigate
the mechanism of action of PCHBS on active transport by System A. These




Figure 7. Kinetics of AIB Transport Followir PCMBS Treatment of H4 Hepatama Cells. The cells were cultured in NaKRB for 5 h to allow enhancement of the System A activity and then incubated in NaERP in the presence ( ) or absence ( ) of 0.2 uM PCMBS for 10 min. The hepatama cells were washed twice in CholERP for 2 min and System A activity monitored for 15 sec at 37*C with a substrate ([3H]-AIB) concentration ranging fron 0.02 M to 50 M. The K and V values were calculated from the Na-dependent velocities by computer'alysis (Cleland, 1979). Where not shown the standard deviation bars are contained within the symbol.




69
PIs
UI I I II 'f 10.2
* PCMBS Km VMax 8.1 W + 3.3=0.2 4.1-0.3
0 1.2:20.1 6A4*0.1
6.2
E
* 4.3
0
E *
0 1.1 2.2 3.3 4.4 5.5
nmol. mg protein15sepmM




studies addressed three areas; 1) the effect of PCMBS on the initial rate of Na-dependent AIB uptake; 2) the effect on the rate for AIB efflux; and 3) the effect on the final steady state distribution ratio of AIB. Fig. 8 shows the results of a series of experiments designed to examine the role of PCMBS on the initial rate of AIB uptake and the steady-state distribution. Shown is a time-course for Na t AIB uptake into H4 hepatma cells follower PCBS treatment. The Na dependent AIB uptake in control cells was essentially linear throughout the time of the experiment (60 min). In contrast, the Na*-depene uptake of AIB was significantly lowered and only linear for approximately 1 min following PCMBS exposure. Even after 60 min, the accumulation of AIB within H4 cells had not reached steady state, however, the accumulation of AIB into IGIBS treated cells had reached steady-state within 30 min.
To quantitate the degree of uncoupling of active transport by System A, the distribution ratio of AIB (i.e., AIBin/AIBut) was determined for both control and IGIBS treated cells. Distribution ratios in excess of 1 for a given solute of neutral charge and limited intracellular trapping are generally taken as evidence for active transport. To calculate distribution ratios the intracellular water volume must first be determined. Using the 3M method described in apter II (Kletzien et al., 1975), an intracellular water volume of 5.7 1/mg protein was measured for the H4 hepatcma cell (Fig. 9). Using this value and the number of poles of AIB accumulated within the cell at 60 min, the distribution ratio for AIB was calculated at




Figure 8. Tim-Course of AIB Uptake into H4 Hepatama Cells Following PCMBS Treatment. H4 hepatama cells were incubated in NaKRB for 5 h to enhance the amount of System A activity by adaptive regulation. The cells were then incubated in NaERP ( ) or NaKRP containing 0.2 b14 PCMBS
( ) for 10 min at 37 C. System A activity was monitored by measuring the Na-dependent uptake of 50 M [3H]-AIB for 0.25 to 60 min. The results ae the averages + S.D. of 4 determinations and are reported as pmol mg protein versus time (min). Where not shown the standard deviation bars are contained within the symbol.




(pmol AIB mg-1 protein) x 10
*,PCMBS
00 0
0
o 0 0 (omol AIB~ma~ protein) x1i0




Figure 9. Effect of IGIBS on the Intracellular Water Volune of H4 Hepatcmna Cells. H4 hepatama cells were incubated in NaERB for 5 h to enhance the amount of System A activity by adaptive regulation. The cells were then incubated in NaERP ( ) or NaERP containing 0.2 zoM PCMBS
( ) for 10 min at 370C. The cells were quickly rinsed twice with NaERP (4.0*C ) and incubated with NaKRP containing radiolabeled 3M as described in Chapter II at 370C. After 1.5 h, the medium was removed and the cells washed four times in CholERP containing 1 IfM phloretin (40 C). The amount of radiolabeled 3MG remaining in the cells was determined as described in Chapter II. The ru are reported as (averages + S.D. of 4 determinations) pool 3MG ng protein verses the concentration of 3M. Where not shown the standard deviation bars are contained within the symbol.




74
15
o 13
-+ PCMBS .5 11
0
L.
T. 9
E
C'7
-5 5
E
3 -PCMBS
1
1.0 3.0 5.0 7.0 9.0
3MG 1, mM




75
appraximately 24 (i.e., AIBij = 1179 M and AIBut = 50 M). Aledort et a. (1968) reported that exposure of erythrocytes to PCEBS caused an increase in the intracellular water volume by approximately 40%. Therefore, the intracellular water volume in the H4 hepatana cells was also measured after PHBS exposure. As seen in Fig. 9, the intracellular volume increased appraximately 2.5-fold (14 1/mg protein). Using this value a distribution ratio for AIB accumulation at steady state (30 min) was calculated to be approximately 0.6 (i.e., AIBin = 28 M and AIBt = 50 M). These data indicate that the primary action of ICMBS on System A was to uncxuple active transport by this carrier.
In the absence of cellular metabolism of the transport substrate, as in the case for AIB and MeAIB (Noall et al., 1957; Christensen and Jones, 1962), the steady state distribution ratio for a given solute arises from the balance between all routes for entry and all routes of exodus. Tb test whether the decrease in the initial rate of uptake could account solely for the lowered distribution ratio for AIB, efflux of AIB was assayed following PCMBS treatment. This was performed by preloading the H4 hepatama cells with radiolabeled AIB (external concentration of 50 M) for 1 h at 370C. The cells were then incubated in NaERP buffer in the presence or absence of 200 M PEBS (10 min) at 370C. Following the treatment with PCOBS, the cells were rinsed rapidly with NaERP buffer and then incubated in 1 ml fresh NaERP buffer. At various times, the amount of AIB remaining in the cells was determined as described in Chapter II. As seen in Fig. 10, the net rate of AIB




Figure 10. Time-Course of AIB Efflux fran H4 Hepatcana Cells Following PCMBS Treatment. H4 hepatcma cells were incubated for 1 h at 37*C with 50 M [3H]-AIB. The cells were then rinsed with ice-cold NaERP and incubated an additional 10 min in NaERP ( ) or NaKRP containing 0.2 mM PC!BS ( ) Efflux of AIB was measured by washing the cells twice in NaERP (4.OC) and then incubating the cells in 2 ml NaERP (37*C). At the indicated times, the cells were rapidly washed four times with icecold CholKRP and the amount of radiolabeled-AIB remaining in the cells was determined as described in Chapter II. The results reported are the averages _q.D. of 4 determinations and are plotted as pol AIB remaining mg protein versus time (min). Where not shown the standard deviation bars are contained within the symbol.




77
5.0
4.0
O N -PCMBS
a 3.0
0
IM
1.0
0
O 3.0 6.0
Time, (min)




78
Table 7. Protection of System A Transport Activity by Amino Acids
Amino Acid % Inhibition of Transport % Protection of Activity
L-Alanine 92 35 D-Alanine 5 11 &-Chloro-L-alanine 86 38 L-Serine 86 68 D-Serine 1 35 2-Amincbutyrate 83 47 L-Proline 81 36 D-Proline 0 18 IL-Norleuine 73 62 N-Acetyl-I4-histidine 52 49 L-Histidlne 46 48 Taurine 5 12 i-Aspartate 2 7
L-Alanin-N-hydroximate 0 0 i-Arginine 0 8
I-Lysine 0 7 H4 hepatcma cells were incubated in NaXRB for 5 h to stimulate transport activity via adaptive regulation and then transferred to NaKRP containing 0.2 M PCMBS in the presence or absence of 5 1M of the indicated amino acid. After 10 min at 370C, the cells were washed in CholKRP for 5 min and the System A activity assayed by measuring the Na+-dependent uptake of 50 M AIB for 1 min at 370C. Cells not treated with PCMBS were tested for substrate inhibition of System A by measuring the uptake of 50 M 13H]-AIB in the presence or absence of 5 rM of the indicated amino acid. The data aje represented as a percent of the initial value (337 + 15 pmol mg protein min ) observed in the absence of the inhibitor.




79
efflux was increased 11-fold for cells treated with 101BS. The calculated t values for AIB efflux were 77.0 + 0.7 and 7.0 + 0.1 min for 101BS treated and control cells, respectively. Thus, the reduction in the steady state distribution ratio for AIB appears to result from both a decrease in the initial rate of uptake as well as an increase in the rate of exodus.
As discussed above, protein-modifying reagents react
indiscriminately with the sulfhydryl groups of many membrane proteins (Rothstein, 1970). A series of experiments were designed to address whether the inhibition by IP4BS was the result of direct carrier modification or due to indirect plasma membrane perturbations. One of the strongest indications that a membrane protein is modified by a chemical reagent is protection of activity by substrates. Therefore, our first analysis examined the ability of substrate amino acids to protect System A activity from P1BS-dependent inactivation. There was a correlation between the ability of substrate amino acids to inhibit System A-mediated AIB uptake and the ability to protect the carrier from 101BS-dependent inactivation (Table 7). For example, a significant amount of protection of System A activity was observed with most amino acids containing small neutral side-chains. System X A, G such as L-aspartate (Makawske and Christensen, 1982) and typical System y+ substrates such as Ir-arginine and Ir-lysine (Ihite and Christensen, 1982) protected only 7-8% of the ICGBS-sensitive activity. The stereospecific nature of the protection by amino acids (Table 4), along with the inability of non-substrates to cause protection (Table 7),




Figure 11. Dixon Plot of the Kinetics of Ir-Norleucine Inhibition of AIB Uptake. The N*-dependent uptake of (H]-AIB was monitored at 37-C for 1 min using AIB concentrations of 0.1, 0.2, and 0.5 M. The results shown are the Na-dependent uptake velocities in the presence of the indicated concentrations of unlabeled I-norleucine as inhibitor. The results are reported as the + S.D. of 4 determinations and are plotted as pmol AIB mg protein versus the concentration of I-norleucine. The estimated Ki value was determined as described by Cleland (1979).




5
[@0.1mM
[AIB] A 0.2mM
4 0 0.5mM
3 Kg 19 2 0. mM
2
-2 0 2 4 6 8 10
LNorleucine]. mM




82=
indicate that the protection is not due to chemical reaction of PCIBS with the amino acid or other indirect effects.
The lack of a more precise correlation between the degree of
inhibition of System A and the protective capability of a given amino acid is not totally understood (Table 7); however, it may result from the considerable amount of trans-inhibition observed with same System A substrates (Kelly and Potter, 1979; Kilberg et Al., 1985). Transinhibition by the protective amino acid complicates the calculation of the degree of protection by altering the control transport rate measured. An attempt to correct for trans-inhibition was taken into consideration by using the rate V (velocity of uptake after incubation in the presence of the protective amino acid only) as described in Chapter II, yet a certain amount of imprecision prevailed. For example, the non-metabolizable analogs, AIB and MeAIB, gave such inconsistent results because of their high degree of trans-inhibition that they were not used routinely as protective amino acids for quantitative studies. On the other hand, Ir-norleucine typically produced both the highest measurable protection and the least amount of trans-inhibition (10% to 15% inhibition of total uptake) of all the amino acids tested. Therefore, it was chosen as the test amino acid for additional protection studies.
Kinetic analyses was conducted to demonstrate that Ir-norleucine was indeed an effective substrate for System A in liver tissue. The Na dependent uptake of 0.1, 0.2 and 0.5 nM radiolabeled AIB was assayed in




Figure 12. Inactivation of System A Transport Activity by PCMBS: Kinetics of IrNorleucine, L-Serine, and Ir-Alanine Protection. H4 hepatcma cells were cultured in NaKRB for 5 h to allow System A activity to increase via adaptive regulation. The cells were then incubated in NaXRP containing 0.2 nM PCMBS for 10 min at 370C in the presence or absence of the indicated amino acid at concentrations ranging frcm 0.25 mM to 25 nM. System A activity was monitored by incubating the cells in CholKRP for 2 min then measuring the Na* uptake of 50 M [3H]-AIB for 1 min at 37-C. The results are represented as the percent of System A activity that was inactivated. The concentration of amino acid that gave half-maximal protection frca PCMBS-dependent inhibition
(Kp) was estimated by coupter analysis of the data.




84
100
Norleucine 80 Kp=0.6S0.1mM
60
4020
0
100
"a Alanine
S 80 Kp 2.4t 1.5mM
S 60
E 40
e
20
W 20
c
Sein
0
w
a.
100
Serine 80- Kp=2.10'G.8mM
60
40
20
0 1 5 10 15 20 25
[Substrate], mM




85
the presence of varying concentrations of unlabeled L-norleucine. The data were then analyzed by the method of Dixon (1953) and are presented in Fig. 11; the results indicate that L-norleucine is a ccapetitive inhibitor of Na*-dependent uptake of AIB and show the usefulness of this amino acid as a model System A substrate. The affinity of L-norleucine for System A (Ki = 1.9 + 0.1 rM) is within the range observed for most other substrates (Oxenxer and Christensen, 1963).
The kinetics of Lr-norleucine protection of System A activity was then determined using concentrations of L-norleucine ranging from 0.25 to 25.0 EM, while the PCMBS was maintained at an external concentration of 0.2 DM. As demonstrated in Fig. 12, r-norleucine protected more than 70% of the System A transport activity that was subject to PCMBS inhibition. The protection was concentration-dependent with halfmaximal protection of AB uptake occurring at an extracellular Lnorleucine concentration of 0.6 + 0.1 nM. Furthermore, the amount of System A activity protected by L-norleucine was essentially the same regardless whether Nat-ions were present or absent during the experiment (Table 6). The protective capability of Ir-norleucine was also tested using H4 hepatama-derived plasma membrane-enriched vesicles (Table 8). The inclusion of 5 M Ir-norleucine during exposure of the vesicles to PCMBS protected approximately 42% of the total Na AMB uptake inhibited by PCMBS. The System A substrates L-alanine and L-serine were also characterized with respect to protection of the carrier from PCMBS inactivation (Fig. 12). Concentration-depedent protection was observed for both these substrates and the external concentrations required for




Table 8. L-Norleucine-Dependent Protection from PCMBS Inactivation
of System A Transport in H4 Hepatoma or Rat Liver
Membrane Vesicles
condition Velocity % Control % Protectiion
H4 Hepatcma
Ocntrol 597 25 100 0.25 M PCMBS 109 18 18C
5 mM IrNorleucine 749 14 125
0.25 M PCMBS +
5 rM L-Norleucine 394 + 14 66c 44 Hepatocytes
Control 1492 69 100
0.5 M POIBS 29 t 53 2c
5 M IrHNorleucine 896 79 60
0.5 WK PCBS+
5 M Ir-Norleucine 115 + 10 8b 10
Rat liver of H4 hepatma membrane vesicles were treated with the indicated concentrations of PCBS in the presence or absence of L-norleucine as described in Chapter II. The System A activity was then assayed by measuring the Na+-dependent uptake of 200 M [(3H]-AI for 1 min aj 370C. The data are reported as the averages of the velocity (pmol mgq protein min ) + S.D. for three determinations. This experiment was performed with the assistance of Kathleen Dudeck-Collart. bThese values are significantly different frame the control values to p values <0.1. These values are significantly different fraum the control values to p values < 0.005.




87
half-maximal protection were 2.4 + 1.5 M and 2.1 + 0.8 mM for Ir-alanine and Lr-serine, respectively.
Hepatic neutral amino acid uptake is mediated largely by four systems (Systems A, ASC, N, and L) each possessing distinct, but overlapping specificities (Kilberg, 1982). A given amino acid may be transported largely by a single agency, such as Irglutamine by System N or by multiple routes, such as Ir-alanine by Systm A, ASC, and L. Therefore, it was of interest to determine whether I-norleucine could protect additional amino acid transport system. If the protection is specific it should be reflected in the system-specificity of Irnorleucine. The results of these experiments are illustrated in Table
9. Several transport systems were first evaluated for their sensitivity to 0.2 M PCMBS and those that were sensitive were tested for protection of each carrier by the inclusion of 5 14 L-norleucine. Systems ASC and L were strongly inhibited by PCMBS (90% and 99%, respectively), yet showed no significant protection of transport activity. System Gly was also inactivated under these conditions (92%), but once again, little or no protection was afforded by L-norleucine (10%). System N-mediated transport was decreased approximately 91% following PCMBS treatment, with only 24% of this activity protected by Ir-norleucine. System N is recognized to be particularly sensitive to non-capetitive inhibition by neutral amino acids that are not substrates (Kilberg al., 1980). The protection observed may be the result of L-norleucine interaction in this manner. The beta amino acid carrier, System 8, was also sensitive to PCMBS, unfortunately the low activity of this system in the H4




88
Table 9. L-Norleucine Protection from PCMBS-Dependent Inactivation of
Several Amino Acid Transport Systems in H4 Hepatoma Cells
SYSTEM CNIOL CORIOL PMBS %INAclVAICN PCBS % PROTECTI + +
Norlemcine Norleucine
A 51623 47225 1166 77b 33515 61C ASC 1896238 1488147 15619 92e 17014 le L 985L37 5694421 254 99e 291 Oe N 4719 46810 417 91e 1443 24e Gly 905 932 70.2 92e 155 9e
x A,G 31418 26118 357 89e 833 21e y +Na 525 494 315 404 434 67b y -Na 525 493 372 29 324 Od a 23 313 62 81 ND ND
Effect of PCMBS en several amino acid transport systems. H4 cells were incubated for 4 h in NaRB to enhance System A activity and minimize trans-effects. The cells were then incubated for 10 min in Na1RP containing 0.2 n PCMB in the presence or absence of 5 uM r-norlecine. The cells were rinsed twice in CholKRP for 5 min and then the specific systems were measured as follows: System A, Na-dependent AIB uptake; System ASC, Nat-dependent I-threenine uptake in the presence of 5 rM MeAIB; System N, Na*dependent L-glutamine uptake in the presence of 5 rrm MeAIB; System y+, saturable Na*irdepenent L-arginine uptake; Syt XA, G, Na -depenent L-glutamte uptake; System Gly, NaI-depeent L-glycine uptake in the presence of 5 rM threonine; and System L, saturable Na-independent leucine uptake. The substrate concentrations were 50 M and all transport assays were performed for 30 sec at 370C, except for System A, which was aasured for min. The data are reported as the averages of the velocity (pnol Mq protein min ) S.D. for three or four assays (N.D. = transport activity not detectable).
~hlese values are significantly different to p values < 0.1. o0hese values are significantly different to p values < 0.01. d~ese values are significantly different to p values < 0.025. elhese values are significantly different to p values < 0.005.




Full Text

PAGE 1

PROl'EIN IDDIFIC'ATION OF 'lHE SYSTEM A CARRIER AND AMINO ACID-DEPENDENI' GENE Rm.JIATION lli HEPATIC TISSUE By 'lHCNAS CRANE OIIIES A DISSERI'ATION PRESENI'ED 'IO 'lHE GRAilJATE SOICX>L OF 'lHE UNIVERSITY OF FIDRim m PARI'IAL FULFill.MENT OF '!HE~ FOR '!HE DErnEE OF IX)CIDR OF FHIIDSOEHY UNIVERSITY OF FIDRim 1988

PAGE 2

A~ I would like to thank all the irernbers of the laboratory for their assistance throughout these studies. I would also like to exterrl a deep appreciation to Mary Harrll.ogten for her help with the amino acid transport and the protein electrq:horesis studies, Elizabeth LA.ldenhausen for her assistance with the reconstitution assays, arrl Kathleen LA.ldeck-COllart for the membrane vesicles studies. In addition, I would like to ackncMledge Neil Shay, William Wonq, arrl catherine Ketdlem for their assistance in the rrolecular biology :r,oase of this research. Finally, I would like to ackncMledge especially my mentor Dr. Michael S. Kilberg for his time, trainin:J arrl guidance throughout all of these studies. ii

PAGE 3

TABIE OF mNrENI'S A~ 11. LlS'r OF FI G.JR:ES V I..ISI1 OF TABI.1!S viii ~TI~S ............................................ ix ~er ................................................. X1 ClIAPI'ER I ClIAPI'ER II AMINO ACID TRANSIORI' IN CELIS 1 MEMBRANE TRANSIORI' MEIHOI:S 10 ClIAPI'ER III DIFFERENTIAL SENSITIVITY OF AMINO ACID TRANSIORI' SYSTEMS 'IO SULFHYDRYirIDDIFYING REAGENI'S 23 Introduction. Results ... Discussion ... .23 25 49 ClIAPI'ER N EVIDENCE FOR '!HE DIRE.Cr OIEMICAL M'.)I)IFICATION BY PCMB5 OF '!HE SYSTEM A CARRIER IN RAT H4 HEPA'l{:MA CELIS 57 Introduction .................. 57 Results .................... 60 Discussion .......................................... 96 ClIAPI'ER V MATERIAIS AND MEIHOI:S FOR Irol'EIN OIEMISTRY AND KJlEClJI.AR BIOLCX;Y ............ 102 ClIAPI'ER VI IDENI'IFICATION AND OIARACI'ERIZATION OF AMINO ACID STARVATION INUJCED HEPATIC ~lli'S .................................. 122 Introduction .. ... 122 Results .129 Discussion ... .219 ClIAPI'ER VII ~y .. 226 APPENDIX I TISSUE ClJilIURE AND TRANSIORI' MEDIUM 230 APPENDIX II SOI1JrIONS FOR GEL EIECI'ROIH::>RESIS 233 APPENDIX III GENERAL MEIHOI:S AND ENZYME ASSAYS 236 APPENDIX N SOI1JrIONS FOR IDIEaJI..AR CIDNING 246 iii

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APPENDIX V GENERAL MEIHOOO FOR M)I.EClJIAR CI.DNING. 249 APPENDIX VI RF.AGENI'S FOR '!HE OIEMICAL IDDIFICATIOO OF ~INS 253 Bim..I~ 254 BI~CAL S~ 271 iv ------------

PAGE 5

LIST OF FIGURES 1. Concentration Deperrlence of the Inhibition of System A Activity in Nonnal Rat Hepatocytes an::l Several Hepatana. Cell Lines by NEM or PCMBS ............... 31 2. MeAIB Inhibition of Na+ -Deperrlent AIB Transport in Several Hepatana Cell Lines ........... 36 3. Concentration Deperrlence of NEM Inhibition of System A Activity in Fao Hepatana. Cells CUltured in Amino Acid Rim ~it.nn 39 4. Concentration Depen:lence of the Inhibition of System A Activity in Rat Hepatocytes or Rat H4 Hepatana Membrane Vesicles by NEM ................. 4 3 5. Reversal of PCMBS Inactivation of System A by Di thioth.rai tol ............................ 62 6. Concentration Depen:lence of the Inhibition of System A Activity in Rat Hepatocytes or H4 Hepatana. Membrane Vesicles by PCMBS ....................... 66 7. Kinetics of AIB Transport Folla,,,ing PCMBS Treat:Irerlt of H4 Hepatana Cells ......................... 69 8. Time-Course of AIB Uptake into H4 Hepatana Cells Folla,,,ing PCMBS 'rrea.t:Irerlt ............................ 72 9. Effect of PCMBS on the Intracellular Water Volt.nne of H4 Hepatana Cells ................................ 7 4 10. Time-Course of AIB Efflux fran H4 Hepatana Cells Folla,,,ing PCMBS 'rrea.t:Irerlt ............................. 77 11. Dixon Plot of the Kinetics of IrNorleucine Inhibition of AIB Uptake 81 12. Inactivation of System A Transport Activity by PCMBS: Kinetics of IrNorleucine, Ir-Serine, an::l Ir-Alanine Pl:"otection 84 13. Time-Course for 3-o-Methyl-D--Glucose Exodus fran H4 Hepatana Cells Folla,,,ing PCMBS Treat:Irerlt ....... 92 V

PAGE 6

14. Elect.rq::horetic Protein Pattern of a Coanassie Blue Stained 'l\vo-Dim:m.sional Polyacrylamide Gel. ..... 131 15. Fluorograrn.s of the Synthesis of Irrli vidual Hepatic Membrane Proteins in Response to Amino Acid starvation 134 16. ~titation of the Rates of Incorporation of Radiolabeled IrLeucine into Membrane Proteins .......... 138 17. Antibody Production Schene for Preparirg f.k)nospecif ic Iol ycl0l1cll .Arltil:x:x:iies .... 141 18. Detection of MP-73 by Irnrm.u1d:)lottirg of Rat Liver Membrane Proteins . . 14 4 19. Ti.Ire-course of Antibody Production Against MP-73 .... 147 20. Senlm Antibody Titer Against MP-73 ....... 149 21. Irnrm.u1d:)lot Detection of MP-73 FollCMirg 'l\vo-Dim:m.sional Polyacrylamide Gel Elect.rq::horesis .. 152 22. Irnmunablot of Imrrune or Noni.nm.me Senlm Against MP-66 FollCMirg One-Dim:m.sional Polyacrylamide Ge,l Elect.rq::horesis ..................................... 155 23. Irnmunablot of MP-66 FollCMirg 'l\vo-Dim:m.sional Polyacrylamide Gel Elect.rq::horesis ......... 157 24. Estimation of the f.k)lecular Weight of MP-73 FollCMirg OneDim:m.sional Soditnn-Ibdecyl-SUlfate Polyacrylamide Ge.l Elect.rq::horesis ......... 160 25. l1mt.lnc:t>lot of MP-73 FollCMirg One-Dim:m.sional Nonreducirg Soditnn-Ibdecyl-SUlfate Polyacrylamide Gel Elect.rq::horesis ....................... 162 26. Estimation of the f.k)lecular Weight of MP-73 FollCMirg 0neDim:m.si0l1cll Nonreducirg Soditnn-Ibdecyl-SUlfate Polyacrylamide Gel Elect.rq::horesis ......... 164 27. Coanassie Blue Stain of Proteins fran the SUbcellular FractiOl'lcltion of Rat Liver ............. 167 28. Silver Stain of Proteins fran the SUbcellular Fractionation of Rat Liver .......... 169 29. SUbcellular localization of MP-73 by Irnmunablot Analysis.171 30. Coanassie Blue Stain of Proteins fran the Fractionation of 'Ra.t Ll ver Mi t:.cx:l'lorrl.ria .......... 17 5 31. Silver Stain of Proteins fran the Fractionation of Rat Ll ver Mi t:.cx:l'lorrl.ria .......... 177 vi

PAGE 7

32. Inum.md:>lot Analysis of Fractionated Rat Li. ver Mi tcx:::llorrlria ....................................... 179 33. MP-73 Irnnrunoreactivity Followin:J Triton X-114 Fbase Separation of Rat Li.ver Mitoplasts 183 34. Fast Green Stain of the Proteins Followin:J Triton X-114 Fbase Separation of Rat Li.ver Mitoplasts 185 35. Imnrunoprecipitation of MP-73 fran [3H]-Ir-I..eucine ~loo Cells ............................................ 188 36. Biosynthesis of MP-73 D.rrin:J Amino Acid Deprivation 190 37. .Adaptive Regulation of System A D.rrin:J Amino Acid Starvation of Rat Hepcttocytes 193 38. Map of the Lambda gtll Genane Showin:J Major Restriction Sites .................................................... 197 39. Inmmoscreenin:J of a Ht.nnan Fetal Li.ver crnA Expression Library with Antiserum Against MP-73 200 40. Molecular Size of the Ht.nnan Fetal Li.ver crnA Insert fran Bacteriq::hage Lambda gtll an:i IVC19 203 41. Molecular Size of the Adult Rat Li.ver crnA Insert fran Bacteriq::hage Lambda gtll an:i JVC19 205 42. Restriction Map of IVC19 Genane 209 43. Restriction Errlonuclease Analysis of the Bacteriq::hage Lambda gtll an:i the SUbcloned Ht.nnan Fetal Li.ver crnA Insert .. 211 44. Hybridization Analysis of Rat an:i Ht.nnan Li.ver crnA's 213 45. Northern Analysis of Polyadenylated RNA Crnplementar:y to the crnA Isolated fran the Adult Rat Li.ver crnA Expression Librar:y 216 46. Northern Analysis of Polyadenylated RNA Crnplementar:y to the crnA Isolated fran the Ht.nnan Fetal Li.ver crnA Expression Librar:y 218 vii

PAGE 8

L.Isr OF TABUS 1. Sensitivity of system A Activity in Normal Rat Hepatocytes am H4 Hepatana Cells to Protein Modifyirg Reagents ... 26 2. Sensitivity of system A Activity in Normal Rat Liver am H4 Hepatana Vesicles to Ihenyl Isothiocyanate ... 28 3. Recx:>nstitution of system A Activity from Rat Hepatocytes or H4 Hepatana Cells Folla,,irg NEM-Treabrent of SOlubilized Plasma Membrane Proteins ...... 45 4. Amino Acid-Depen:lent Protection from Inactivation by PCMBS of system A-Mediated Trans:(X)rt 47 5. Sensitivity to NEM or PCMBS of Several Amino Acid Transport systems in Rat Nonnal Hepatocytes or H4 Hepatana Cells .. 48 6. I.rNorleucine-Deperrlent Protection from PCMBS Inactivation of system A Transport in H4 Hepatana or Rat Liver Membrane Vesicles ......................................... 64 7. Sensitivity of system A Activity in H4 Hepatana Cells to PCMBS in the Presence am Absence of Na+-ions .... 78 8. Protection of system A Transport Activity by Amino 'Ac.ids 8 6 9. I.rNorleucine Protection from PCMBS-Deperrlent Inactivation of Several Amino Acid Trans:(X)rt systems in H4 Hepatana Cells ...................................... 88 10. Effect of PCMBS on lactate Dehydrogenase Release from H4 Hepatana Cells ............................................ 94 11. Effect of PCMBS on the Na+ -Deperrlent Uptake of I.rPyntvate, Uridine, arrl AIB in H4 Hepatana Cells ....... 95 12. Enzyioo Marker Analysis of Isolated Rat Liver Mi tc:x::llooor ia . . . . . . . . . . . 173 13. Enzyioo Marker Analysis of Rat Liver SUl:initc:x::llooorial Fractio11S ......... 181 viii

PAGE 9

ACT AIB AMP ASN BSA Se Eagles Minimal Essential Meditnn microgram microliter ix

PAGE 10

M IrM mrrol min MW NaCl NaKRB NaKRP rnool NaOH NEM PAGE PBS PCMBS poc>l S.D. SI:6 Tris X-gal micrc:m::>lar milinolar milinole minute nolecular weight sodhnn chloride Na+ -containin;J Krebs-Rin;Jer bicartx:>nate buffer Na+-cx>ntainin;J Krebs-Rin;Jer Iilosii'late buffer nancrcole sodium hydroxide N-ethylrnaleimide polyacrylamide gel electrq:iloresis Iilosii'late buffered saline p-chloraoorcuribenzene sulfonate picarole starrlard deviation sodiurn-dodecyl-sulfate tris(hydroxyrrethyl)ami.naoothane 5-brat0-4-chloro-3-irdolyl-B-galactoside X

PAGE 11

Abstract of Dissertation Presented to the University of Florida in Partial F\llfillrrM=nt of the Requireirents for the D9gree of lbctor of fhilosq:hy morEIN M:>DIFICATION OF 'IHE SYSTEM A CARRIER AND AMINO ACID-DEPENDENI' GENE RErrJI.ATION IN HEPATIC TISSUE BY 'IH(J,1AS CRANE OIIIBS April, 1988 Olainnan: Michael S. Kilberg Major Deparbtelt: Biochemistry arrl Molecular Biology '!he transport of amino acids by system A in nonnal rat hepatocytes arrl several hepatana cell lines has been sha,m to be inhibited by m:xiification of free sulfhydryl group(s). system A-mediated uptake in all of the hepatic cell types tested was inhibited by the organic mercurial p-<"".hloranero.rribenzene sulfonate (PCMBS). In contrast, the sensitivity of the system A carrier in hepatana cells to inactivation by N-ethylrnaleimide (NEM) varied fran m:xierate to no inhibition. '!he inactivation of system A in the H4 hepatana cells by PCMBS was rapidly reversed by treabrent of the cells with dithiothreitol. '!he PCMBS inhibited the initial rate of AIB uptake, whereas AIB efflux was stimulated. '!he net effect was a decreased steady state distribution ratio for AIB accumulation (AIBir/AIBout) fran 24 to 1. Kinetic analysis revealed that the effect of PCMBS was a reduction in the rnax.illlal. translocation rate as well as a decrease in the affinity for system A substrates. SUbstrate level protection fran inhibition by x i

PAGE 12

PCMBS was observed in H4 hepatorna cells whereas none of the System A substrates tested protected transport activity in nonnal hepatocytes. Amino acid-deperrlent protection was stereospecific arrl laI"gely restricted to System A. Similar results were ootained usirq plasma lOOlllbrane vesicles isolated fran intact hepatorna cells arrl rat liver. '!he biosynthesis of several rat liver lOOlllbrane proteins was denonstrated to be enhanced by amino acid deprivation of hepatocytes in pr.imary culture. One of these proteins, MP-73, has an estimated roc>lecular weight of 73 kia arrl an isoelectric point of 7. O. Monospecific polyclonal antisennn prepared against MP-73 was used to localize the protein to a rat liver suboellular fraction highly enriched for inner mitochorrlrial lOOlllbranes. Based on Triton X-114 Ifuise separation, the protein appears to be a hydrophooic inte:Jral lOOlllbrane protein. '!he antisennn was used to screen both an adult rat liver arrl a htmian fetal liver lambda gtll crnA expression library. Of five rat arrl foor hmnan irrleperrlent clones that were plaque-purified, two rat (2000 arrl 1000 bp) arrl one hrnnan (1100 bp) were subcloned into the bacterial plasmid p.JC19. crnA probes prepared fran either the rat or hmnan inserts identified two transcripts in rat arrl hmnan tissue of awroximately 1900 nucleotides arrl 2400 nucleotides in lerqth. xii

PAGE 13

CliAPl'ER I AMINO ACID TRANSIORI' m z.w.t.1ALIAN CELIS All known cellular o:rganisrn.s utilize L-amino acids to comuct many essential processes (Meister, 1965) '!he liver is a major site for amino acid metabolism in the body with rrost of the metabolism occurring in the hepatocyte. 'lhese metabolic processes include protein synthesis, gluco:neogenesis, utilization by both the urea arrl tricarlx>xylic acid cycles, as well as conversion into a wide variety of metabolites (I.ehninger, 1982). In order to can:y out these processes, however, cells nust maintain intracellular levels of both essential arrl nonessential amino acids. Non-essential amino acid levels are maintained by a variety of mechanisms which include transport fran the extracellular medium, de novo biosynthesis, interconversion of a-keto acids, arrl intracellular protein degradation. On the other harrl, the essential amino acids are supplied largely by intracellular protein degradation or by transport into the cell; however, the plasna membrane acts as a barrier to polar arrljor charged rrolecules. consequently, in order to acamul.ate amino acids fran the extracellular medium, rrost eukacyotic cells possess a series of plasna membrane carriers capable of selectively transporting amino acids. M:Jvenent of o:rganic solutes, such as amino acids, fran the extra to the intracellular environment necessitates their passage through the plasna membrane. Transport of amino acids across the plasna membrane

PAGE 14

2 ocx::urs by two distinct rout.es, either passive diffusion or facilitated uptake (protein-madiated). Passive diffusion is a nonprotein madiated process goven10d largely by the m:>lecular size arrl net dlazge of the solute (Collarrler, 1949). Flux across the nanbrane is goven10d largely by the direction of the solute's concentration gradient arrl does not saturate at high substrate concentrations. In contrast, protein madiated transport of amino acids ocx::urs via two nmes, designated as either facilitated transport or active transport. Facilitated transport is an energy-in:leperrlent processes by which solutes are transported in the direction of their concentration gradient. Net flux ceases at electrochemical equilibril.nn, resultirq in a distribution ratio of 1:1 for the amino acid with respect to the inside arrl outside of the cell (Houslay arrl Stanley, 1982). Facilitated carrier proteins show both stereo-specificity arrl saturability with respect to their substrates. Many energy-in:leperrlent amino acid transport systems denonstrate the property of trans-stimulation, i.e. the stimulation of a cis-to-trans unidirectional flux of labeled substrate brought about by the presence of unlabeled substrate at the trans-face. Cells possess two fonn.s of active transport designated as either primacy or secoroary. Primacy active transport by definition requires the hydrolysis of ATP; examples of this fo:rm of transport include the ion-deperrlent ATPases, such as the Na+~ -ATPase arrl the ca++ -ATPase. Secornary active transport is an energy-deperrlent processes which allows transport of organic solutes against their concentration gradient. Energy is usually 5UI=Plied in the fo:rm of an ion-driven gradient. In

PAGE 15

3 the case of hepatic amino acid transport, solute flow is coupled with that of Na+-ion flux, resulting in the sinultaneous transfer of one or ioore Na+-ions with an amino acid ioolecule (on atcms, especially when the side-chains

PAGE 16

4 were branchEd. N-methylation of IOC>St of the neutral amino acids restricted their uptake solely to System A (Orristensen et al. 1965) Hepatic System A activity is nonitored by the lOOdel amino acid substrate 2-aminoisd:Jutyric acid (AIB) Both AIB ani its N-methylated analog, 2-(methylamino) -isd:Jutyric acid (MeAIB), are not metabolized to a significant extent due primary to the lack of an al,a-hydrogen on the al:i;ila-carbon (Orristensen an:i Jones, 1962; Noall et al.~ 1957). 'Ihe carrier is also strorgly deperxient on Na+-ions; however, the hepatic System A carrier can accept Li+-ions as a replacerent for Na+-ions (Kilberg, 1982). System A is subjected to trans-inhibition by substrates, inhibited at Eif values below 7. O, an:i is controlled by both ho:rnones an:i substrate availability. 'lhe later process is known as adaptive regulation (Kilberg et al., 1982). 'Ihe adaptive regulation process was originally described in duck embryo heart cells (Gazzola et al., 1972) an:i diai;irragm muscle (Riggs an:i Pan, 1972), an:i nore recently in hepatocytes (Kelly an:i Potter, 1978) as well as the H4 hepatana cell line (Kilberg et al. 1985) In hepatic tissue, the substrate-deperxient regulation of System A consists of two distinct p,ases. 'Ihe first ,ase (tenned derepression) occurs when hepatocytes are stazved for extracellular amino acids an:i results in a stimulation of transport activity. 'lhe secorrl ,ase (tenned repression) results fran the addition of System A substrates to hepatocytes that have been previously cultured in the absence of amino acids; this results in a rapid decrease in System A activity. 'Ihe stimulation of transport activity which occurs durirg amino

PAGE 17

5 acid starvation is aa:x:npanied by an increasein the substrate translcx::ation rate (Vmax) by the carrier, without a dlan:Je in the awarer-it affinity of the carrier for substrates (Kelly et al., 1982). 'Ihe initial increase in transport activity occurs in:leperrlent of protein synthesis arrl has been ascribed to a release of the carrier fran transinhibition. Although the basis for trans-inhibition remains unresolved, it is thought to arise primarily fran the aocurrulation of active System A carriers in a cytosolic orientation due to increased intracellular levels of amino acids. Upon amino acid starvation, the intracellular amino acid concentration is lowered, thereby allowin;J nore active System A carriers to be oriented on the extracellular surface (i.e. the carriers are "released" fran trans-inhibition). Release fran transinhibition accounts for only a small increase in transport activity followin;J amino acid deprivation; the ma.jority of the enhanced transport activity, however, requires the synthesis of both RNA arrl protein (Kilbmg et al., 1985; Kelly arrl Potter, 1978). More recently, tunicamycin, an inhibitor of asparagine-linked glycoprotein biosynthesis, was shown to inhibit the imuction of System A activity upon amino acid deprivation of hepatccytes (Barber et al., 1983), suggestin;J the involvement of a glycoprotein in adaptive regulation. 'lhe secom p,ase of adaptive regulation is the reversal of the stimulation of transport activity. Sinply, inhibition of transport occurs when cells that have been previously starved for amino acids are placed in a meditnn containin;J System A substrates. '!his decay of transport activity can be imuced by the addition of a sin;Jle System A

PAGE 18

6 substrate (Harrllogten arrl Kilberg, 1984; Harrllogten et al., 1985; Bracy et al., 1986). For exanple, within 6 h after the addition of Ir asparagine to primary cultures of rat hepatocytes, System A-nmiated transport is lc:,,,,,ered to basal rates. 'lhe decay is dlaracterized by a half-ti.me of 1.5 h. Although the initial decrease in transport activity is protein synthesis-in:leperrlent arrl ascribed to trans-inhibition, the majority of the decay requires the synthesis of both RNA arrl protein arrl is considered to be the result of gene repression. System N3C. was originally described in the Ehrlich ascites cell (Cl'lristensen, 1967). Its activity was defined as that :portion of the Na+-depernent neutral amino acid uptake which is insensitive to the System A specific substrate MeAIB. A significant aIOOllllt of Ir-alanine, Ir-serine, am Ir-cysteine uptake by the Ehrlich cell was foum to be nmiated via System 'ASC.. '!he carrier prefers neutral amino acids with :polar side-chains containing oxygen or sulfur. System N3C. is not inhibited at pl values below 7, yet is subject to trans-stimulation by substrates. Although many of the dlaracteristics of System N3C. frc.m the Ehrlich cell are retained in the rat hepatocyte, Kilberg et al. (1979) foum that the Na+-deperrlent uptake of Ir-cysteine into rat hepatocytes was totally insensitive to MeAIB, suggestirg that all of the Na+deperrlent uptake of Ir-cysteine was nmiated by System 'ASC.. '!his is not the case, however, for the H4 (Kilberg et al., 1985) arrl the HI'C (Harrllogten et al., 1981) hepatana cell lines. Ir-threonine awears to represent a DK>re selective substrate for System N3C. in these transfonood liver cells. In contrast to the Ehrlich cell, however, the Na+-

PAGE 19

7 depen:lent uptake of AIB is not solely mediated by System A. In both freshly isolated hepatocytes {I.eCam arxi Freydlet, 1977) arxi saoo hepatana cells (Kilberg et al. 1985) the hepatic Na+ -deperrlent uptake of AIB has a small System ASC canponent. System L, a Na+ -i.meperrlent carrier, was dem:>nstrated by Oxen:ier arxi Cllristensen (1963) to prefer apolar amino acids, such as those with branched arxi aranatic side-chains, as well as Ir-histidine arxi Ir methionine. A decrease in affinity for amino acid substrates was fourrl to oocur sharply as the rn.nnber of carbon atans fell below five. System Lis dlaracterized by its high capacity for trans-stinulation arxi can be assayed selectively with the IOOdeled substrate BCH, 2-aminabicyclo (2,2,1)-heptane-2-carboxylic acid (Cllristensen et al., 1969). 'lhe activity of System L has also been dlaracterized in isolated hepatocytes (Ma;ivan arxi Bradford, 1977). Although the hepatic System L retains IOOSt of the characteristics first described in the Ehrlich cell, recent studies have denonstrated the presence of a previously urrletected Na+irrleperrlent system (Weissbach et al. 1982) 'lhe identification of this secorrl canponent system was based largely on kinetic analysis. In these studies, the initial rate of uptake of Ir-leucine arxi BCH was fourrl to be biiilasic in nature as a function of time in primary culture. 'lhe first canponent {Ll) has a high affinity for System L substrates arxi a low capacity for transport, whereas the secorrl canponent (L2) has a low affinity for substrates, yet a high capacity for transport. F\Jrthenoore, when adult rat hepatocytes were plac.ed into primary culture the activity of the L1 canponent increased durin;J time in culture,

PAGE 20

8 whereas the activity of the L2
PAGE 21

9 insensitive, was subject to trans-stinulation by substrate amino acids, am was inhibited by neutral amino acids when incubated in the presence of Na+-ions. Transport systems specific for the anionic amino acids have also been identified in the Ellrlich cell (Gazzola et al., 1981) am rat hepatocyte (Ballatori et al., 1986). In isolated rat hepatocytes, anionic amino acid uptake occurs largely via a Na+-deperrlent agency which is inhibitable by the nooel substrates Ircysteate am Ir cysteinesulfinate. '1he transport system has been designated as System X-A G am is subjected to trans-stimulation by substrate amino acids, possesses a low~ (0.016 nM) am is irrluced by dexairetbasone (Gebhardt am Mecke, 1983). In hepatic tissue, this carrier a:wears to be acccnpanied by a lower affinity system, specific for Ir-glutamate(~= 3.24 nM). A system for the transport of .B-alanine am taurine has been described for the Ellrlich cell (Olristensen, 1964). Hardison am Weiner (1980) have described .B-alanine transport in freshly isolated hepatocytes. In these studies, the uptake of .B-alanine was shown to be strorqly deperrlent on Na+-ions, subjected to trans-stimulation, am cati)etitively inhibited by taurine am hypotaurine.

PAGE 22

OIAPl'ER II MEMBRANE TRANS:roRI' MEIHOC6 Male Sprague-I:awley rats weighin:J 100-200 g were obtained fran a colony maintained by the University of Florida, Division of Animal Resoorces. '!he [methyl-3H]-2-aminoisotutyric acid, L[G-3H]-threonine, B-[3-3H(N)]-alanine am [methyl-14c]-3-0-methyl-D-glUCX>Se were fran ICN Rlarmaceuticals (I:rvine, ca). 'Ihe [l-14c]-pyruvate, L[G-3H]-glutamine, L[5(n)-3H]-arginine, L[2-3H]-glycine, [5,6-3H]-uridine am L[4,5-3H]leucine was ?J]'."Chased fran Amersham Co:rp (ArlinJton Heights, IL). 'Ihe unlabeled amino acids, protein-notifying reagents, Type I collagenase, am antibiotics were obtained fran Sigma Cllemical Co (St. I..a.ri.s, Mo). 'Ihe F.agles minimal essential medium (MEM) am fetal .bovine sennn (FBS) were ?J]'."Chased fran Flow laboratories (!tt::lean, Va) am the scintillation cocktail (No. 3a70B) was obtained fran Research Products International (Mt. Prospect, IL). Filters for the vesicle uptake studies were Gelman type GN-6 (0.45 micron). Tissue culture dishes am the 24 well cluster trays were fran Fisher Science (Orlamo, Fl). All other tissue culture suwlies were ?J]'."Chased fran Corning. Highly purified glucagon was a gift fran Dr. Ronald E. Olance of Eli Lilly laboratories. Hepatana Cell CUlture 'Ihe rat hepatana cells H4-II-EC3 (H4), Fao, Hl'C, am the human hepatana Hei2 were grown in 75 cm2 culture flasks (Falcon 3023, tissue culture flasks) at 370c urrler a humidified atJrosEOere of 5% ~95% air. 'Ihe hepatana tissue culture cell lines were maintained in 75 cm2 culture 10

PAGE 23

11 flasks in MEM, pi 7 4, supplemented with 25 l1'M NaHCD:3 2. 5 l1'M glutamine, 10 g/ml :penicillin, 5 g/ml streptanycin, 28.5 g/ml gentamicin, 0.2% bovine sennn alb.nni.n (BSA) arrl 5% FOO. Two to three days prior to the transp::>rt assays, the hepatana cells -were rinsed with (PBS) arrl rem:,ved fran the culture flasks by trypsinization (0.5 ml of a solution contai.nin;J 0.05% trypsin arrl 0.02% EIJI'A in PBS [10 l1'M scx:tium ~te, 150 l1'M NaCl, pi 7. 4] ) 'lhe cells -were then diluted with 100 to 150 ml of MEM contai.nin;J 5% FOO arrl transferred to 24-well cluster trays (Costar #3524, 24 well/16 mm well dia.) at a ratio of 4 to 6 trays contai.nin;J approximately 1 ml cells/well. sterile Na+ -contai.nin;J Krebs Ringer bicartx>nate (NaKRB) buffer supplemented with antibiotics was used as the amino acid-free (AAF) medium for the substrate starvation experiioonts. 'lhe crnposition of the Krebs-Ringer bicamonate is listed in AWerrlix I. 'lhe origin arrl characterization of the hepatana cell lines used in these studies is described below. 'lhe rat H4 hepatana cell line, H4-II -EC3 (Pitot et al., 1964), was derived fran the original Reuber H4 or Reuber H35 hepatana (Reuber, 1961) 'lhe Reuber H4 hepatana was obtained by feedirg rats a diet supplemented with N-2-fluorenyldiacetamide, which resulted in a bile secreting transplantable hepatocellular carcinana. 'lhe Fao hepatana is a well differentiated cloned cell line obtained after exposure of H4 hepatana cells to 8,-azaguanine (Deschatrette arrl Weiss, 1974). 'lhe Fao cell line expresses a rnnnber of liver specific proteins as well as the production arrl secretion of sennn alb.nni.n, arrl ho:nIDnal imuction of both tyrosine aminotransferase arrl alanine

PAGE 24

12 aminotransferase. Interestin;Jly, the Fao hepatana cell line does not contain readily detectable levels of alc:x:nol dehydrogenase, glucose-6-Jnospiate dehydrogenase or aldolase. 'lhe HK! hepatana cell line was derived originally fran feedin;J male buffalo rats a diet containin;J N,N'-2,7-fluorenylenebis-2,2,2-trifluoroacetamide ('Iharp;on et al., 1966) 'lhe h\.Illlan hepatana, H~2, was obtained fran liver biopsies of a male exhibitin;J primary hepatoblastana arrl hepatocellular carcinana (Aden et al., 1979). '!he H~2 is histologically a well differentiated parenchymal cell, capable of biosynthetically producin;J many liver specific proteins, includinJ ceruloplasmin, albJmin arrl transferrin (Knowles et al., 1980). Hepatocyte Isolation arrl CUlture Hepatocytes were isolated fran male Sprague-C8.wley rats (100-200 g) as described by Kilberg ( 1988) Briefly, rats were anesthetized by intraperitoneal injection of pentobart>ital (65 ngjkg body wt.). '!he right renal vein arrl inferior vena cava were then rapidly cannulated arrl retrograde perfusion was began at 3 ml/min. '!he perfusion solution was maintained at 37C arrl consisted of 25 nM sodium Jnosphate, Ii{ 7 .4, 3.1 nM potassium chloride, 119 nM sodium chloride; 5.5 nM glucose, arrl 5 ng/1 p.lellOl red. After perfusion was initiated, the hepatic artecy arrl portal vein were severed, the superior vena cava was clanp:rl with the aid of a hen:>stat arrl the p.mp speed was then increased to 10 ml/min. When liver was perfused with approxima.tely 100 ml of the perfusion solution, 75 units/ml of collagenase (Type I fran Sigma, C-01301) dissolved in 10 ml of perfusion buffer was added to the remainin;J 100 ml

PAGE 25

13 of perfusion buffer. Followi1~ perfusion, the liver was reroc,ved arrl placed in ice-cold NaKRB (30 ml). '!he liver was dispersed by gentle agitation arrl the cells separated by filtration through a 75 m nylon cloth. 'lhe mixture was then centrifuged at 100 g for 2 min. 'Ihe supernatant was discarded arrl the pellet oonta~ hepatocytes was resusperrled in 40 ml NaKRB arrl centrifuged at 100 g for 2 min ( 4 C) '!he washirg arrl centrifugation prcx::mure was repeated an additional three tilres. Hepatocyte viability was detennined by tcypan blue exclusion arrl was typically 85% to 90%. 'Ihe pellet conta~ hepatocytes was then diluted to 8 x 105 cells/ml with wann culture nmium (MEM or NaKRB) arrl the cells 'iN'ere placed (0.33 ml/well) into the bottan of 24-well cluster trays which had been previously coated with collagen. Collagen coat~ of the cluster trays was perfonned by a~ 0.5 ml of a stock solution consist~ of 20 g/ml sterile acid-soluble collagen (Sigma Type III, C-3511) to each well. After 12 h, the solution was rerroved arrl the trays stored at roan te.nq:>erature. 'Ihe stock solution of collagen was made by dissolv~ the collagen in 0.5% acetic acid ( 1 rrg collagen/ml) 'Ihe hepatocytes 'iN'ere maintained in an incubator at 37C with a humidified atJoosiiiere of 5% cn2 arrl 95% air. Detennination of Intracellular Water Voltnne Intracellular water was ireasured by the 3-0-methyl-D-glucose method of Kl.etzien et al. (1975). For exanple, H4 hepatana cells 'iN'ere placed in 24-well cluster trays conta~ MEM arrl 5% FBS for two days as described above. 'Ihe nmium was reroc,ved by rins~ the cells with NaKRP buffer (Na+-conta~ Krebs-Ringer i:nosp1ate buffer, AWerrlix I) arrl

PAGE 26

14 then placed in 1 ml of NaKRP buffer (37"C) CX>ntainin;J vacyin:J anounts of unlabeled 3-0-methyl-D-glucose (3M;) at concentrations of 1 ITM, 5 ITM, an:i 10 ITM. 'lhese 3M; solutions also CXllltained a trace anount (3.5 rnrole) of [methy1-14c]-3K; (S.A. = 40 Ci/nole). 'lhe 3M; is used to nonitor the glucose carrier, because it is not metabolized to a significant degree by cells (Stein, 1986). 'lhe cells -were then incubated with the 3M; solution for 1. 5 h. '!his incubation period was intemed to allow the 3M; to equilibrate across the plasma nanbrane; the cells -were then washed quickly four times with 2 ml of ice-CX>ld Olollrx:::entration is equivalent to the extracellular CX>rx:::entration at equilibrium. 'lherefore, by detenninin:J the rnnnber of noles of 3M; intracellularly per ng protein at a given extracellular CX>rx:::entration the intracellular water content per ng protein can be ascertained. Whole Cell Transoort Assay Transport of whole cells was measured by a notification (Kilberg et al., 1988) of the method originally described by Gazzola et al. (1981). Briefly, cells plated in 24-'.Nell cluster trays -were incubated in Ololl
PAGE 27

15 minimize possible trans~ffects due to intracellular Na+-ions am substrate amino acids. To initiate transport, the OlolKRP ruffer was rercoved am the awrcpriate radioactive amino acid solution in NaKRP or OlolKRP ruffer was added. 'lhe carp'.)Sition of the radiolabeled uptake ruffers is described in AWerrlix I. Exposure of the cells to the uptake solution was acx:x:mplished simultaneously for all wells by using the canplementary fitting lid to the 24-well cluster trays. 'lhe lids were m:xtified so that they contained Beem embedding capsules (#00). Within 30-60 sec the radioactive solution was discarded by irwerting the cluster tray am the cells washed four tines with 2 ml/wash of ice-cold OlolKRP ruffer (5 sec/wash) 'lhis was also acx:x:mplished simultaneously for all wells by m:xtifying the canplementary fitting lids with 12 x 75 mm plastic test tubes. After canpletion of the transport assay, the hepatocytes were solubilized by the additional of 0.2 ml of a solution containing 0~2 N NaOH am 0.2 % soo. Within 30 min, the anount of radiolabeled amino acid was detennined by rercoving o .1 ml of this solution am adding it to 3 ml of scintillation fluid containing 0.1 ml of O. 2 N HCl. To estimate the anount of protein in each well a m:xtification of the I1:Mry method was -ercployed (Kil.bag et al. 1983) For the hepatocytes, O. 6 ml of a copper reagent solution containing o. 58 nM EDI'A (copper-disoditnn), 189 nM Na2COJ, 100 nM NaOH, am 1% soo was added to the remaining 0.1 ml solubilized material. After 10 min, 60 1 of a Folin-Ciocalteau reagent (diluted 1:1) was added to each well. 'lhis mixture was then incubated at roan tercperature (30 min) am the

PAGE 28

16 abso:rbance along with a bovine serum albumin st:an:Jard curve was neasured at 750 nm. Upon carpletion of transport assays with the hepatana cell lines, 0.22 ml of a solution containing 10 % trichloroacetic acid ('ff:A) was added to each well. '!he trays were then incubated for 1 h at 4 C. 'lb estimate the ann.mt of radiolabeled amino acid traR)d within the cells, approximately 0.2 ml of this solution was rerroved am added to 3 ml of scintillation fluid. Radioactivity for all cell types was detennined by scintillation spectrcplotametry. 'lb estimate the ann.mt of protein in the wells following the rerroval of the 'ff:A lysate, 0.1 ml of the 0.2 N NaOO am 0.2% soo solution was added. After 15 min, the protein concentration was assayed as described for the hepatocytes. Iata Analysis 'lhe Na+-deperx:lent transport was taken as the difference in the uptake rate of labeled amino acid observed in the presence of Na+containing Krebs-Ringer J;ilosiilate ruffer (NaKRP) am in the absence of Na+ (OiolKRP). 'lhe saturable Na+-irrleperrlent transport was taken as the difference in the uptake rate of labeled amino acid observed in CllolKRP ruffer am in CllolKRP ruffer containing 10 nM of the ai:p:rq:,riate unlabeled amino acid. 'lhe transport data were calculated am analyzed with the aid of a micrc:x::x::mp.rt utilizing ccmp.rter programs that incorporated st:an:Jard statistical analyses. 'lhe transport kinetic paran-eters were calculated with FORl'RAN programs that estimated am subtracted the basal activity. '!he corrected data were then analyzed by a non-linear least squares method (Clelam, 1979). Unless otherwise

PAGE 29

17 irrlicated, the results for all whole cell experiments are reported as the averages ( the stamard deviations, S.D.) of 3 to 4 detenninations for a sin;Jle pop..uation of cells. Nearly all experiments were repeated usin;J different preparations of cells. Olemical M:xtification of Plasma Membrane Proteins in Whole Cells Cells were plated an:l cultured in 24;.iell cluster trays containin;J MEM an:l 5% FBS as described above. 'lhe cells were then rinsed twice with NaI or NaI
PAGE 30

18 measured (Chiles am Kilberg, 1986). For those experinents that enployed organic IOOrCUrials as protein m::xlification reagents, the buffer solution was suwlemented with an equal-nnlar concentration of EIJI'A. '!his precaution was inten::led to chelate any free mercury. 'Ihe arramt of transport activity protected by an amino acid durir)3' exposure of the cells to protein reagent was calculated by substitutir)3' the awrc.priate transport velocity into the followir)3' equations: % Inactivation= [ (V-Vi)/(V)] x 100 % Protection= [ (Vaa+i-Vi)/(Vaa-Vi)] x 100 Plasma Membrane Vesicle Isolation from CUltured Cells Plasma membrane-enriched vesicles were prepared from H4 hepa:tana cells by culturir)3' the cells in 150 nun x 25 mn Falcon 3025 tissue culture dishes in MEM am 5% FBS (rudeck et al. 1987) About 6 to 8 h prior to the membrane isolation, the cells were incubated in NaKRB to increase the arramt of iooasurable System A activity by adaptive regulation (Kilberg et al. 1985) 'Ihe cells were then rinsed twice with Buffer A (0.25 M sucrose, 0.2 nM M:;JC12 10 nM HEPES-KOH, Eif 7 .5), scraped from the culture dishes with a rubber policeman into 15 ml of Buffer A, am then collected by centrifugation at 3,000 g for 5 min (4C). 'Ihe cell pellets from 16-20 dishes were canbined, resusperrled in approximately 20 ml Buffer A containir)3' 1 nM EIJI'A, 1 nM }.ilenylmethanesulfonyl floride (R-EF) am 5 nM benzamidine (4C) am disrupted with a Potter-Elvehjem haoogenizer usir)3' a tight-fittir)3' pestle (100 to 125 nntor-driven strokes at 930 :rpn). 'Ihe haoogenate was

PAGE 31

19 then centrifuged for 10 min at 500 g to removed unbroken cells am nuclei am the resultirg supernatant was centrifuged (30 min at 39,000 g) to obtain a pellet containirg plasma membrane-enridled vesicles. '!he final vesicle preparation was stored in 50 1 aliquots at a protein concentration of approximately 10 ng/ml in Buffer A (-700c). 'lb minimize the loss of transport activity, eadl aliquot was tha'W'Erl only once. Plasma Membrane Vesicle Isolation from Nonnal Rat Liver Nonnal hepatocyte plasma membrane vesicles were isolated from intact rat liver tissue as described by Pl:pic' et al. (1984). In order to increase the measurable System A transport activity in the resultirg rat liver vesicles, the rats were injected with 1 ng of glucagon per 100 gm body weight 5 h prior to membrane isolation (Harrllogten am Kilberg, 1984). In brief, male Sprague-0:iwley rats, weighirg 100-200 g were anesthetized am the liver perfused as described above with the exception that the liver was si.nply blanched free of blcx:x:l with ice-cold PBS. Followirg perfusion, the liver was removed, weighed, am placed in an equal volume (w/v) of ice-cold Buffer B (0.25 M sucrose, 10 TIM Trisbase, pl 7. 5) containirg 1 TIM EDI'A. '!he liver was then minced am hatOJenized by ham usirg a glass rnmce hatOJenizer (10 strokes with a loose-fittirg pestle followed byan additional 4 strokes with a tight fittirg pestle) '!he hatOJenate was diluted to 6% (w/v) with Buffer B containirg 1 TIM EDI'A, the weight (grams) was based on the original anount of tissue. '!he mixture was then centrifuged at 120 g for 2 min ( 4 C) to remove unbroken cells am nuclei. '!he supernatant was

PAGE 32

20 centrifuged at 1500 g for 10 min (4"C) to obtain a :pellet enriched for plasma membrane. 'Ibis pellet was resusperrled in a final volume of 30 ml (Buffer B/1 nM EDI'A), filtered thra.lgh dleese-cloth; diluted with Buffer B/1 nM EDl'A to 31.2 ml an1 then added to Percell (Sigma, P-1644) to give a final volume of 35. 4 ml. '!he solution was mixed with the aid of a glass rod an1 11. 8 ml was transferred into each of three 15 ml Corex tubes. '!he membranes were centrifuged at 34,500 g for 30 min (4C). After carefully reJOOVin;J the top lipid-containin;J layer, the plasma membranes were reJOOVed (first bani of membranes um.er the lipid layer), diluted 1:6 (v/v) with Buffer B an1 centrifuged at 34,500 g for 30 min ( 4 C) 'lhe final plasma membrane-enriched :pellet was resusperrled in Buffer Bat a concentration of approximately 10 rrg/ml an1 stored in 50 1 aliquots at -700c. To minimize the loss of transport activity, each aliquot was thawed only once. Transport 'Assay for Vesicles System A-nmiated transport by either membrane vesicles or reconstituted proteoliposanes was assayed as described by Bracy et al. (1987). Briefly, 20 1 (50 g) of membrane at 40c was added to an equal volume of a 2X uptake buffer (200 nM of either KCl or NaCl, 10 nM M3C12 an1 10 nM HEPES-KOH, Iii 7 .5, 370c) containin;J 0.4 nM of AIB an1 [3H]-AIB (1.0 Ci/ml). 'lherefore, the final canposition of the uptake mixture was 0.125 M sucrose, 5.1 nM M3C12 10 nM HEPES-KOH, Iii 7.5, 100 nM NaCl or KCl, an1 0.2 nM [3H]-AIB. 'lhe vesicles or proteoliposanes were incubated with the uptake mixture for 1 min in a water bath at 220c. Transport was tenninated by the addition of 1 ml of ice-cold step-buffer

PAGE 33

21 (125 ITM NaCl, 0.2 ITM M;JC12 10 ITM HEP.ES-KOH, pi 7.5). 'lhe mixture was rapidly vortexed am filtered over a 0.45 m nitrocellulose filter. '!he filter was rinsed twice with 4 ml of ice-cold stq>-ruffer am then the trawed radioactivity :re.asured by scintillation spectrcnetcy. Chemical M:xtification of Membrane Vesicles Treatment of isolated mernbrane vesicles with protein-m:x:lifyi.rg reagents was perfonned at 220c for 10 min. Typically, 400 g of membrane protein was resuspen1ed in Buffer A am irx::ubated with the protein-m:x:lifyi.rg reagent at the in:licated concentration ( 10 min) 'lhe final volume am protein concentration duri.rg treatlrent was o .15 ml am 2. 7 ng protein/ml, respectively. '!he mernbranes -were centrifuged in a Becknm1 airfuge at 150, ooo g for 5 min. '!he supernatant was decanted am the mernbrane pellet was rinsed with o .1 ml of Buffer A witha.zt resuspension. Followi.rg another centrifugation, the membrane pellet was resuspen:led in Buffer A at a protein concentration of 2. 5 rrg/ml. System A-madiated uptake was ilmnediately assayed as described above. Modification am Reconstitution of Solubilized Membrane Proteins Plasma mernbrane proteins fran either rat liver or H4 hepatana cells, at a concentration of 1.5 ng/ml in Buffer A, -were solubilized by diluti.rg the mernbrane to 0.5 rrg/ml (1:2) with solubilization-ruffer (0.1 ITM EDI'A, 100 ITM KCl, 1 M FMSF, 2.5% cholate/4 M urea [Pierce, sequanal grade] am 5 ITM HEP.ES-KOH, pi 7 .5). 'lhe insoluble nenbrane material was reooved by centrifugation at 100,000 g for 30 min. 'lhe solubilized membrane proteins -were irx::ubated in the presence of 1 ITM NEM for 10 min at roan terrperature am then the reaction was st:q:prl by the addition of

PAGE 34

22 2 nM D-cysteine. For the control incubations, 1 nM KCl replaced the NEM but the 2 nM D-cysteine was ack:led as usual. 'lhe solubilized proteins were precipitated. by the ack:lition of p::,lyethylene glycol (M.W. = 8,000) as described by Gal et al. ( 1983) 'Ihe precipitated. proteins were recx>nstituted. into artificial proteoliposanes by the mathod of Bracy et al. (1987). A stock solution (40 ng/ml) of asolectin {Asscx::iated. Concentrates, BA #1267 soybean phosinolipid) was prepared by susperrlin;J the lipid in :i<+"-containirg uptake-buffer arrl sonicatin:J the mixture in a bath-type sonicator until a clear solution was obtained (awroximately 10-15 min). Reconstitution of system A activity was perfonned by mixirg 1 ng of solubilized membrane proteins, 20 ng of sonicated. asolectin, arrl 1 ng of potassimn cho late (the stock solution of twice-recrystallized cholate was 10% w/v ) 'lhe mixture, awroximately 1 ml total voltnne, was frozen in liquid nitrogen, thawed at roan tercperature,arrl then diluted. with 4 ml of 2X :i<+" -containirg uptake-buffer. 'lhe mixture was then sonicated. for 20 sec in a bath-type sonicator. 'lhe proteoliposanes were pelleted. by centrifugation at 125,000 g for 45 min at 40c arrl then resuspe.rrled in 200 1 of Buffer A with gentle vortexin:J.

PAGE 35

CliAPI'ER III DIFFERENTIAL SENSITIVITY OF AMINO ACID 'IW\NSroRI' SYSTEMS 'IO SULFHYCRYI.rMIDIFYING RF.AGENI'S Intrcx:luction Amino acid uptake by animal cells is madiated by several distinct transport systems with overlappin;J specificities. To date, the infonnation concernirq the twelve or llX)re carriers described for amino acid transport has been largely descriptive. '!he basic characterization of transport systems includes infonnation on the ion~eperrlency, substrate specificity, J;.il-sensitivity, trans-effects am regulation, if any, by ho:nrones or substrate availability (Kilberg, 1982). At the llX)lecular level far less is known about iniividual amino acid transport systems. None of the membrane proteins responsible for the hepatic uptake of amino acids has been identified or isolated. In general, proteins that catalyze ion-coupled transport of organic solutes have rema.ined refract.my to llX)lecular characterization. '!his is due mainly to the lack of specific affinity probes am the necessity to employ detergents durin;J isolation am purification. Detergents sanetilres irreversibly inactivate carrier function to an extent that the only functional assay, reconstitution, is nonfunctional. Recent studies by Hayes am Mc:Gi van ( 1983) have provided sane evidence for a protein involved in Na+-deperrlent Ir-alanine transport into isolated rat hepatic plasma membranes vesicles. 'lhese authors 23

PAGE 36

24 deoonstrated that awroximately 50% of the total Na+~ uptake of It-alanine into hepatic meJnbrane vesicles could be inactivated by 1 nM NEM, suggestin:J the necessity of free sulfhydcyl(s) rane. Preincubation of the vesicle pcp.il.ation with 2 nM It-alanine prior to am durin:J the NEM treaOlelt reduced the inactivation by 21%, leadin:J the authors to speculate that NEM was inactivatin:J an It-alanine carrier protein. When vesicles were incubated with radiolabeled NEM in the presence or absence of It-alanine am the labelled proteins analyzed by soditnn-dodecyl sulfate polyaccylamide gel electrq:noresis (Sr::6-PAGE), six major proteins were fourxl to be covalently roodified by racliolabeled NEM. In contrast, when the same vesicle pcp.il.ation was treated with unlabeled NEM am It-alanine, washed to reroc,ve the excess NEM am alanine, am then incubated with racliolabeled NEM, one major protein of m::>lecular weight 20,000 daltons was labeled. Unfortunately, the authors e.nployed L alanine as the substrate amino acid in these protection studies, because It-alanine is transported in rat liver by two different Na+-deperrlent agencies, System A am ASC., as well as the Na+-irx:ieperrlent System L, it is unclear whidl carrier protein was labeled. Recently the Na+-depement L-proline carrier (i.mino carrier) of intestinal brush border nembranes was identified am dlaracterized with respect to possible amino acids involved in substrate birrlirq usin:J rrethods similar to that of Hayes am :t-k:Givan (1983). Wright am Peerce ( 1984) were able to take advantage of two related primacy amino group roodifyin:J reagents, fluorescein isothiocyanate (F:m:) am pienyl

PAGE 37

25 isothiocyanate (P~) 'Ibey foun:l that the Na+ -deperrlent uptake of Ir proline was equally sensitive to both FITC am~ am in the presence of sodium, Irproline protected the carrier fran inactivation. Specific labelin;J by FITC was detected after first nolifyin;J unrelated proteins with P~ in the presence of sodium am I.r-proline, the meni:>ranes were then washed free of unJ::x:mn P~ am then in::ubated with FITC. '!he proteins were analyzed by sr:s-PAGE, scanned with a fluorescence detector, am a FITC labeled polypeptide of 100,000 daltons was detected. '!he authors further characterized the carrier with respect to its Na+-bintin:J site(s). In this respect, the authors detected specific confonnational manges upon alkali ion binlin:J, which were blocked by N acetylimidazole nolification of the carrier. '!his i;i1ase of my research was directed towards addressin;J questions concenrirg the dlenical prq>erties of the hepatic System A protein. '!he experinents disa.is.sed below employed the use of protein nolifyin;J reagents to detennine whidl dlenical reactive groups present on the carrier, resulted in a loss of transport activity 'When nolified. 'Ihe prq>erties of System A with respect to protein nolifyin;J reagents was also cx::npared between nonnal am transformed liver tissue. Materials am Methods See the transport method procedures aitl.ined in Cbapt:er II. Results In preliminary studies the sensitivity of System A-mediated transport, as nonitored by assayin;J the Na+ -deperrlent uptake of AIB, toward a host of group-specific protein-m:xtifyin;J reagents was tested.

PAGE 38

26 Table 1. Sensitivity of System A Activity in Normal Rat Hepatocytes and H4 Hepatoma Cells to Protein-Modifying Reagents Inhibitor None Acetic anhydride SUccinic anhydride Chloroacetate Iodoacetate Iodoaoetamide N-ethyl.male:unide P-chloratero.1ril:lenzene sulfonate P-chloratero.1ril:lenzoate Fluoresoein isothioc:yanate Rlenyl isothioc:yanate N-acetyl:unidazole N-bran::isucx::inimide Trini tra:ienzene sul fonate Rlenylglyoxal Ethoxyformic anhydride Hepatocytes H4 Hepatana Cells 100 100 89 116 66 86 0 31 30 124 101 106 109 75 157 77 ND Percent of Control 100 85 109 121 134 96 99 30 17 106 85 78 0 112 130 ND 7 Hepatocytes or H4 hepatana cells were o.lltured in an amino acid-free tredium (Nal
PAGE 39

27 A variety of protein-m::xtifying reagents examined so as to prooe for several ftmctional amino acid side-dlains present in proteins (Table 1). A list of all the protein m::xtifying reagents use in these studies arrl their chemical reactivity is listed in AWerrlix VI. '!he cell lines used for these studies~ nonnal rat liver tissue (freshly isolated hepatocytes) arrl a rat liver-derived ttntor cell line, H4-II-EX:3 (H4). Nonna! hepatocytes arrl the H4 hepatana cell line dlosen in order to caipare the sensitivity of System A between nonnal arrl transfo:rmed liver tissue. Many laboratories have reported distinct chanJes which ocx::ur in neutral amino acid transport following transfonnation (Harrllogten et al., 1981; Kelly arrl Potter, 1979). 'lhus, it was of interest to detennine if similar chanJes ocx::urred with respect to chemical m::xtification. Reagents that show preferential reactivity with amino groups includin;J fluorescein isothiocyanate (FTIC), i;::henylisothiocyanate (PITC), (Edman, 1950), arrl trinitrooenzene sulfonate, '!NBS (Kotaki et al., 1964), produced only nodest chanJes relative to control cells not exposed to the reagent. 'lhese initial studies perfo:rmed in the presence of Na+-ions (i.e., NaKRP ruffer), so the lack of inhibition by FTff: arrl PITC may have resulted either fran protection of essential lysyl-amino graip(s) by Na+-ions or fran the low Iii enployed. Wright arrl Peerce (1984) obsel:ved that inactivation by PITC of the imino carrier was blocked by the presence of Na+-ions. In reference to the Iii effects, these protein-nodifying reagents are tha.lght to react with primary amino groups at an alkaline Iii I was reluctant to test their

PAGE 40

28 Table 2. Sensitivity of System A Activity in Normal Rat Liver and H4 Hepatoma Veicles to Phenyl Isothiocyanate Cooditia, Hepatocytes H4 Hepatana caitrol ( 1% CMF) 1151 52 954 36 10 11N Ir-norleuc:ine 1080 192 885 123 l JIM~ 45 4b 209 ab 10 JIM Ir-norleuc:ine +lnN~ 7 4b 215 13b Mentlrane vesicles 'iil'E!r8 incubated with 0,25 M sucrose, 10 JIM Tris, pi 9.2, 100 JIM NaCl containirq 1 JIM in the presen::e or absenoe of 10 rrM Ir-norleucine. 'lhe PITC was dissolved in dimethylformamide (I:MF) such that the final percentage of I:MF exposes to the ire.nt,rane vesicles was 1%. After 20 min the ment>ranes were centrifi..xJed at 150,000 g for 5 min, the supeznatant was discarded, arrl the ire.nt,rane pellet washed arrl resusperrle::l in 8.Lffer A. System A activity was then measured by m::>ni.tori.ng the Na+deperdent uptake of 200 M (~L-t,m for 1 mii!1at 37c. '!he data are expressed as the velocity of AIB uptake (pool ng protein min ) arrl represent the averages standard deviations of foor determinatia,s, nus experiment was performed with the assistan::.e of Rathleen OJdeck-COllart. bstatistically different fran the control values at the P<0.005 level.

PAGE 41

29 effects at pi values above 8, because damage to the integrity of the plasma meni>rane or cell viability might occur, thereby resultin:J in considerable non-specific inhibition of Na+-clepenjent System A transport. When the System A activity in rat liver or H4 hepatana plasma meni>ranes vesicles was assayed followin:J treatment with 1 nM PI'IC (pi 9.2), transport was inhibited awroximately 96% am 78%, respectively (Table 2). N-aoetylim.idazole is an acylatin:J reagent that reacts with both amino arrl tyrosyl groups; however, the reaction with tyrosine has been reported to be nore selective (Riodan arrl Vallee, 1963; Riodan et al., 1965). Inactivation of the Na+-depernent Ir-proline carrier has been reported to occur upon treatrcent of brush border meni>ranes with N acetylim.idazole (Wright arrl Peerce, 1984). AlthcA.1gh this reagent decreased System A activity by 22% in the H4 hepatana cell line, both cell types "Were largely resistent (Table 1). Fhenylglyoxalic acid, a specific reagent for guanidine arrl tenninal amino groups (Takahashi, 1968), actually caused an increase in the d:>se:rved System A activity in both cell types. Ethoxyfonnic anhydride (diethylpyrocarbonate), an im.idazole specific reagent (Miles, 1977) produced a small decrease in System A--naiiated transport (23%) in the nonnal hepatocytes (Table 1). 'lhe activity of both nonnal hepatocytes arrl H4 hepatana cells derronstrated a similar degree of sensitivity to the sulfhydrylpreferrin:J reagents p-dll.orooercuribenzoate (I:om) arrl p dll.orooercuribenzene sulfonate (f0fi3S). Inhibition rarged frcm 70% to 83% for the Na+-clepenjent uptake of AIB. In contrast, the sulfhydryl-

PAGE 42

Figure 1. Concentration Deperxience of the Inhibition of system A Activity in Nomal. Rat Hepatocytes am Several Hepatana Cell Lines by NEM or IOmS. '!he cells 'W'ere incubated in an amino acid-free medium (NaKRB) for 5 h to enhance System A transport activity by adaptive regulation am 'W'ere then transferred to NaKRP contai.nirg IOmS ( ) or NEM ( ) for 10 min at 37C. 'lhe inhibitor corx::entration was varied between o. 025 am 1. o :rrM. After ri.n.sinJ the cells with Ololl
PAGE 43

100 75 50 0 .. 25 .. c 0 V ... 0 .. C G.> 100 .. G.> 7S so 2S Hepatocyte e PCMBS ANEM K1,. = 55:26uM 2 K 1,. :46:19uM 2 H4 2 4 6 8 10 31 HTC FAO K 1,: =40:t4uM 2 2 4 6 8 10 Onhibitor], uM (xl0.2 ) HepG2 2 4 6 8 10

PAGE 44

32 preferrin:J reagent NEM produced carplete inhibition of System A in the hepatocytes, while the correspc:niin;J transport activity in H4 hepatana cells was resistant to the inhibitor (Table 1). '!he stron;J inactivation of System A in hepatocytes by NEM, but not by iodoacetate, is in agreement with ooservations by others (Kilberg et al., 1980; Hayes arrl M:Givan, 1983; Sips arrl van O:nn, 1981) Further discrimination of the System A activity present between these two cell types was achieved through the use of N-braoosuccin.imide (NBS); this reagent abolished AIB uptake in the H4 hepatana cells, but was largely ineffective as an inhibitor of the activity in nonnal. hepatocytes. Altha.igh NBS is known to cleave :peptide boms at trypt:qi'lan, tyrosine, arrl histidine residues, oxidation of sulfhydcyl residues is reported to occur nore rapidly (Fontana, 1972). Methionine arrl cystine are also subject to oxidation by NBS (Means arrl Feeney, 1971). Regardless of the medlanisms involved, there is a clear difference in the reactivity of the System A carrier in hepatocytes arrl H4 hepatana cells treated with either NEM or NBS .Additional experiments were perfo:rmed to ascertain whether the resistance to NEM by the H4 hepatana cells was a unique prq:>erty of that hepatana cell line or a general characteristic shared by other liverderived turoor cells. In this study a variety of hepatana cell lines i.ncludirg both hmnan (He;2) arrl rat (Fao, me, arrl H4) were examined for sensitivity of System A to inactivation by NEM or PCMBS (Chiles arrl Kilberg, 1986). 'Ihe concentration-deperrlence of inactivation was nonitored usin:J concentrations of reagent ran;Jm:J fran 0.025 nM to 1.0 nM. PCMBS treatment was foon:i to decrease System A-mediated transport

PAGE 45

33 by 70% to 80% in all cell types examined; concentrations that produced half-maxilnal. inhibition (for ease of disaJSSion this value will be referred to as is_12) ran:Jed fran 40 to 119 M (Fig. 1). NEM-depement inactivation of System A in the hepatocytes was nearly carplete. Greater than 95% of the total Na+ -deperrlent AIB uptake was inhibited relative to non-treated cells with is_12 of 46 19 M. In a::mparison, the kinetics am extent of inactivation in the hepatana cell lines was strikirqly different. For exanple, the Hl'C-associated System A activity was inhibited by NEM, yet required concentrations of 1 n'M before the degree of inhibition was c:x:mparable to that cx:,served in nonnal hepatocytes. Although the level of NEM required to produce half maximal inhibition in the rat Fao am hmnan Hep:;2 hepatanas was relatively lCM cis_12 = 53 20 Mam 271 32 M, respectively), less than 50% of the total Na+ -deperrlent transport was inactivated at the m::st effective concentrations of inhibitor. Consistently, the System A activity in the H4 hepatana cells was largely unaffected by NEM at mncentrations exceeding 1 n'M (Fig. 1). '1he saturable inhibition of only 40% to 50% of the total Na+deperrlent AIB uptake by NEM in the Fao am Hep:;2 hepatana cells raised the :(X)SSibility that additional routes for Na+-depenient AIB uptake might exist in these transfo:rm:d liver cell lines. Saturable inactivation of only 40% to 50% of carrier activity has been reported for a limited rnnnber of transport systems. For exanple, the sensitivity of rnICleoside transport in rat ecythrccytes has been investigated using PCMBS (Jarvis am Ya.IDJ, 1986). PCMBS produced a concentration-

PAGE 46

34 depement inhibition of uridine uptake, yet inactivation of transport readied a plateau of 50% at 0.1 nM PCMBS. No further inhibition of transport was observed usinJ concentrations of PCMBS in excess of 1.0 nM. Detailed analysis later revealed that uridine transport into the rat ecythrocytes occurred via two distinct rart:es. One cacponent was highly sensitive to nitrd:>enzylthioinosine (NIMPR), an active site inhibitor of uridine transport. '1his cc:nponent exhibited a for uridine uptake of 163 18 M, whereas the other cc:up:>nent was NIMPR insensitive possessinJ a for uridine of 50 18 M. 'lhe authors observed that the NIMPR-insensitive portion of uridine uptake was inactivated by PCMBS, whereas the NIMPR-sensitive catpOnent was unaffected. A sanewhat similar observation has been seen for Na+-:imepen:lent uptake of Irleucine. Irleucine transport into Olan;J liver cells can be resolved into two cacponents. One cx:uponent possess a low ( 44 M) for Irleucine uptake, whereas the other cc:nponent has a of 8.0 nM. Takadera am Mdlri (1982; 1983) reported that NEM actually stinulated Ir leucine uptake (2-fold) via the high-affinity system. In ce>ntra.st, transport-mediated by the low-affinity cc:nponent was actually decreased 2-fold. Based on these observations arrl the results of Fig. 1, AIB uptake was tested for inhibition by the System A-specific substrate, ~B (Olristen.sen et al., 1965). ~tionally, the portion of the Na+depement AIB uptake that is inhibited by 2-(methylamino)-isaaltyric acid (~B) is assumed to be mediated by System A; any remaininJ Na+-

PAGE 47

Figure 2. Me.AIB Inhibition of Na+-Deperrlent AIB Tran.sport in Several Hepatana Cells Lines. Hepatana cells were incubated in an amino acidfree na:tium (NaKRB) for 5 h to stinulate System A activity by adaptive regulation an.:l were then rinsed with Olollncentrations of unlabeled Me.AIB (O.Ol to 20.0 n'M). '!he results are reported as the peraent of the transport rate in the absence of Me.AIB. '!he control velocities (averages S:..'fl: of 3 ~~tions) were 434, 673, 514 an.:l 409 prol ng protein min for the rat H4 ( ) me ( ) Fao ( ) an.:l human HepG2 ( ) hepatana cells, respectively. '!he Ki values were calculated by catp.Iter analysis as described by Clelan.:l (1979).

PAGE 48

36 100 A HepG2 Ki:0.4~0.07 mM FAO Ki :0.4!0.04 mM 0 80 I. HTC Ki :0.1 tO.O 1 mM .. C y H4 Ki=0.2!0.04 mM 0 u 60 0 .. 40 C Q) I. Q) 20 A. 0 0 4 8 12 16 20 ~eAI~, mM

PAGE 49

3 7 depement AIB transport is na:liated by System ASC. Al though AIB uptake is largely restricted to System A, it has been reported that System A5C can contribute significantly to the uptake of AIB in hepatocytes (I.eCam arrl Freydlet, 1977) arrl H4 hepatana aalls (Kilberg et al., 1985) when the activity of System A is fully repressed by substrates. In contrast, when the activity of System A is stinulated by substrate stai:vation i.e., adaptive regulation, the relative contrili.rtion of System A5C diminishes rapidly. 'lherefore, the hepatana cells -were incubated in amino-acid free na:lium for 5 h to enhance both the ano.mt of iooasurable System A activity arrl to diminish any contrili.rtion by System ASC; these incubation corrlitions oorresp::>rrl to those used to obtain the data shown in Fig. 1. As seen in Fig. 2, nearly all of the Na+ -deperrlent AIB uptake was inhibited by an excess of MeAIB in the Fao arrl HIC hepatanas with Ki values of 400 40 M arrl 110 10 M, respectively. Greater than 85% of the Na+ -deperrlent uptake of AIB in the IieEG2 arrl the H4 hepatana aalls was inhibited by MeAIB (Ki = 400 70 M arrl 200 40 M, respectively). 'lhe st.nnJ inhibition by MeAIB denalstrates that System A na:liates nearly all of the Na+-depernent AIB uptake in all four hepatana aall lines. 'lhe results irrlicate that the saturable rut incacplete inhibition by NEM in both the Fao arrl HeEG2 aalls does not appear to represent selective inactivation of one of nultiple rcutes for AIB transport. Rather, the data suggest two possibilities: First, only a subset of the System A carrier proteins in these cells is sensitive to NEM (i.e., the sensitivity may be restricted to those carriers which -were synthesized durinJ adaptive regulation, whereas the basal carriers

PAGE 50

Figure 3. Corx::entration Deperrlence of NEM Inhibition of System A Activity in Fao Hepatana Cells CUltured in Amin:> Acid-Ridl Medimn. 'Ihe Fao oells incubated in an amino acid-ridl medimn (MEM) for 24 h to decrease the neasurable ann.mt of System A transport activity. 'Ihe oells then transferred to NaKRP containirg NEM for 10 min at 37C. 'Ihe inhibitor concentration was varied between 0.01 arrl. 1.0 nM. After rin.sin:J the oells twice with CholKRP (37C), System A activity was assayed by neasurin:J the Na+ -depeooent uptake of 50 M [ JH]-AIB for 1 min at 37C. 'Ihe results are reported as the percent of the rate 2{ transport i!!1the non-treated oells havin:J a velocity of 39.4 pwl ng protein min (averages s.o. of 4 detenninations). 'Ihe K, 12 value was calculated by catp.Iter analysis as described by Clelarrl. (1979).

PAGE 51

39 100 FAO 80 K112: 0.05 mM -0 .. .. 60 a u "la 0 40 .. C Q) .., .. Q) 20 A. 0 .__ ...... __ ...._ __ ..._ __. __ _. 0 0.2 0.4 0.6 0.8 1.0 [NEM], mM

PAGE 52

4o are unaffected or vice-versa). Seoorrl, all of the carriers are :roodified, but the activity of the transporter is slc:,r,.,ied rather than catpletely prevented. In reference to the later sug;Jestion, the basal or fully repressed System A activity in the Fao hepatana cells, which deloonstrated a saturable inhibition of 40% to 50% of total activity upon amino acid starvation, was tested for sensitivity to NEM. 'lhe system A activity was repressed by culturirg the cells.for 24 h in a meditnn rich in amino acids (MEM). 'lhe basal activity was then assayed for sensitivity to NEM usirg corrlitions correspoming to those used to obtain the data for the amino acid starvation-in:luced activity. As seen in Fig. 3, only 40% to 50% of the total Na+-deperrlent AIB uptake was inhibited by NEM. Althcugh the measurable K 1 ; 2 value was scm:!What higher (50 0.1 M), the results are qualitatively similar to the data obtained when System A activity was stinulated by amino acid deprivation. '1hese results sug;Jest that at least for the Fao hepatana oell line all of the carriers are :roodified, yet the activity is slc:,r,.,ied rather than cc:npletely prevented. It is interestirg to note, however, that studies of system A transport durirg amino acid deprivation have sug;Jested that the newly synthesized carriers may be distinct fran the basal carriers in sane tissue types. Klip et al. (1982) noted that Ir-proline uptake followirg amino acid starvation of I.6 myci:>last cells was inhibited by 1 nM NEM ( 50%) whereas uptake by System A in amino acid--suwle.nented cells (basal System A activity) was unaffected. Presumably, the System A

PAGE 53

41 carriers that are synthesized durin;J adaptive regulation contain sulfhydryl grrup(s) essential for activity that are not displayed by the carriers present prior to substrate stal'.Vation. It was also noted that the basal System A carrier :p:>ssessed a higher~ for substrates relative to the newly synthesized carrier. '1he pl cptinum for transport activity was shifted fran 7.8 to 7.5 for the amino acid-fed arrl amino acid stru:ved cells, respectively. '1he authors concltded that the carriers synthesized durin;J adaptive regulation -were chemically distinct fran the basal System A carrier. In reference to the sensitivity of System A transport to NEM between the nonnal hepatocytes arrl the hepatana cell lines, these differences could be attriruted to: 1) selective m::xiulation of the carrier by NEM-sensitive cytoplasmic elements present at varyin;J degrees in the in:tividual cell types, 2) structural differences in the carrier protein itself, or 3) differences in the meni>rane envirornnent that may exist between the cell types (i.e., lipid m1p:sition am,lor organization) To test for cell-specific differences in sensitivity to NEM m::xlification in the absence of cellular integrity, System A activity was assayed in isolated plasma membrane-enriched vesicles prepared fran either nonnal liver tissue or H4 hepatana cells (r:udeck et al., 1987). Fig. 4 shows the results of a series of experiloonts designed to examine the effects of varyin;J concentrations of NEM on System A-mediated uptake. When the Na+ -deperxlent uptake of AIB was assayed follCMin;J treatJnent of the :membrane vesicles with NEM, the activity of System A in the nonnal liver tissue was inactivated effectively (K1 ;2 = 370 M), but

PAGE 54

Figure 4. Concentration Depen:ience of the Inhibition of System A Activity in Rat Hepatocyte or H4 Hepatana Membrane Vesicles by NEM. 'lhemeni:>rane vesicles were prepared as described in Olapter II am then were treated with NEM for 10 min at 22c. '!he inhibitor concentration was varied between 0.5 am 5.0 nM. After rinsin] the meni:>ranes with Buffer A, System A activity was assayed by measurin] the Na+-deperrlent uptake of 200 M (3HJ-AIB for 1 min at 22c. '!he results are reported as the :percent of the rate of transport in the ID11-treated vesicles; those velocities (av~ges s.q_"l of 3 detenninations) were 1518. 7, am 984 ptDl ng protein min for the nonnal hepatocyte am H4 hepatana neti:>rane vesicles, respectively. '!his experiment was perfo:rmed with assistance fran Kathleen D.ldeck-COllart.

PAGE 55

43 100 FAO 80 K1;2 : 0.05 mM -0 I. 60 a V 0 40 C 4) "" I. a, 20 Q. 0 .... __. __________ ___. __ ... 0 0.2 0.4 0.6 0.8 1.0 [NEM], mM

PAGE 56

44 transport by the H4 hepatana-derived vesicles was tmaffected at concentrations up to 5 nM (Fig. 4). Althcugh the concentration of Nm required to produce half-maximal inhibition in the nerbrane vesicles is sanewhat higher than that measured for intact hepatocytes (370 M verses 46 M) the results are consistent qualitatively with those reported for the intact cells (Figure 1). '!he selective inactivation by Nm was further substantiated by t.reatin;:J detergent-solubilized plasma nenbrane proteins with Nm. Followin;:J solubilization of either nonnal or H4 hepatana plasma nenbranes, the protein mixture was e>qx)Sed to 1 nM Nm for 10 min at rtXll\ tercperature. '!he excess Nm was then quenched by the addition of 2 nM D-cysteine arxi the proteins reconstituted into artificial proteoliposaoos (Bracy, 1987) As shown in Table 3, Nm was effective in partially inactivatin;:J the System A activity solubilized fran nonnal liver tissue, although the inhibition was not as strorq as that observed for intact nenbrane vesicles or whole cells. In contrast, Nm had little or no effect on the activity of the solubilized carrier fran H4 hepatana cells. Additional evidence for the marked heterogeneity of System A between the nonnal rat hepatocytes arxi the transformed liver tissue was obtained by exami.ninJ the ability of neutral amino acid substrates to protect System A activity fran KMBS-deperrlent inactivation (Chiles et al., 1988). In these studies KMBS was dlosen as an inhibitor, because this protein mxtifyin::J reagent inactivated System A-nv::rliated transport by awroximately 80% to 90% in both cell types. Ir-serine, Irproline,

PAGE 57

45 Table 3. Reconstitution of System A Activity from Rat Hepatocytes or H4 Hepatoma Cells Following NEM-Treatment of Solubilized Plasma Membrane Proteins Membrane Preparatioo Assay System cattrol + NEM Reconstituted No:rmal Liver 22c:KC1.;NaC1 933 46 696 1gb H4 Hepatana Cell 22c:KC1.JNaC1 1042 202 1056 100 No:rmal Liver 37c:Iranes isolated ard the proteins solubilized in dlolate/urea as described in Cl'lapter II. Solubilized memt>rane proteins i.n;::ubated with &.lffer A in the preserce or absence of 1 n'M NEM for 10 min at roan telll)erature ard then subjected to reoonstitutioo. Proteolip:lSQ'lleS fran t\ different memt>rane preparations were assayed for System A by measurirg the uptake of 200 M [3HJ-AIB for 1 min at either 22c with the transport ruffers listed in Cl'lapter II (KCl or NaCl contai..nirq b-.lffer) or 37c with I
PAGE 58

46 arrl L-norleuci.ne were chosen as protector substrates because a large percentage of their uptake by liver tissue is mediated by system A (Kilberg et al. 1985) 'As seen in Table 4, when H4 cells were incubated with 0.2 nM PCM8S in the presence of the neutral amino acids, the inactivation of system A activity was blocked significantly. l)eperninJ on the amino acid tested, the aIOCA.mt of transport activity protected ran:JEl(i f:ran 41% to 72%. In each case the cx,rresporxtin:J Disaoor yielded lc:,.,,,er transport activity. In ex>ntrast, the PCMBSinactivation of system A f:ran the nonnal liver cells (freshly isolated hepatocytes) was not blocked by the presence of any of the neutral amino acids tested. '!he remai.nin;J major amino acid transport systems in nonnal arrl H4 hepatana cells were also assayed for sensitivity to NEM arrl PCMBS. '!he results of these inactivation studies are shown in Table 5. In the H4 hepatana cells, the Na+-depernent amino acid transport Systems ASC, N, arrl the Na+-imepernent system L were inhibited effectively by PCMBS, whereas the Na+ -imeperrlent system y+ activity was decreased by only 28%. In contrast, the reagent decreased transport via Systems ASC, N, arrl y+ in nonnal liver cells, hc:Mever amino acid uptake by system L was slightly elevated in these cells. 'lhese data imicate that all of the Na+ -deperrlent transport systems tested are sen.siti ve to PCM8S in both cell types, 'Whereas the Na+-imeperrlent agencies for amino acid uptake were differentially inactivated in the nonnal arrl transfonned cells. Heterogeneity betv.ieen these transport systems in the nonnal arrl hepatana tissues was also noted through the use of NEM. All of the

PAGE 59

47 Table 4 Amino Acid-Dependent Protection from Inactivation by PCMBS of System A-Mediated Transport Amin:> Acid cait.rol PCMBS-treated cells %Protection Amino Acid Amin:> Acid Amin:> Acid Hepatocytes present absent present Irproline 78 13 36 5 15 5 0 Irproline 93 7 20 9 13 3 0 D-serine 222 4 53 4 58 4 3 Irserine 226 25 59 7 12 4 0 Irnorleucine 106 4 25 5 26 4 1 Hepatana Cells Irproline 889 58 219 19 267 11 7b Irproline 357 U 152 6 236 11 41C D-serine 333 24 lU 14 191 5 35C Irserine 634 4 162 10 502 9 72C Irnorleucine 372 15 92 15 237 27 52C Hepatocytes or H4 hepatana .oells were cultured in amino acid-free meditnn (NaKRB) for 5 h to enhance System A activity arrl were then transferred to NaKRP contai.nirq o. 2 I!'M NDf or PCMBS in the presence or abeence of the in:licated amino acid (5 n'M) for 10 min at 370c. Cells were washed extensively with 01oll
PAGE 60

48 Table 5. Sensitivity to NEM or PCMBS of Several Amino Acid Transport Systems in Rat Normal Hepatocytes and H4 Hepatoma Cells System Control NEM %of Caltrol Control PCMBS %of Testa:l Control H4 Hepatana A 652 60 767 77 118 372 15 92 3 25C ASC ;3188 109 3183 263 100 2988 292 294 19 1ob N 362 11 589 34 163 468 10 41 7 9C L 1672 100 1689 111 101 6900 510 25 4 1C y+ 262 18 277 15 106 174 15 125 4 74C Hepatocytes A 140 5 22 4 16 100 1 27 3 27 ASC 50 1 17 2 34C 40 2 13 2 32c N 375 7 373 12 99 282 27 81 9 29C L 111 11 67 10 60 108 2 131 10 121b y+ 38 11 33 11 87 47 1 9 11 19c In:lividual amino acid transport systems were tested for activity after in::,..ibatinq the cells for 10 min with NaI
PAGE 61

49 amino acid transport systems tested in the hepatana cell line were resistent to inhibition by NEM (Table 5); System N was actually increased to a significant degree. Even after increasin;J the NEM concentration above 1 nM, no significant inhibition of these carriers was dJsel:ved (Table 5). 'lhe results contrast ooservation.s by others who have reported partial inactivation of Systems A, ASC, am N in an alternate strain of H4 hepatana cells (Vad:Jama am Olristensen, 1983). Tests for NEM-deperrlent inactivation of Systems ASC am L in the no:nral hepatocytes sha..red transport rates decreased by 66% am 40%, respectively, whereas Systems N am y+ were relatively resistent to the inhibitor. Discussion '!he aim of this Ji}ase of my research was to detennine which amino acids are .inportant for System A carrier function. Investigations centered ara.un testin;J a wide variety of protein nmifyin;J :reagents so as to imividually nmify specific amino acicl side-chain grrups which are present on the carrier protein. Followin;J a brief exposure (10 min) to the p:rotein--nmifyin;J reagent, whole cells or plasma meni>rane vesicles were assayed for System A activity by nonitorin;J the Na+deperrlent uptake of AIB. '!he results of Table 1 clearly de.non.strate that System A transport activity is largely resistent to covalent nmification by both alkylatin;J reagents, includin;J iodoacetate, iodoacetamide am dlloroacetate; am to acylatin;J reagents, such as acetic anhydride am succinic anhydride. '!he reagents specific for ami.no-graJp, fluoresoein isothiocyanate am ?'lenyl isothiocyanate, were

PAGE 62

50 ineffective in whole cells, presumably due to the low pl at which these reagents 'ilvere tested (i.e. pl 7. 4) Inactivation of System A transport activity was, however, ci:>Sel:ved in plasma me.nt>rane-enridled vesicles isolated fran both nonnal rat hepatocytes am H4 hepatana cells when the pl was raised fran 7.4 to 9.2 (Table 2). Unforbmately, no System A substrate tested was effective at blocki.n;J this inactivation. 'lhese reagents may be m:xlifyinJ plasma meni:>rane proteins other than System A or may be m:xlifyinJ the carrier protein at a location re.noved fran the amino acid bi.rrlinJ site am thus not protected by substrates. System A-mediated uptake of AIB was highly sensitive to the no:ncx:,val.ently interactinJ mercurials, HgC12 PCMB am PCMBS. Transport activity was carpletely abolished in the H4 hepatana cells by HgC12 irrleed, this mercurial was the nnst :potent inhibitor of transport activity. 'lhese results carplement the studies of stirlinJ (1975) who also denonstrated that HgC12 was 10 to 20 tll'OOS nore potent than PCMBS in blocki.n;J galactose am amino acid uptake into rabbit brush border plasma membrane vesicles. 'lhe organic mercurial catpOUOOS PCMB am PCMBS 'ilvere equally effective as inhibitors of transport in both the nonnal hepatocytes am H4 hepatana cells. Mercurial catpOUrrls have loIXJ been known to per.tum membrane stnicture am function (Rothstein, 1970). For exarcple, sane of the membrane systems susceptible to sulfhydryl agents include: alkali metal :penreability (Rega et al., 1967); a variety of active transport processes for sugars (Van Steveninck et al. 1965) amino acids (Schaeffer, et al., 1973), am nucleosides (Hare, 1975);

PAGE 63

51 ho:rnone bi.rrlirg to receptors incl~ insulin (Dixit arrl Iazarow, 1967) arrl acetyldloline (Karlin arrl Bartels, 1966). It was also denonstrated that the System A carrier fran normal arrl transfo:nood liver tissue has umergone significant~ with respect to its sensitivity to dlemical no:lification by protein-no:lifyin;J :reagents (Table 1). '!be inherent~ in the carrier are dem:>nstrated by the differential inactivation by NEM arrl NBS. NBS carpletely abolished System A-mediated transport in the H4 hepatana cell whereas this reagent was ineffective as an inhibitor of transport in the normal hepatocytes. Partia.ilarly strikinJ is the differential sensitivity of System A to the covalent alkylatin;J agent NEM. All of the transfo:nood liver cell lines tested -were either weakly inhibited or unaffected by NEM. In contrast, the System A activity in the normal hepatocytes was carpletely inactivated (Fig. 1). Several possibilities can be postulated to account for sudl differences in dlemical reactivity. '!here DBY be an alteration in the primacy sequence of the carrier protein that eliminates or masks a highly reactive cysteine group. In this case, a secorxi less reactive cysteine DBY be responsible for the partial inhibition that is cl::lserved in the HD:, FAD, arrl fleE:G2 cell lines. Alternatively, the carrier protein in the normal arrl transfo:nood tissues DBY assune different confonnation states as a result of differences in the primacy sequence of the protein that do not eliminate the cysteine residue but do alter its reactivity. It was possible that either cytosolic factors or the membrane lipid enviromoont itself ca.Il.d account for the differential

PAGE 64

52 sensitivity observed with NEM. To eliminate any seoorrlary effects by the cytosol, NEM inactivation of system A transport was assayed in plasma membrane-enridled vesicles isolated fran both no:rmal hepatocytes an:l H4 hepatana cells (Fig. 4). Clearly, the inability of NEM to inhibit System A transport was retained in the H4 hepatana membrane vesicle pcpll.ation, whereas the membrane vesicles derived fran no:rmal hepatocytes dem:>nstrated sensitivity. In an effort to reduce the possible influence of the plasma nanbrane lipid envirornnent, total plasma membrane proteins fran both no:rmal hepatocytes an:l H4 hepatana cells were detergent-solubilized, treated with NEM, an:l then reconstituted into artificial liposomes prepared fran soybean ?'106?'10lipid (Table 3). Analysis of AIB uptake revealed that the solubilized System A activity had retained the differential sensitivity to NEM. It is i.nportant to note that while the reconstitution methodology was designed to renove nudl. of the rulk lipid, tightly bourxl annular lipid may be present an:l c:x:>nferrirg the differential sensitivity on the carrier. Finally, we nee1.ed to eliminate the possibility that AIB was transported via an agency other than System A in the hepatana cell lines. In this case, the new route for AIB uptake would be largely resistent to NEM. To test if additional routes of entry existed in the hepatana cells, the effects of MeAIB on the uptake of AIB was ascertained (Fig. 2) an:l MeAIB was shown to inhibit greater than 90% of the AIB uptake in all hepatana cells, except the human cell line HepG2 (80%). If either the tran.smembrane Na+gradient or the nanbrane potential had been differentially affected in

PAGE 65

53 the nonnal hepatocyte by NEM, System A 'WOUld have been inhibited. '!his is tml.ikely because both System N am System y+ ,;.iere not affected by NEM in the hepatocyte. 'Ihese cbsel:vations argue strorgly again.st the prc:p:>Sal. that cytosolic factors am ment>rane envil::'omoont alter the sensitivity of the carrier protein am .irrlicates that the disparate inactivation between nonnal am tran.sfonned liver tissue is due to inherent stn.Ictural. differences in the System A carrier protein itself. Recently, I.ea et al. (1987) de.roc>nstrated strikirg differences between rat liver am a variety of rat hepatana cells with respect to the uptake am incorporation into protein am I:NA of Ir-leucine am thymidine, respectively. In their study, the action of the camancylatin;J agents 2-chloroethylisocyanate, ethylisocyanate am sodium cyanate was carpared in nonnal rat hepatocytes am the ?-t:>rris hepatana cells 7288CR::, 7777, 5123C, am 8999. In all of the hepatana cell lines tested, the camancylatin;J agents stro:rgly inhibited the uptake of Ir-leucine am thymidine, whereas there was little or no effect with nonnal hepatic tissue. Similar cbsel:vations ,;.iere noted for the incorporation of Ir-leucine into protein am the incorporation of thymidine into I:NA. Additional evidence for the inherent dlanges in the System A carrier was afforded by amino acid-depen:ient protection fran inactivation (PCMBS)studies. Protection of transport activity by amino acids was observed only in the H4 hepatana cells am membrane vesicles. Althalgh it is urx:lear why System A substrates provide no observable protection of transport activity in either nonnal hepatocytes or

PAGE 66

54 membrane vesicles isolated fran rat liver tissue, there are a mnnber of p::JSSible explanations. 1) PCMBS may be nmifyinJ the carrier protein at a sulfhych:yl groop distant fran the amino acid bi.n:linJ site; in this case, bi.n:linJ of amino acid substrate nust have little or no effect on the interaction of PCMBS with the carrier protein. 2) PCMBS may react with an amino acid residue in or near the substrate bi.n:linJ site, yet, stnictural features within this area do not allow the boon:l amino acid to block the reaction. 3) In cx>ntrast to its action in the transfonned oells, PCMBS may inactivate transport activity in the nonnal hepatocytes by means other than direct dlemical nmification of the carrier protein. For exanple, any interaction of PCMBS with plasma membrane carponents whidl -wcw.d pertw:b the trans-membrane soditnn electroc:hemical gradient, such as inactivation of the Na+~ ATPase, -wcw.d result in inhibition of active transport. Irrleed, other laboratories have sug:Je5ted that the action of PCMBS on many Na+ -depen::lent transport processes is due to a general in:rease in the plasma membrane penneability to small ino:rganic cations (Will am Hq>fer, 1979) rather than direct transporter nmification. 'lhese results extern am catpliment the "WOrk of several laboratories that have reported inherent differences in the dlaracteristics of amino acid transport between nonnal rat ( fresh! y isolated hepatocytes) am tran.sfonned liver epithelial oells. 'lhese chanJeS in::lu:le the disawearance of system y+ durin] maturation of fetal hepatocytes to adult am its reawearance upon transfonnation to a hepatana oe11 line (White am arristensen, 19s2). 1.r-g1utamine uptake in

PAGE 67

55 mature hepatocytes is neiiated largely by the Na+-depement system N carrier (Kilberg et al., 1980); hov.'ever, greater than 80% of glutamine uptake in~ hepatana cells is sensitive to the System A-specific substrate MeAIB (Vadgama am Crristensen, 1983). In nonnal. hepatocytes, Na+-depement Ir-cysteine uptake is considered a selective test for system 'ASC (Kilberg et al. 1981) whereas in the hepatana cell line threonine represents a better selective substrate for the 'ASC carrier (Hanllogten et al. 1981) In the mature rat hepatocyte, the Na+ depement anionic carrier' system r A[;, transports Ir-aspartate am Ir glutamate equally \tJel.l (Gazzola et al. 1981) However, in both fetal hepatocytes am~ hepatana cells, the substrate specificity of this system has been altered sudl that only the shorter (i.e., Ir-aspartate or Ir-cysteate) anionic amino acids are ac:oeptable, there awea,rs to be little or no uptake of either Ir-glutamate or haoocysteate (Makowske am Crristensen, 1982). Irrespective of the mechanisms involved, the ci:>servations described above provide additional evidence that distinct structural charges in amino acid transport systems result fran transfonnation of nonnal. rat hepatocytes. Although only four hepatana cell lines have been tested to date (Chiles am Kilberg, 1986) the fact that amino acid carrier proteins in all of them show the same~ in dlerni.cal reactivity when oarpared to nonnal. hepatocytes suggests that sane fun::1amental alteration in transport systems cx:x:,.n:'S follow:i.rg developrent of a dlerni.cally-irxiuced transfo:nood state. '!he differential sensitivity of System A to inactivation by NEM has been examined in a stable SV40-

PAGE 68

56 transfonned liver cell line. '!he laborato:cy of Cha.I develq>ed this cell line by infectin;J fetal rat hepatocytes with a SV40 nutant that is tenperature-sen.sitive with respect to growth am to the transfonned :r;:nenotype (Cha.I am Schlegel-Haueter, 1981; Chai am Ito, 1983; Chai, 1985). 'lhese cells are referred to as RIA209-15 am exhibit prq,erties dlaracteristic of transfonned cells at 330c, rut at the restricted tenperature of 400c the cells behave like nontransfonned cells. To detennine if the RIA209-15 hepatocytes retained dlaracteristics similar to the nonnal rat am transfonned hepatana cells, their sensitivity to either NEM or PCMR5 was ascertained followirg growth at pennissive or restricted tenperatures. Han:Uogten am Kilberg (1988) noted that System A activity was inhibit:Erl by PCMR5 regardless of the incubation tenperature; however, treatJoont with NEM result:Erl in no noticeable inactivation of transport activity at the restricted tenperature, suggestirg that the SV40-transfonned cells behave similar to that of chemically-irrluced transfonned cells.

PAGE 69

ClIAPI'ER IV EVIDENCE FOR '!HE DIRECI' OIEMICAL K>DIFICATION BY PCMBS OF '!HE SYSTEM A CARRIER m RAT H4 HEPA'KMA CELIS Introduction '!he results presented in Oiapter III deloonstrated that the hepatic System A carrier protein contains a sulfhydcyl group(s) whidl awears to be essential for transport activity. Although the carrier shows sensitivity to dlemical noiification by PCMBS in all liver-derived cells tested, substrate level protection was d::lserved in the H4 hepatana cell line but not in nonnal. hepatocytes. 'Ihese results suggest that the inactivation of the H4 hepatana System A activity is a result of direct chemical noiification. '!he goal of this Iilase of my research was to a) dlaracterize the IOOde of PCMBS inactivation with respect to active transport by System A in the H4 hepatana cell an:l b) to detennine to what extent the inactivation was due to direct carrier noiification as CJWC)Sed to general membrane pertw:bation. CUltured cells am tissues have proven extremely useful in the study of organic solute transport; however, a primary draw back with whole cells is intracellular metabolism of the transported solute. '!his also awlies to the use of protein noiifyin:J reagents, especially when these c:x:trp:X.JmS are used to study plasma neti:>rane ~Typically cells are exposed to a protein IOOdifyin:J reagent for a given period of time an:l then the carrier activity ascertained. Although the specific 57

PAGE 70

58 carrier ftmction umer study may be significantly altered due to direct chemical m:xlification, secorrlary effects whidl are the result of the m:xlifyin:J reagent interaction with intracellular carp:>nents on which carrier ftmction is depernent can also oc:x:::ur. SUdl secorrlary effects can lead to false CX>nclusion.s attrib.Itin:J the d1arge in a particular carrier prq;>erty to direct m:xlification. An exanple is the inactivation of glucose transport in yeast cells (Van steveninck et al. 1965) Iodoacetate inhibits the active transport of glucose into yeast cells by 90%. 'lhe inhibition was initially attriruted to direct chemical m:xlification of the glucose carrier by iodoacetic acid. SUbsequent analysis revealed, harJever, that iodoacetate was not inhibitin:J transport, rut was instead inhibitin:J glyCX>lysis via m:xlification of triooe ~te dehydrogenase. '!his resulted in a rapid depletion of cellular ATP production arrl ultimately lead to inhibition of active transport. It has been suggested that the inactivation of the mammalian kidney tissue Na+,n<+-ATPase activity (Taylor, 1963) by iodoacetate may also be due to seCX>rrlary interactions of the chemical reagent with intracellular proteins-(Rothstein, 1970). One means of distirguishin:J between nenbrane verses intracellular effects is to assay internal metabolic markers. For exanple, followin:J exposure of cells with a sulfhydl:yl m:xlifyin:J reagent, one may choooe to nonitor cellular respiration or assay for the presence of oxidized glutathione. 'lhe ioost direct method for avoidin:J secorrlary effects, however, is to ercploy either plasma nenbrane vesicles or reCX>nStituted proteoliposanes derived fran the tissue urxler study. Given that plasma

PAGE 71

59 ment>rane vesicles are essentially devoid of cytosolic elements, their use eliminates the potential for secorrlary effects. Unforb.mately, they do not allow the differentiation between nonspecific inhibition due to interactions of the m:xlifyin;J reagent with unrelated ccrrponents within the plasma membrane. For exanple, active transport by System A is strorX}ly depement on the trans-membrane soditnn electrochemical gradient (Kilberg am Cliristensen, 1980; Kristensen, 1980). 'Aey interaction of a protein m:xlifyin;J reagent which would allow rapid dissipation of the artificially-inposed soditnn electrochemical gradient would result in inhibition of System A-nmiated uptake. SUlfhydl:yl m:xlifyin;J reagents have been known to pertum the soditnn electrochemical gradient across the plasma ment>rane (Van steninck et al., 1965; Aledort et al., 1968 Knauf am Rothstein, 1971). KMBS inactivates the ecythrocyte plasma ment>rane Na+ ,n<+" -ATPase, resultin;J in an increase in soditnn influx am a general inhibition of all ion-coupled transport processes (Aledort et al. 1968) Will am Hepfer ( 1979) dem:nstrated recently that KMBS increased the soditnn penneability of isolated rat brush border membrane vesicles. 'lllese authors concluded that the increased Na+-ion penneability was sufficient to acootmt for the observed inhibition of both valine am glucose active transport. Sale infonnation conc::ernin;J KMBS-deperrlent inactivation of System A transport has been reported in the literature.
PAGE 72

.. 60 concentrations in excesses of 10-5 M. Klip et al. (1980) deronstrated in L6 myd>lasts that PCMBS inhibited adaptive regulation irrluced System A transport activity in a concentration deperrlent manner. Unfortunately, no infonnation ex,~ the actual IOOde of inactivation by PCMBS was reported in these stu:lies. It remains unclear if the inhibition was due to direct carrier IOOdification or IOOdification of tmrel.ated plasma membrane ccrrp::>nents. Pemaps the IOOSt extensive stufy mercurial perturbation of neutral amino acid uptake was reported by stirlirg (1975). 'lhis investigator used Hg-++ am PCMBS to inhibit uptake of Ir-alanine (80% to 90%) in brush bo:rder membrane vesicles. Elemental mercury was about 10-fold nore effective than PCMBS in abolishirg transport. Dithiothreitol (Drr) :reversed Hg-++ am PCMBS inhibition by 40% am 100%, respectively. '!here aweared to be no noticeable~ in Na+-penneability across the membrane, suggestirg that a significant ann.mt of the inhibition was due to direct carrier IOOdification. Materials am Methods See the methods am experimental procedures cutlined in Olapter II. Results '!he results of Table 1 imicate a general refractoriness of the System A protein in H4 hepatana cells to inhibition by covalent IOOdification, yet, the noncovalently interactirg mercurials inhibited System A activity by 85% to 90%. 'lb characterize further the inactivation of System A, a ti.me-c:arrse of PCMBS inhibition was measured. As seen in Fig. 5, greater than 85% of the total Na+ -

PAGE 73

Figure 5. Reversal of PCMBS Inactivation of System A by Dithiothreitol. H4 hepatana cells -were incubated in NaKRB for 5 h to allOW' irrluction of System A activity by adaptive regulation. '1he cells -were then incubated in NaKRP containin;J o. 2 11M PCMBS ( ) for the irrli.cated times. '1he H4 cells -were rinsed twice in NaKRP for 2 min to rem::,ve the inhibitor am then incubated in NaKRP containin;J l nM ( ) or 5 nM ( ) dithiothreitol (OIT). At the irrlicated times, the cells -were rinsed in OlolKRP for 2 min am~ A activity tested by ~irg the Na+-depement uptake of 50 M [3H]-AIB for 1 min at 37C. '1he results are reported as the percent of the transport rate in .!:J:1e absence 2f PCMBS treatllle.nt; the rontrol velocity was 386. prol :ng protein min

PAGE 74

62 100 200.0 uM PCMBS 1.0mM DTT 0 I. 80 & S.OmM DTT .. C 0 u ... 60 0 .. 40 C 4) I. 4) 20 CL 0 5 10 15 20 25 Minutes

PAGE 75

63 depenient AIB uptake was inactivated within 8 min after exposin;J the H4 hepatana cells to an external. concentration of 0.2 n'M I0100; the t1;2 for inhibition was 3 min. 'lhe inhibition of System A was not reversed by extensive washirg with NaKRP buffer, however, a significant ano.mt of the total activity was recovered when the cells were exposed to 5 n'M dithiothreitol (DIT). 'lhe rapidity of inhibition by I0100 am the reversibility by DIT suggested a direct interaction of the dlemical reagent with plasma membrane carp:>nents. Al though all of the experiments irwolvin;J I0100-depen:lent inactivation were performed in the presence of 170 n'M sodhnn, the inactivation process does not~ to require sodium durin;J the treatm:mt period (Table 6). Given the considerable difference in affinity of I0100 for sulfhydryl groups versus other ligarrls (Means am Feeney, 1971), the data presented above point to the presence of an essential cysteine residue ( s) contained within the System A carrier. F\lrthenoore, the negatively dlarged nature of I0100 does not allow this CXl1p)UJ'rl to cross the plasma membrane readily (Al.edort et al., 1968); hence, the I0100-sensitive group on System A is likely located on the extracellular surface of the plasma membrane. In order to confi.nn that the I0100-sensitive sulfhydryl group(s) was located within the plasma membrane, the effect of I0100 on System A activity was tested usin;J H4 hepatana-derived plasma membrane vesicles (D.ldeck et al., 1987). Membrane fractions enriched for plasma membrane vesicles were isolated am then exposed to increasin;J concentrations of I0100 usin;J coniltions corresporrlinJ to those used to obtained the data

PAGE 76

64 Table 6. Sensitivity of System A Activity in H4 Hepatoma Cells to PCMBS in the Presence or Absence of Na -ions o:n:litioo Velocity %0::ntrol \ Protectioo NaKRP 648 52 100 NaKRP + Ir-norleucine 505 30 78 NaKRP + PCMBS 138 8 21b NaKRP + lrnorleucine + PCMBS 337 16 52b 54 CllOll
PAGE 77

Figure 6. Concentration Deperrlence of the Inhibition of System A Activity in Rat Hepatocyte or H4 Hepatana Meitt>rane Vesicles by :E01BS. Plasma neti:>rane vesicles were prepared as described in Olapter II am. were treated with :E01BS for 10 min at 22c. '1he inhibitor con::entration was varied betvJeen o .1 am. 1. O n'M. After rinsin;J the neti:>ranes with Buffer A, System A activity was assayed by maasurin;J the Na+-deperxlent uptake of 200 M [3HJ-AIB for 1 min at 22c. '1he results are reported as the :percent of the rate of transport in the non-treated vesicles; those velocities (ayFges S.D.:. 1 of 3 detenninations) were 2291, am. 590 ptDl rrg protein min for the rx:>nnal hepatocyte am. H4 hepatana membrane vesicles, respectively. 'Ihis experiment was perfonned by Kathleen D.xleck-COllart of this laboratory.

PAGE 78

66 100 -80 0 .. H .. C 0 Hepatocyte u 60 0 .. C 40 a, .. a, A. 20 0 0.2 0.4 0.6 0.8 1.0 [PCMBS], mM

PAGE 79

6 7 shown in Fig. 5 for intact cells. As shown in Fig. 6, PCMBS treatlnent of H4 meni:>rane vesicles, as well as vesicles derived f:ran rx:>nnal rat liver, decreased the total am::x.mt of measurable Na+-deperrlent AIB uptake by 80%-90%. 'Ihe inactivation of transport was concentration-deperrlent arrl the external concentration of PCMBS required to prcx:iuce half-maximal inhibition was awroxbnately 200 M. Collectively, these results sug:JeSt that the action of PCMBS is due to direct m:x:lification of plasma meni:>rane carp::>nents. Additional information about the inactivation of system A-mediated transport was gained by studyirg the initial-rate kinetics of Na+depen:lent AIB uptake before arrl immediately followirg PCMBS treatlnent. In this experiment, H4 cells were treated with 200 M PCMBS (10 min) at roan tenperature, the cells were washed rapidly with Choll
PAGE 80

Figure 7. Kinetics of AIB Transport FollowiD1 J?CMBS Treatlllent of H4 Hepatana Cells. 'lhe cells were all tured in NaKRB for 5 h to allow enhanoeloont of the System A activity am then inaibated in NaKRP in the presence ( ) or absence ( ) of 0.2 nM J?CMBS for 10 min. 'lhe hepa:tana cells were washe1 twice in CholKRP for 2 min am System A activity m:>nitored for 15 sec at 37C with a substrate ( [3H]-AIB ) concentration ran:JID1 fran o. 02 nM to 50 nM. 'lhe am v values were calallated fran the Na+-depen:lent velocities by carprt:er~ysis (Clelam, 1979). Where not shown the starnard deviation bars are contained within the symbol.

PAGE 81

.. -~ 69 -. "' G) "' an 10.2 ... PCMBS Km vmax C + 3.3~0.2 4.1 :!: 0.3 G) 8.1 1.2:t 0.1 6.4:0.1 .. -0 0. 6.2 'i a, E 4.3 al -c 2.1 -0 E C 0 1.1 2.2 3.3 4.4 5.5 I nmo mg protein 15seYmM

PAGE 82

"J'J studies addressed three areas; 1) the effect of PCMBS on the initial rate of Na+-deperxient AIB uptake; 2) the effect on the rate for AIB efflux; am 3) the effect on the final steady state distrirution ratio of AIB. Fig. 8 shows the results of a series of experiments designed to examine the role of PCMBS on the initial rate of AIB uptake am the steady-state distrirution. ShCM1 is a ti.me--ccJurse for Na+-deperrlent AIB uptake into H4 hepatana cells followi.nJ PCMBS treatment. '!he Na+ deperrlent AIB uptake in cx>ntrol cells was essentially linear t.hralghout the time of the experiment (60 min). In contrast, the Na+-deperx:ient uptake of AIB was significantly lowered am only linear for a,wroximately 1 min followi.nJ PCMBS exposure. Even after 60 min, the accunulation of AIB within H4 cells had not reached steady state, hOlrleVer, the accunulation of AIB into PCMBS treated cells had readled steady-state within 30 min. To quantitate the degree of uncoupli.nJ of active transport by System A, the distrirution ratio of AIB (i.e., AIBi.J/AIBa.rt) was detenni.ned for both control am PCMBS treated cells. Distrirution ratios in excess of 1 for a given solute of neutral charge am limited intracellular trawl.DJ are generally taken as evidence for active transport. To cala.ll.ate distrirution ratios the intracellular water vol\.lll'e nust first be detenni.ned. Usi.nJ the 3M:; rrethod. described in
PAGE 83

Figure 8. Tine-Course of AIB Uptake into H4 Hepatana Cells Following PCMBS Treatroont. H4 hepatana cells ,;,.,ere in::ubated in NaKRB for 5 h to the ano.mt of System A activity by adaptive regulation. '!he cells ,;,.,ere then in::ubated in NaKRP ( ) or NaKRP containing O. 2 rcM PCMBS ( ) for 10 min at 37C. System A activity was m:>nitored by measurin] the Na+~t uptake of 50 M [3H]-AIB for 0.25 to 60 min. '!he results~ the averages s.o. of 4 detennination.s am are reported as pool ng protein versus tine (min). Where not shown the stan:1ard deviation bars are ex>ntai.ned within the syrrool.

PAGE 84

72 4.0 7.0 r;, 0 0 ... .. >< >< --C 3.0 C G) 5.0 .. "' 0 0 a. &a I. Q. -. 0, u 2.0 0, E A. E ... 3.0 &a &a C 1.0 C -0 0 E E Q. 1.0 Q. -0 30 60 Time, (min)

PAGE 85

Figure 9. Effect of PCMBS on the Intracellular Water Vol'l.llle of H4 Hepatana Cells. H4 hepatana cells were incubated in NaKRB for 5 h to enhance the annmt of System A activity by adaptive regulation. '!he cells were then incubated in NaKRP ( ) or NaKRP contai.nin;J o. 2 nM PCMBS ( ) for 10 min at 37C. '!he cells were quickly rinsed twice with NaKRP ( 4. o c ) am incubated with NaKRP contai.nin;J ractiolabeled 3M; as described in Cllapt:er II at 37C. After 1.5 h, the medium was rercoved am the cells washed four tiroos in Clloll
PAGE 86

,, 0 .. >< -C Q) .. 0 .. a. -. en E C) C'? 0 E a. -15 13 11 9 7 5 3 1 7 4 -PCMBS 1.0 3.0 5.0 7.0 9.0 [ 3MG ),mM

PAGE 87

7 5 awroximately 24 (i.e., AIBin = 1179 Mam AIBa.zt = 50 M). Aledort et al. ( 1968) reported that exposure of ecythrocytes to PCMBS caused an increase in the intracellular water voltn'Ie by awroxilnately 40%. '!herefore, the intracellular water voltn'Ie in the H4 hepatana cells was also measured after PCMBS exposure. As seen in Fig. 9, the intracellular voltn'Ie increased awroxilnately 2.5-fold (14 1/ng protein) Usin;J this value a distrirution ratio for AIB aca.mulation at steady state (30 min) was calculated to be awroximately 0.6 (i.e., AIBin = 28 Mam AIBazt = 50 M). 'lhese data in:ticate that the primacy action of PCMBS on System A was to uncx:,uple active transport by this carrier. In the absence of cellular matabolism of the transport substrate, as in the case for AIB am Me.AIB (Noall et al. 1957; Olristensen am Jones, 1962), the steady state distrirution ratio for a given solute arises fran the balance between all routes for enb:y am all routes of excxius. 'lb test whether the decrease in the initial rate of uptake could aooc,.mt solely for the lowered distrirution ratio for AIB, efflux of AIB was assayed followin;J PCMBS treatment. 'Ibis was perfonood by preloadin;J the H4 hepatana cells with radiolabeled AIB ( external ooncentration of 50 M) for 1 hat 370c. '!be cells were then incubated in NaKRP buffer in the presence or absence of 200 M PCMBS (10 min) at 370c. Followin;J the treatment with PCMBS, the cells were rinsed rapidly with NaKRP buffer am then incubated in 1 ml fresh NaKRP buffer. At various times, the ann.mt of AIB remainin;J in the cells was detennined as described in Cllapt.er II. As seen in Fig. 10, the net rate of AIB

PAGE 88

Figure 10. Time-carrse of AIB Efflux fran H4 Hepatana Cells Followin;J KMOO Treatment. H4 hepatana cells -were incubated for 1 h at 37C with 50 M [3H]-AIB. 'lhe cells -were then rinsed with ice-cold NaKRP ar:rl incubated an acxlitional 10 min in NaKRP ( ) or NaKRP containin;J 0.2 nM KMOO ( ) Efflux of AIB was neasured by washin;J the cells twice in NaKRP (4.0"C) ar:rl then incubatin;J the cells in 2 ml NaIld OlolKRP ar:rl the anount of radiolabeled-AIB remainin;J in the cells was detenn.ined as described in Cllapter II. '!he results reported are the averages -~ D. of 4 determinations ar:rl are plotted as pool AIB remainin;J rrg protein versus time (min) Where not shown the stamard deviation bars are contained within the synool.

PAGE 89

C") 'o pa >< -C Q) .. 0 I. a. -I 0, E ,:a C -0 E a. -s.o 4.0 3.0 2.0 1.0 77 -PCMBS PCMBS 3.0 Time, (min) 6.0

PAGE 90

78 Table 7. Protection of System A Transport Activity by Amino Acids Amiro Acid \ Imibitia1 of Transport \ Protectia1 of Activity IrAlanine 92 35 D-Alanine 5 11 8-0lloro-Iralanine 86 38 IrSerine 86 68 D-Serine 1 35 2-Aminc:b.Jtyrate 83 47 IrProline 81 36 D-Proline 0 18 Ir-Norleuc:ine 73 62 N-Aoetyl-Irhistidine 52 49 IrHistidine 46 48 Taurine 5 12 IrAspartate 2 7 IrAlanine-N-hydroximate 0 0 IrArginine 0 8 Irlijsine 0 7 H4 hepatana cells were incubated in NaKRB for 5 h to stinulate transport activity via adaptive regulatioo ard then transferred to NaKRP oontaining o. 2 1!M POoffiS in the presence or absence of 5 1!M of the irdicated amino acid. After 10 min at 370c, the cells were washed in Olol..KRP for 5 min ard the System A activity assayed by measur1.n1 the Na+-depenjent uptake of 50 M AIB for l min at 370c. Cells not treated with POoffiS were tested for substrate inhibition of system A by measur1.n1 the uptake of 50 M [3HJ-AIB in the preseroe or absence of 5 !TM of the irdicated amino a~id. 'Ille data represented as a percent of the initial value (337 15 poc,l ng protein min ) cbserved in the absence of the inhibitor.

PAGE 91

79 efflux was increased 11-fold for cells treated with PCMBS. '!he calculated t 1 ; 2 values for AIB efflux -were 77. o o. 7 arrl 7. O O .1 min for PCMBS treated arrl control cells, respectively. 'lhus, the reduction in the steady state distribution ratio for AIB awea,rs to result fran both a decrease in the initial rate of uptake as 'iNell as an increase in the rate of exodus. As disalSSE!d above, protein-Iocx:tify~ reagents react in:liscriminately with the sulfhydcyl groops of many nenbrane proteins (Rothstein, 1970). A series of experiments -were designed to address whether the inhibition by PCMBS was the result of direct carrier :roodification or due to in:lirect plasma nenbrane :pertw:bations. one of the strorgest imications that a nenbrane protein is :roodified by a chemical reagent is protection of activity by substrates. 'lherefore, our first analysis exam:ine1 the ability of substrate amino acids to protect System A activity fran PCMBS-depement inactivation. '!here was a correlation beb.ieen the ability of substrate amino acids to inhibit System A-naliated AIB uptake arrl the ability to protect the carrier fran PCMBS-deperrlent inactivation (Table 7). For exanple, a significant annmt of protection of System A activity was observed with IOOSt amino acids conta~ small neutral side-chains. System :X-A G substrates sudl as Iraspartate (Makowske arrl Olristensen, 1982) arrl typical System y+ substrates such as Irarginine arrl Irlysine (White arrl Olristensen, 1982) protected only 7-8% of the PCMBS-sensitive activity. 'lhe stereospecific nature of the protection by amino acids (Table 4), along with the inability of non-substrates to cause protection (Table 7),

PAGE 92

Figure 11. Dixon Plot of the Kinetics of Ir-Norleucine Inhibition of AIB Uptake. '1he Na+-deperrlent uptake of [3H]-AIB was m::>ni.tored at 37C for 1 min usin;J AIB concentrations of 0.1, 0.2, arxi 0.5 nM. '1he results shown are the Na+ -deperrlent uptake velocities in the presence of the in:licated concentrations of unlabeled Ir-norleucine as inhibitor. '1he results are reported as the S.D. of 4 d.etenninations arxi are plotted as :i;::m:>l AIB ng protein versus the concentration of Ir-norleucine. '1he estimated Ki value was determined as described by Clelarxi ( 1979)

PAGE 93

C? 0 ... >< c -E C Q) .. 0 .. Q. 0, E 5 4 3 2 81 [ e O.lmM (AIB] A 0.2mM O.SmM Ki: 1.9::t0.1 mM -2 0 2 4 6 8 10 [Norleucine], mM

PAGE 94

82= in:ticate that the protection is not due to dlemical reaction of PCM8S with the amino acid or other in:tirect effects. 'llle lack of a nore precise correlation between the degree of inhibition of System A am the protective capability of a given amino acid is not totally urrlerstcxxi (Table 7); however, it may result fran the considerable arrount of trans-inhibition c:t>served with sane System A substrates (Kelly am !utter, 1979; Kilberg et al., 1985). Transinhibition by the protective amino acid carplicates the calculation of the degree of protection by alterin;J the control transport rate measured. An attenpt to correct for trans-inhibition was taken into consideration by usin;J the rate vaa (velocity of uptake after incubation in the presence of the protective amino acid only) as described in Cllapter II, yet a certain arrount of inprecision prevailed. For exanple, the non-metabolizable analogs, AIB am MeAIB, gave such inconsistent results because of their high degree of trans-inhibition that they -were not used raitinely as protective amino acids for quantitative studies. On the other ham, Irnorleucine typically prcx:iuced both the highest measurable protection am the least anount of trans-inhibition (10% to 15% inhibition of total uptake) of all the amino acids tested. 'lherefore, it was dlosen as the test amino acid for additional protection studies. Kinetic analyses was corrlucted to deroc>nstrate that Irnorleucine was in:leed an effective substrate for System A in liver tissue. 'llle Na+ deperxient uptake of o .1, o. 2 am o. 5 ITM radiolabeled0 AIB was assayed in

PAGE 95

Figure 12. Inactivation of System A Tran.sport Activity by PCMBS: Kinetics of IrNorleucine, IrSerine, an::l IrAlanine Protection. H4 hepatana cells were cultured in NaKRB for 5 h to allow System A activity to increase via adaptive regulation. '!he cells were then irx::ubated in NaI
PAGE 96

.,, Q) .. 0 > -.. "' 0 C C E Q) .. Ill ),. II) .. C Q) "' "' Q) a. 100 80 20 84 Norleucine Kp=0.6* 0.1mM o ..... ..... __. __ ..._ _____ 100 Alanine 80 Kp 2.4t 1.SmM 60 40 20 0 100 Serine 80 Kp.l ~0.8mM 60 40 20 0 1 5 10 15 20 25 [Substrate], fflM

PAGE 97

85 the presence of varyin;J concentrations of unlabeled Irnorleucine. '!he data \ere then analyzed by the method of Dixon (1953) am are presented in Fig. 11; the results irrlicate that Irnorleucine is a cut~titive inhibitor of Na+-deperrlent uptake of AIB am show the usefulness of this amino acid as a m:xiel. System A substrate. '!he affinity of Irnorleucine for System A (Ki = 1 9 0.1 nM) is within the rarge observed for IOOSt other substrates (OXerxier am th these substrates am the external concentrations required for

PAGE 98

Table 8 L-Norleucine-Dependent Protection from PCMBS Inactivation of System A Transport in H4 Hepatoma or Rat Liver Membrane Vesicles Corxl:itioo Velocity 'caitrol 'Protectial H4 Hepatana caitrol 597 25 100 0. 25 11M F01BS 109 18 18C 5 11M IrNorleucine 749 14 125 0.25 11M F01BS + 5 11M IrNorleucine 394 14 66 44 Hepatocytes control 1492 69 100 0. 5 11M F01BS 29 53 2C 5 11M L-Norleucine 896 79 60 0.5 11M F01BS+ 5 nM L-Norleucine 115 10 ab 10 Rat liver of H4 hepatana mesrbrane vesicles were treated with the in:ticated concentratioos of F01BS in the presence or absence of Lrnorleucine as descri.be::l in Cllapter II. 'Ihe ?YStan A activity was then assayoo by measurirq the Na+-deperoent uptake of 200 M [JHJ-A.I~ for 1 min ~l 370c. 'Ihe data are reported as the averages of the velocity (rol ng l)rotein min ) S.D. for three determination.s. '!his experiment was perfoI11l8d with the assistaocie of Kathleen D.ldeck-COllart. IlleSe values are significantly different fran the oaitrol values to p values
PAGE 99

87 half-maximal protection we.re 2.4 1.s nM am 2.1 0.8 nM for Iralanine am Irserine, respectively. Hepatic neutral amino acid uptake is mediated largely by foor systems (systems A, 'ASC, N, am L) eadl possessin;J distinct, rut overlappin;J specificities (Kilberg, 1982). A given amino acid may be transported largely by a sin;Jle agency, sud1 as Irglutamine by System N or by nultiple ra.ites, such as Iralanine by systems A, 'ASC, am L. 'Iherefore, it was of interest to detennine whether Irnorleucine could protect a
PAGE 100

88 Table 9 L-Norleucine Protection from PCMBS-Dependent Inactivation of Several Amino Acid Transport Systems in H4 Hepatoma Cells SYSTEM cx:NIR:>L a:NlroL P01BS \IN1\Cl'IVATIOO P01BS % :mJI'ECI'IOO + + Norleucine Norleuc:ine A 516 472 116 77b 335: 61c ASC 1896 1488 156 92 8 170: le L 985: 5694 25: 998 29:tl oe N 471. 468 41 918 144 24 8 Gly 90: 93 7,2 928 15: 98 X-A,G 314 261 35: 898 83 218 y++Na+ 52 49:t4 31 4od 43 67b y+-Na+ 52 49:t3 37 2gd 32 od 8 2 31 6 818 ND ND Effect of P01BS a, several amino acid transport systems. H4 cells were incubated for 4 h in Nalniese values are significantly different top values< 0.01. lhese values are significantly different top values< 0.025. &nlese values are significantly different top values< 0 005.

PAGE 101

hepatana cell did not allow an acx:::urate iooasurement of the degree of protection. '!he Na+ -deperrlent anionic carrier, system XA G, was also I sensitive to P01BS (89% inhibition) am aw:roximately 17% of this activity was protected when the P01BS treatment was perfo:rmed in the presence of Irnorleucine (Table 9). '!he dicartx:cylic carrier is usually defined as the portion of Na+-deperrlent I.r-qlutamate uptake that is inhibited by Ir-cysteate (Gazzola et al., 1981; Makowske ard Christensen, 1982). '!his amino acid analog represents an excellent nmel. substrate for system XA G because of the low PKa value of the I side dlain ( aba.rt 1. 5) Because characterization of system XA G in the I rat H4 hepatana cell has not been reported in the literature, the Na+deperrlent uptake of 50 M I.r-qlutamate was IOOaSUr0d in the presence ard absence of Ir-cysteate. AE:t>roximately 96% of the total Na+ -deperrlent Ir glutamate uptake was inhibited by 10 nM I.rcysteate (i.e., Na+-depement uptake in the absence ard in the presence of I.rcysteate is 890 74 ard 36 8 pt0l rrg-l protein min-1 respectively), As a result, system X-A,G may be assayed by sinply m:>nitorin] the Na+-deperrlent uptake of Ir glutamate. As seen in Table 9, the saturable uptake of arginine, ne:liated by system y+, was inhibited awroximately 37% by POm.S; a large portion of this transport activity was protected by Irnorleucine (67%), rut only in the presence of Na+. 'Ihe protection of system y+ by Irnorleucine ard Na+ suggested that the neutral amino acid was bin:lirg to the carrier in a Na+-deperrlent fashion. '!he transport of cationic amino acids by system y+ requires a positively dlarged side-dlain structure (White,

PAGE 102

90 1985). In the Ellrlidl cell, the :positive dlarge can be a grCA.Jp other than a quaternary base ('lhanas et al., 1971). In hepatana cells, White et al. (1982) have dem:>nstrated Na+-depen:ient inhibition of arginine influx by a variety of neural amino acids. For exanple, those authors foum that the activity of System y+ in im:::: hepatana cells was inhibited awroxilnate1y 40% by 5 nM L-haooserine am 145 nM Na+, whereas rem::,va1 of the Na+ eliminated the inhibitory effect by haooserine. Based on these ciJseI:vations, arrl the results presented in Table 9, it seems certain that the Na+ -deperx:ient protection of system y+ by L-norleucine probably results fran an occupation of the carrier's cationic amino acid birrlinJ site by the neutral amino acid am Na+. 'As discussed in the Introduction, PCMBS the penneability of the brush border plasma membrane to small inorganic cations, there.by resultin;J in a general inhibition of many Na+ -depenjent transport processes (Will am Hopfer, 1979). F\.lrthenoore, PCMBS is known to inhibit the plasma membrane Na\1~-ATPase (Aledort et al., 1968). '!his inactivation results in a loss of the trans-ire.mbrane :potential am a general urx:::ooplin;J of IOOSt Na+ -depenjent transport processes. To address the question of plasma membrane integrity followin;J PCMBS treatment, several control experinents "'1ere con:lucted. '!he first experinent aa:lressed whether PCMBS produced any dlanJes in the inherent semi-permeable nature of the plasma membrane. 'lhe awroadl was to cacpare the rates of efflux of 3M; fran control am PCMBS treated cells. '!he H4 hepatana cells "'1ere incubated for 1 hr with [ 14c]-3Mi, then incubated in absence or presence of 200 M PCMBS for 10 min. Followin;J

PAGE 103

Figure 13. Time-Course for 3-o-Methyl-D-Glucx:>Se Exodus fran H4 Hepatana Cells Followin:J PCMBS Treatment. H4 cells -were incubated 1 h at 37C with 5 nM unlabelled 3M:; contai.nin;J 3. 5 rmoles of labeled 3M:;. '!he cells -were then quickly rinsed with ice-cold NaKRP am incubated an additional 10 min at 37C in NaKRP ( ) or NaKRP cx:>ntai.nin;J 0.2 nM PCMBS ( ) Exodus of the sugar was initiated by washin:J the cells free of PCMBS (twice with 2 ml of NaKRP at 4.0C), then incubatin:J the cells in 2 ml of NaKRP ( 37 C) At the in:ticated tines, the cells -were quickly washed three tines with ice-cold Choll
PAGE 104

92 35 C 4) 0 28 I. +PCMBS Q, -t1 =10min 'm 21 ~2 E C) 14 M -PCMBS -t1 =O.Smin 0 7 E V2 C 0 2 4 6 8 10 Time, (min)

PAGE 105

9 3 this period, the cells were washed rapidly with NaKRP ( 4 Cc) arxi resusperrled in 2 ml of fresh NaKRP. '1he rate of 3M; exodus was measured by dete.rmininJ the arcnmt of 3M; remainin:J in the cells as a :furction of time. '1he rate of 3M; exodus fran PCMBS-trea:ted cells was actually slower (t112 = 10 min) than the correspon::lm:J rate in control cells (t112 = 30 sec), denonstratin:J that the plasma meni:>rane had retained its inherent inpenneability with respect to small nutrient IIX>lecules (Fig. 13) Irxieed, these data support previous reports of inhibition of Na+ irrleperrlent glucose transport by organic mercurials in dlick errbryo fibroolasts (Smith.,:rctiannsen et al., (1976). 'lhe H4 hepatana cell membrane also~ to retain its inherent inpenneability to macraoolecules. When the same pcp.llation of cells were exposed to 200 M PCMBS (10 min) at 37C., washed, am then incubated in NaKRP buffer for 5 min, no measurable arcnmt of lactate dehydrogenase (IllI) activity was d:lserved in the NaKRP medil.nn (Table 10). F.qually inp::>rtant, however, was the d:>servation that quantitatively equal levels of IIlI activity remain in both the PCMBS treated arxi control cells, denonstratin:J that little, if arr:/, IIlI has leaked fran the H4 hepatana cells. Cell viability was also detennined at 5 arxi 15 min followin:J PCMBS exp::lSUre. Greater than 98% of the cells remainin:J attadled to the culture dishes were viable as judged by exclusion of trypan blue. '1he effect of PCMBS treatlnent on the Na+-d.epernent uptake of pyruvate by H4 cells was also measured. '1he uptake of pyruvate into rat hepatocytes is stron;Jly depen:lent on the Na+ electrodlemical gradient

PAGE 106

94 Table 10. Effect of PCMBS on Lactate Dehydrogenase Release from H4 Hepatoma Cells Extracellular Medium N.D. Cell lqsate 5.43 X 10-3 pa,e; Treated N.D. 5.15 X 10-3 H4 hepatana cells were plated in 100 lllll petri dishes ard a.utured in MEM containi.rg 5.\ F8S for 2 days. 'Ihe cells were then rinsed twice in NaI
PAGE 107

95 + Table 11. Effect of PCMBS on the Na -Dependent Uptake of Pyruvate, Uridine and AIB in H4 Hepatoma Cells Velocity, pool nq-l protein 30 sec -l SUbstrate cart:rol PCMBS \ cart:rol AIB 123 10 29 4 24\b I.rPyruvate 152 13 209 11 137\b Uridine 161 15 106 10 66\b H4 hepatana cells -were ino.ibated in NaKRP b.lffer for 3 hard then transferred to NaKRP ca,tainin;J 0.2 nN PCMBS (370c). After 10 min, the hepatana cells -were rinsed twice in Cl)ol.KRP ard then assayed for specific transport as follows: System A, Na+deperdent uptake of AIB; a-Keto acid carrier, Na -deperdent uptake of Irpyruvate; Nucleoside carrier, Na+-depenjent uptake of uridine. 'Ihe substrate cxn::entrations -were 50 M ard the uptake was performed for 30 sec at 370c. 'Ihe data are presented as the averages stardard deviatioos of four detenninations. brhese values are significantly different top values< 0.005.

PAGE 108

96 (Kilberg am Gwynn, 1983). 'As seen in Table 11, the Na+-deperxlent pyruvate transport in the H4 hepatana cells was not inhibited substantially by IOmS treatment an:l only 34% of the total Na+-deperxient uridine uptake was lowered. In addition, the initial rate of uptake am the steady-state distribution ratio of Na+ was not affected by IOmS ( data not shown) 'lhese transport systems provide a functional test for the presence of a Na+ gradient across the plasma meni:>rane followin:] IOIOO-treatment. In agreem:mt with the studies on 3M; exodus am the lactate dehydrogenase assays, these results illustrate that the plasma membrane has not been rerrlered freely penneable to either small nutrient nolecules or macrarolecules, nor has the trans-membrane Na+ electrodlemi.cal gradient collapsed. Discussion A portion of our current infonnation concernin;J the dlemi.cal basis of protein am enzyme function has been obtained t:hralgh the use of protein-noclifyin:J reagents. Although there are many limitations am warranted resenrations to this type of analysis, dlemical m:x:lification remains the sin'plest am nn;t direct :nethod to identify "essential amino acid groups". Essential groups are those amino acid structures which are clirectly or in:lirectly required for a particular function, sudl as protein stability or active site catalysis. ~tionally, Means am Feeney (1971) have defined essential groups as those for which dlemical m:x:lification brims about a loss of a dlaracteristic p:tq)erty. However, equally i.nportant are those groups tenned "false essential groups", which are those amino acid residues for which m:x:lification results, by

PAGE 109

97 virtue of secx:n:la1:y effects, in el inti.nation of a particular prq>erty. In an attenpt to identify either "essential grcq:is" or "false essential grcq:is" on the system A carrier protein, a variety of chemical group specific reagents -were tested for their ability to inactivate transport. '!he result of the experiments described in Olapters III am IV sug;JeSt that one or ll)re free sulfhydryl grcq:is (cysteine) are required for functional transport of amir> acids by System A. 'Ihis is based largely on the results obtained fran usin:J NEM am :EOIBS as sulfhydryl-specific protein-m:xtifyin:J reagents. :EOIBS is an ai:yl mercurial whidl was introduced as an enzyme inhibitor by Hellennan (1937). Organic mercurials are ll)I10functional in that they can react with only one ligam. 'lhe opti.m.nn rate of the reaction with proteins ocx:::urs at p:I 5.0. Mercurial ccq:x:,urrls react to fonn stronJly ionic boms with ligam atans capable of donatin:J electron pairs, such as, dlloride am hydrogen ions. '!he disscciation constants of thio-mercury oarplexes are on the order of 10-15 M to 10-20 M (Sinp;on, 1961; Boyer, 1954; Means am Feeney, 1971). As a result, the affinity of mercurials for sulfhydryl grcq:is is far greater than it is for any other chemical protein group, however, it has been reported that :EOIBS may react with residues in proteins that do not contain free sulfhydryl grcq:is. For exanple, apocart:,oxypeptidase A canbines strorgly with PCMB resultin:J in a loss of catalytic activity (Vallee et al., 1960). Recent studies have shown that carl:>oxypeptidase lacks sulfhydryl grcq:is (Li.pscarb, 1967). As discussed in the introduction, several proole.ms exist with interpretin:J inhibition data usin:J whole cells or plasma membrane

PAGE 110

98 vesicles. First, organic merarrial effects on ment>rane transport maybe secx:n:Jacy due to inactivation of unrelated ment>rane systems upon whidl transport is deperx:lent. Secom., inhibition of transport maybe secorrlary to inactivation of cellular netabolic pathways, rather than a primacy effect on the membrane protein. A rnnnber of criteria have been established to detennine whether the effects of sulfhydcyl reagents can be attriruted to direct interactions with sulfhydcyl groops in the membrane versus interactions within the cell. As sug:-Jested by Rothstein (1970) one means is to assay markers of cellular netabolism. For exanple, d'lan:Jes in frog skin Na+ -ion penneability by organic merarrials have been localized to the plasma membrane by dem:xlstratin:J that cellular respiration was not affected (Lin::lem.olm, 1951). other criteria might involve nonitorin:J the disawearance of reduced glutathione. Tsen arrl Collier (1960) d.Jserved that NEM reduced glutathione to very le,,/ levels in red blocxi cells, rut did not prcxiuce cellular lysis. In contrast, PCMBS produced rapid cellular lysis with little effect on reduced glutathione, ~in:J that the primacy action of PCMBS was at the level of the plasma membrane, whereas that of NEM was intracellularly. One criterion that I have errployed whidl suc;Rests a direct action of the sulfhydcyl-m::>difyin:J reagents on System A activity is the use of PCMBS rather than PCMB. Although, both CX1YpOllI'rls have been shown to react in a similar fashion with soluble proteins arrl with human erythrocyte ghosts (Van Steveninck et al., 1965; sutherlarrl et al., 1967), PCMBS is markedly hydrqnilic, because it contains a sulfonate

PAGE 111

99 groop whidl is ionized (!>Ka = 3. 4) arrl carries a formal dlarge of -1 at Jilysiological pl values. Al.edort et al. (1968) carpared the rates of uptake for both PCMB arrl PCMBS into human platlets. '!heir results dem:>nstrated that the uptake of PCMB was linear for at least 1 h, while PCMBS exhibited i.Imeiiate birnin;J to the plasma meni>rane with little further uptake Oller the same :period of time. 'lhus, within the time frame of these experiments (i.e., cells are exposed to PCMBS for 10 min), it is unlikely that PCMBS is penneatirg the plasma meni>rane. Irrleed, the rapidity with whidl the inactivation develq)S, i.e., System A activity is effectively inactivated within 2 min, am the rapid reversal by DIT also suggest the extracellular surface of the plasma meni>rane as the site of action. 'lhe reversal of inhibition by DIT also irrlicates that the carrier protein has not been irreversibly denatured. An additional criterion to differentiate between plasma meni>rane or intracellular effects on transport, is to enploy the use of plasma. meni>rane vesicles isolated fran intact cells. It is evident fran the data presented in Fig. 6 that System A transport has retained its sensitivity to PCMBS in membrane vesicles obtained fran H4 hepatana cells, as -well as those isolated fran normal hepatocytes. In reference to the prd::>lem.s associated with inhibition of transport due to m:xtification of membrane proteins whidl may not be the carrier itself, am in absence of protein sequence data for the active site, substra~eperxient blockirg of the inactivation provides the strongest evidence for direct carrier m:xtification. '!he results of Tables 4, 8, 9 am Fig. 12 clearly dem:>nstrate both stereo-specific am

PAGE 112

100 system-specific substrate-deperrlent protection fran the FOiffi-depen:lent inactivation of System A. Transport mediated by System A was retained followi.nJ F0100 exposure in the presence of Ir-oorleucine, yet lost in its absence, so it is possible that the "essential" sulthydl:yl group(s) is located within or near the amino acid bin::lin:J site of the carrier. Altha.lgh F0100 is likely to be nn:tifyi.nJ many membrane protein sulthydl:yl groups unrelated to System A activity, the substrate deperrlent protection in:li.cates that the inhibition is the result of direct chemical nn:tification of this carrier protein. '!he awarent decrease in System A affinity for AIB uptake followi.nJ F0100 treatment substantiates these absel:vations. '!he cc:npetiti ve inhibition of System A-mediated transport by Ir-oorleucine denonstrates that the amino acid is bin::lin:J within the "substrate" or amino acid bin::lin:J site of the carrier. 'Iherefore, the substrate-deperrlent protection by Ir-oorleucine may be interpreted as evidence that F0100 is nn:tifyi.nJ a sulthydl:yl group(s) within the amino acid bin::lin:J site. It is interesti.nJ that Ir-oorleucine protects, at best, only 60%-70% of the total transport activity. '!he possibility exists that a secom class of sulthydl:yl groups may exist that are nn:tified, am cause inactivation, but at a site distant to the active site. 'Ihese "nonc:aTpetitive sites" may explain the decrease in the tran.slocation rate of the remaini.nJ System A activity. '!he location of these groups may be on the carrier protein itself, distinctly separate fran the amino acid bin::lin:J site am not affected by substrate bin::lin:J. Alternatively, these sites may be located on a System A-associated protein whose

PAGE 113

101 m:xlification results in inhibition of carrier function. Finally, it is possible that these "nc:n::acpetitive" sites are localized to a protein unrelated to system A, yet dlemi.cal m:xlification results in nonspecific inhibition of 20% to 30% of carrier activity.

PAGE 114

ClIAPl'ER V MATERIALS AND MElH:)00 FOR PROl'EIN ClIEMIS'IRY AND MJIECUIAR BIOLCGY Nitrocellulose roombrane (0.45 micron) was purchased fran Schleicher am Schuell (Keene, NH) arrl the Gene Screen hybridization meni:>rane (NEF-972) was obtained fran New :En;Jlarrl Nuclear research products (Boston, MA). Highly pure agarose was purchased fran SeaKem Co (Rocklarrl, ME). All bacterial arrl iage growth media -were obtained fran Difa:> laboratories (Detroit, MI). '!he Alrplolites -were purchased fran I1
PAGE 115

lo3 NaKRB alone. Followi.rq a 2 h incubation, L[4,5-3H]-leucine was added (40 Ci/ml) an:i the incubation was ex>ntinued for 6 harrs after wru.ch the cells -wrere rinsed in NaKRP buffer an:i a 10,000 g neri:>rane fraction prepared as foll0v.1S. '!he cells -wrere rinsed three times in PBS an:i then incubated at 4C for 5 min a hypotonic buffer oonsisti.rq of 11TM sodium bicartx:>nate, pl 7.5, 10 1TM EDI'A, 5 1TM benzamidine, an:i 1 ITM IMSF. '!he cells -wrere then :reroc,ved fran the culture dish with a rul:::ber policeman an:i haoogenized with a Potter-Elvehjem haoogenizer usi.rq a tight-fitti.rq pestle (100 to 125 noter-driven strokes at 930 :rpn). '!he haoogenate was centrifuged for 10 min at 200 g to reroc,ved unbroken cells an:i nuclei an:i the resulti.rq supernatant centrifuged (30 min at 10,000 g) to obtain a pellet enridled for mitochorrlria, lysoscmas, an:i plasma neri:>ranes. '!he membrane pellet was resuspen:led in the hypotonic buffer an:i stored at -1oc. Two-Dinen.sional Polyacrylamide Gel Electrgi1oresis (2IrPAGE) Hepatic membrane proteins (10,000 g pellet) -wrere solubilized by a m:xlification of the alkaline-urea method described by Horst et al. (1980) an:i Roberts et al. (1984). Proteins (1-2 ng) -wrere susperrled in 900 1 of 5 1TM potassium bicartx:>nate ex>ntaini.rq 9.3 M urea (pl 10.3) an:i incubated for 5 min at roan tenperature. Nonidet P-40 (NP-40) an:i dithiothreitol (DIT) -wrere added to final concentrations of 2.0% an:i O. 5%, respectively. '!his mixture was then incubated on a rockirq platform for 2 hat roan tenperature. After centrifugation for 10 min (15,000 g) to reroc,ve the insoluble material, approximately 300 g of solubilized protein was applied to the cathcx:le en::i of an isoelectric focusi.rq 10 an polyacrylamide gel (2.5 nm x 12 an glass tubes). '!he polyacrylamide gel was prepared as foll0v.1S: 5.5 g of urea was added to a

PAGE 116

104 solution consistin;J of 1.5 ml of Acrylamide stock SOln 1 (see Apperrlix II) am 2 ml of 10% (v/v) NP-40. '!his mixture was diluted to 9 ml with distilled water am incubated at 37C until the urea was CX1tpletely dissolved. Arrplolites were then added (0.25 ml of pl 3.5-10.0; 0.18 ml of pl 5.0-7.0; 0.08 ml of pl 9.0-11.0) alorg with 0.38 ml of the riboflavin-TEMED mixture. '1he solution was then diluted to 10 ml with distilled water. To initiate polymerization of the aci:ylamide, 1.4 ng of amoonium persulfate dissolved in 0.3 ml of distilled water, was added. '1he gels were inmediately parred with the aid of a 10 ml plastic syrirge fitted with tygon tubin;J. '!he aci:ylamide solution was then overlaid with 0.03 ml of 8 M urea am allowed to polymerize urxier fluorescent light. After 30 min, the urea was renoved am o.o5 ml of NP-40 DIT (SOln 4, see Apperrlix II) was added am the gels incubated for an additional 1.5 h. Proteins were then focused to the ancxie (75 V for 0.5 h; 150 V for 2 h; 300 V for 17 h; 450 V for 3 h) in a ruffer system consistin;J of degassed 40 !TM sodium hydroxide (cathode) am 60 !TM sulfuric acid ( aoode) After fcx::usin;J, the gels were renoved with the aid of a water filled plastic syrirge fitted with a small spinal needle. '1he needle was used to carefully rim the gel away fran the walls umer constant water pressure. Followin;J renoval, the gels were equilibrated for 10 min in a solution of 65 !TM Tris-HCl, pl 6.9, 1% sodium dodecyl sulfate (SOO), am 1% 2-nercaptoethanol. 'lhe tube gel was then placed on top of a polyaci:ylamide-SOO slab gel con.sistin;J of a 2 an stackin:J gel am a 12 an separatin;J gel (I..aemnli, 1970; Roberts et al., 1984), am overlaid with melted tq>-gel-sealer (Soln 9, see Apperrlix II). Electrq:horesis was carried out at 15 mA durin;J the tine in whi.dl. the dye front was in

PAGE 117

105 the stackin;J gel am at 20 mA while the trackin;J dye was in the separating gel. 'Ihe seoon:l dimension separating gel consisted of the following: 9. 5 ml of Running Gel Buffer B (Soln 7, see II) 9. 5 ml of Running Gel Acrylamide Soln 6 (7.5% final concentration), 0.19 ml of 20% srs, 18. 7 ml of distilled water, am 21. 3 ng of anm:>nium persulfate. 'Ihe stackin;J gel consisted of the following: 1.0 ml of Stackin;J Gel Buffer E (Soln 8, see~ II), 1.2 ml of Running Gel Buffer Soln 7 (4% final concentration), 0.04 ml of srs, 5.8 ml of distilled water, am 4. 2 ng of amroni.um persulfate. Following electrqi1oresis, the separated proteins were fixed for 1 h by placing the gel in 300 ml gel-fixative (see II). 'Ihe gels were then incubated in distilled water (300 ml at 4 x 5 min), transferred to a solution cx::mtaining 1 nM sodium salicylate (?I 5.7) for 30 min, am then dried urrler vacuum am subjected to fluorograply at -70C (Cralri:>erlin, 1979). one-Dimensional Polyaczylamide Gel Electrg:noresis (1D-PAGE) Membrane proteins (5 to 100 g) were solubilized by diluting (1:5 to 1:10, v/v) the proteins in sarrple dilution ruffer (see~ II). After 20 min, the solubilized proteins were separated on a 7.5% srs polyacrylamide slab gel system as described above. SUbcellular Fractionation of Rat Liver All prcx::edures described below were carried out at 4 c. 'Ihe final membrane fractions were stored at -70C. A fraction highly enriched for plasma ment>rane (P.rpic' et al., 1984) was isolated as described in the methods section of Olapter II. Isolation of mitcx::horrlrial-enriched membrane fractions by the method of Greenawalt ( 197 4) is described below. Isolation of enioplasmic reticulum, golgi, nuclei, am cytq>lasm

PAGE 118

106 was perfonned as described by Fleischer an:l Kervina (1974). In brief, two male Sprague-Iawley rats (100-120 g) 'were fasted overnight an:l sacrificed by decapitation. 'lhe livers 'were rapidly excised, imnersed in ice-cold 0.25 M sucrose, pl 7.4, a.It into small pieces, 'Neighed an:l blotted dry with filter paper. 'lhe liver was diluted with 5 volumes (w/v) of haoogenization buffer (0.25 M sucrose, 10 ITM HEPES, pl 7 .4) an:l haoogenized in a Potter-Elvehjem haoogenizer fitted with a not.or-driven teflon pestle (3 strokes with a IOOditnn pestle, 3 strokes with a tight pestle). 'lhe haoogenate was filtered thralgh five layers of dleese cloth an:l centrifuged at 960 g (10 min). 'lhe supernatant (Sl) was renoved, filtered thralgh dleese cloth an:l used to isolate golgi an:l "light an:l heavy" microsanes as described below. 'lhe pellet was resusperrled with the aid of a loose-fitting r:o.mce haoogenizer in 33.5 ml of 0.25 M sucrose, 10 ITM HEPES (pl 7 .4), an:l 1 ITM ~12 'lhe density of the mixture was adjusted to 1.6 M (45% w/w) with 10 ITM HEPES, pl 7.4 containi.n;J 2.4 M sucrose am 1 ITM ~12 .AWroximately 23 ml of this suspension was placed in the bottan of a ultracentrifugation tube (&'W28 rotor), overlaid with 15 ml of 0.25 M sucrose, 10 ITM HEPES (pl 7.4), am centrifuged at 70,900 g for 70 min. 'lhe mitochorrlria an:l plasma membranes at the interface (0.25 M/1.6 M) 'were renoved an:l cliscarded. 'lhe pellet, containi.n;J the nuclei, was resusperrled in 25 ml of 2. 2 M sucrose, 5 ITM HEPES (pi 7 .4), 3 ITM ~12 with the aid of a loose-fitting r:o.mce haoogenizer. '!his mixture was then diluted to 76 ml am centrifuged in two &'W28 tubes at 70,900 g (60 min) to ootain a membrane pellet enridled for nuclei. 'lhe pellet was diluted to 38 ml with the buffer an:l centrifuged at 76,000 g (60 min) to ootain a nuclear

PAGE 119

107 pellet, whidl was resusperrled in 3 ml of 2.2 M sucrose am 1 ITM M;JC12 ?J 7.4. To isolate golgi am microsanes, the lc:M speed supernatant (Sl), was centrifuged at 25,000 g (10 min). '!he supernatant was then centrifuged at 34,000 g (30 min) to cbtain: 1) a meni>rane pellet containing ''heavy'' microsanes am golgi am 2) a supernatant (S2) containing "light" microsanes. '!he pellet was gently resusperrled with the aid of a loose-fittirg Dounc:le haoogenizer with 52% sucrose in 100 ITM ph05li'late ruffer, pl 7.1 to achieve a final density of 43.7% .AWroxilnately 5 ml was added to the bottan of a SW28 tube, overlaid with 38.7% (5 ml), 36% (5 ml), 33% (5 ml), am 29% (6 ml) sucrose, pl 7.4. 'Ibis gradient was then centrifuged at 70, 900 g ( 60 min) Meni:>ranes enridled for golgi recovered at the 29%/33% interface, diluted with an equal volune of ice-cold water am centrifuged at 120, ooo g ( 40 min) '!he resultirg pellet was resuspenjed in 1 ml of O. 25 M sucrose, pl 7. 4 am stored at -1oc. '!he "heavy" microsanes recovered from the bottan of the sucrose gradient, diluted with 2 volumes of ice-cold water am centrifuged at 124,000 g (60 min). '!he final "heavy'' microsanal pellet was then diluted with 0.25 M sucrose, ?I 7.4. To separate "light" microsanes from the cytosol, the supernatant (S2) was centrifuged at 124,000 g for 60 min. '!he pellet containing "light" microsanes was resusperrled in 2 ml of o. 25 M sucrose, pl 7, 4 am canbined with the "heavy" microsanal fraction to cbtain a mixture of both roogh am snooth microsanes. '!he supernatant containing the cytosolic fraction was stored at -1oc.

PAGE 120

108 sutxnitod'lorrlrial Fractionation Rat liver mitodlon::Jria -were subfractionated by a nmification of the methcxl described by Greenawalt (1974). Livers -were rero.red, minced, arrl placed in Buffer M whidl contains 70 nM sucrose, 200 nM D-mannitol, 4 o nM HEPES, pl 7. 4 arrl BSA ( o. 5 ng/ml) '1he suspension was then haoogenized in a Potter-Elvehjem haoogenizer fitted with a noter-driven loose-fitt.ug teflon pestle (4 strokes). '1he haoogenate was diluted 1:3 in Buffer M arrl centrifuged at 1450 g for 10 min. '1he result.ug supernatant was then centrifuged at 2000 g for 10 min. '1he pellet was diluted to one-half the original haoogenate voltnne arrl centrifuged at 7000 g for 15 min. '1he pellet was resuspenjed in Buffer M to one-fourth the original haoogenate voltnne arrl centrifuged at 7000 g for 15 min. '!his pellet, contain.ug the intact mitochorrlria, was adjusted to 100 ng protein/ml arrl then diluted with an equal voltnne of a 1. 0% digitonin stock solution. '1he digitonin (Sigma D-5628) stock solution was prepared by br.ug.ug Buffer M (without BSA) to a near boil, then addin;J the digitonin while stirr.ug rapidly. '1he solution was allowed to l to roan tenperature before addin;J the BSA. '1he solution contain.ug the resuspemed 11:!Ilt>rane pellet arrl digitonin was mixed gently on ice for 15 min arrl then diluted 1:10 with Buffer M. After centrifugation at 10, ooo g for 10 min, the result.ug supernatant contained solubilized outer mitodlon::Jria 11:!Ilt>rane arrl released internenbrane-space proteins. '!his fraction was stored at 70C. Fractions enridled in either mitoc:horrlrial inner 11:!Ilt>rane proteins or matrix-space proteins -were prepared by the sonication of the mitcplast-contain.ug pellet. '1he mitcplasts -were adjusted to 40 ng protein/ml arrl sonicated with a probe sonicator (35 watts for 4 x 15

PAGE 121

109 sec) in an ice-water bath. 'Ibis suspension was diluted 1:20 with Buffer M arrl centrifuged at 250,000 g for 50 min. '!he supernatant, oontai.ninJ released mitodlorrlrial matrix proteins, was stored at -1oc. '!he resultinJ pellet was washed in 10 volmnes of Buffer M. '!he final nenbrane pellet, contai.ninJ mitochomrial irmer nenbranes, was stored in 1 ml of Buffer Mat -1oc. Isolation of a Membrane-Enridled Fraction fran Tissue CUl.ture Cells Primary cultured hepatocytes or hepatana cells 'iNere washed three times in PBS arrl then irx::ubated at 4C for 5 min in a hypotonic buffer consistinJ of 1 ITM soditnn bicart>onate, pl 7 .5, 10 ITM EIJI'A, 5 ITM benzamidine, arrl 1 ITM IMSF. '!he cells 1iNere rem:wed fran the culture dish with a ruli:>er polioernan arrl haoogenized in a Potter-Elvehjem haoogenizer with a tight-fittinJ teflon pestle (100 strokes) '!he haoogenate was centriftged at 500 g for 10 min to reoove unbroken cells arrl nuclei, arrl the resultinJ supernatant was centrifuged at 10,000 g for 30 min. '!he neri)rane pellet was resusperrled in hypotonic buffer arrl stored at -1oc. Typically, 1-2 ng protein per 25 x 106 cells was recovered in the nenbrane pellet. Antibody Production '!he follc:Mi_nJ mathodology is a m:xli.fication (ariles et al., 1987) of a p.lblished report by Knudsen, (1986). Membrane proteins of interest 'iNere separated by 2D-PAGE arrl electrcp'loretically transferred to nitrocellulose. '!be proteins 'iNere localized by stai.ninJ the nitrocellulose paper with 1% Fast Green dye (5 min) arrl destained in mathanol:water:acetic acid (50:40:10, v/v). '!he same protein spot was excised fran several replicate nitrocellulose blots (6-10), pooled, arrl dissolved in dimathylsulfoxide (J:MSO) as described by Knudsen, (1986).

PAGE 122

110 An equal volume of F:reurx:l's adjuvant was added to the I:MSO mixture, the solution was enul.sified, am then injected sul:cutanea.lsly into six sites alo:D3' the back of a male New Zealam White rali>it. All subsequent secorrlary .ilmunizations -were :perfonned by inplant~ sul:cutanea.lsly nonsolubilized protein .i.ntoobilized on nitrocellulose. '!he protein spot was excised fran Fast Green-stained nitrocellulose blots, rolled into the shape of a cylinier am inserted into the bore of a 16 gauge hyp:xlermic needle (16 x 1-1/2). '!he nitrocellulose was inplanted in the rali>it by expel!~ it with a stainless steel rod. '!he rali>it was bled fran a lateral ear vein 10 days follow~ eadl secomary (boost) inplantation am antibody production was measured us~ .i.nm.moolott~ techniques. IgG was ?,Jrified fran whole sennn as described in AWerrlix III. Detection of Protein Antigen Imnmilized on Nitrocellulose Hepatic membrane proteins -were separated by 1Dor 2D-PAGE. '!he gel was renoved am soaked in transfer buffer (25 nM Tris-base, pi 8.3, 192 nM glycine, am 20% [v/v] ioothanol) for 30 min. '!he gel was then ca:npletely su1:merged in transfer buffer, nitrocellulose was placed on one side of the gel, the gel was then sarrlwiched between two pieces of WhatJnan #1 filter paper, two sporge pads am asseni:>led inside the plastic hals~ of the transfer unit (Ta.vbin et al., 1979). Proteins -were electrqiloretically transferred to nitrocellulose paper (40V, 16 h or 300 mA, 5 h) Follow~ transfer, the nitrocellulose paper was carefully renoved fran the gel, rinsed in water am the proteins stained with either 0.1% Fast Green or 0.1% .Amido Black in ioothanol:water:acetic acid (50:40:10, v/v). After the nitrocellulose paper was destained with ioothanol:water:acetic acid (50:40:10 v/v), the blot was either placed on a glass plate, wrap taut with saran wrap am stored at -2o<>c or the

PAGE 123

111 incubated (2-4 h) with TBS (20 nM Tris-base, 0.5 M NaCl, pl 7 .4) containing 3% BSA, 0.5% 'lween-20, am 0.01% sodium azide (blot ruffer). Alternatively, if a significant annJnt of nonspecific anti.body bin:lin;J occurred, the nitrocellulose paper was blocked with TBS containing 5% non-fat dry milk am o. 5% Tween-20. Purified IgG ootained fran inm.mi.zed ral:i:>its was diluted in blot ruffer (typically 1:250) am incubated with the nitrocellulose paper for either 2 hat roan tenq:>erature or 16 h at 4.oc. '1he antiserum was renoved am stored at -2oc; antiserum catld typically be used 4-5 tilnes wit.ha.rt loss of specificity. '1he nitrocellulose paper was then washed 1 h with several changes of TBS containing 1% BSA am o. 5% soo. Boum anti.body was detected by incubatin:J the nitrocellulose paper with ( 125rJ-Protein A or [ 125rJ-sheep anti-ral:i:>it IgG (106 CEJtVml) in blot ruffer. After 90-120 min, the nitrocellulose was washed as described above am subjected to autoradiograiily. Immunoscreenim of an Adult Rat Liver am a Human Fetal Liver qt.11 cr:NA Expression Li.brazy An F.scheridria coli rn;. coli) strain Y1090 was used to inc:x:,.l1_ate 50 ml of sterile YT nmium (see AWemix IV) containing 0.2% maltose am 50 g/ml anpicillin. '1he strain Y1090 was used durin:J the initial scree.rtin;J of the library, because it is deficient in ion protease am thus, exhibits a reduced rate of degradation of expressed eukaroytic antigens (Youn;J am IBvis, 1983). '!his culture was incubated overnight at 370c with constant aeration (i.e., 100 :q:.m on a :rotatin:J wheel). '1he followin:J 100rning the culture was centrifuged at 2,500 g for 10 min. '!he supernatant was discarded am the cell pellet was resuspemed in 10 ml of SM ruffer (see AWemix IV) containing 0.2% maltose, 50 g/ml

PAGE 124

112 anpicillin an:l stored at 4. 0C. To maintain high platin;J efficiencies, the cells "ttJere stored no le>DJer than 7 days ( 4. o C) an:l are referred to throughout the text as a 5X ]j;. coli stock of cells. A 1:10,000 dilution of a suspension containin;J a htnnan fetal cCNA expression libracy was made in SM ruffer an:l 0.1 ml was then incubated with 0.3 ml of the 5X E. coli stock (37C for 15 min). '!his step was inten:led to allow adsol:ption of the }ilage to the cells. Bacteria grown in the preserx:::e of maltose adsorb bactericpiage lanixla nDre efficiently because maltose in:luces the maltose qJerOn, whidl contains the gene (lamb) oc:x:lin3 for the lanrda receptor. In the iooantine, soft agar (0.8% agarose in L.B. medium, see Awerrlix IV) was nel ted in a boilin;J water bath an:l allowed to cool to 47C, while six 150 X 15 nm agar petri dishes (1.5% agarose in L.B. medium) "ttJere wanned to 42c to allow surface nDisture to evaporate. 'lhe piage:]l;. coli suspension was rapidly mixed with 9 ml of the soft agar solution an:l then poured onto one 150 X 15 mn agar petri dished. '!his step was repeated separately for the remainin;J five agar petri dishes. 'lhe dishes "ttJere then incubated at roan t:.enperature (R. T.) for 20 min to allow the soft agarose to gel. 'lhe dishes "ttJere then incubated at 420c until aba.rt: 50,000 }ilage plaques/dish~ (4 to 6 h). Meanwhile, six 0.22 micron nitrocellulose filters (Micron Separations Inc., #E02HY08250) "ttJere incubated in 10 nN isoprcpyl-B-D thiogalactopyranoside (!PIG) for 2 h. '1he filters "ttJere then air dried, il'nrood.iately overlaid on tq> of the bacteria, marked to corresponi with the marks on the petri dishes an:l incubated ( inverted) for 3. 5 h at 370c. Followin;J this period, the dishes "ttJere cooled to 4.0C so that

PAGE 125

113 the tq> soft agarose 'WOul.d not adhere to the nitroJel.lulose paper an.1 the filters carefully renoved an.1 air dried. 'lhe nitroJel.lulose filters were then incubated with TIE (see IV) contai.nin:J 5% nonfat cb:y milk (carnation) for 1.5 hat R.T. '!he filters were rinsed with TIE to renove excess milk an.1 incubated (4.00c) with pn-ified antiserum (see III) raised to MP-73 (1:100 dilution in TIE contai.nin:J 3% BSA an.1 0.05% 'l\Neen-20). 'lhe followirg nDrnirg the nitroJel.lulose filters were washed 3 times for 15 min each with 50 ml of TIE contai.nin:J 0.05% 'l'vJeen-20. Goat anti-ral:i)it IgG conjugated to horseradish peroxidase (Bio-Rad, inm.mo-blottirg grade # 170-6515) was diluted 1:1,000 in TIE contai.nin:J 2% BSA an.1 incubated with the filters at R. T. for 2 h. Drrirg this tine, the dishes contai.nin:J the filters were covered with foil to reduce light interference of color fonnation. '!he nitroJel.lulose filters were then washed as described aoove with TIE contai.nin:J 0.05% 'l\Neen-20 an.1 the primary antioody detected by incubatirg the filters in 10 ml of TIE contai.nin:J 2 ml of 4-chloro-1-nai:hthol (Sigma C-8890) at a concentration of 3 ng/ml in nethanol an.1 0.01 ml 30% hydrogen peroxide. B1age plaques which produced stronq positive reaction were localized on the original petri dish an.1 renoved with the aid of a pastair pipet (bhmt em 15 nm c.peninJ) In order to allow the piage particles to diffuse out of the agarose, the plugwas incubated overnight in 1 ml of SM buffer contai.nin:J 1 cb:q> of chlorofonn. In general, one plaque contains approximately 106 to 107 infectious piage particles (Maniatis et al., 1982). '!he followirg nDrnirg, 0.01 ml of a 1:100 dilution (in SM buffer) of the piage suspension was incubated with 0.075 ml of the 5X ~coli stock (37C). After 15 min, this suspension

PAGE 126

114 was diluted with 5 ml of melted soft agar (42C) an::l poored onto one 75 nm petri dish oontainin;J 1.5% agarose in L.B. medium. '!he Iiiage were then rescreened as described above, with the exception that only 20 to 100 :(ilage plaques/dish were allc:Med to develop before the dishes were overlaid with the nitrocellulose filters. Followin:J this secorrl room of screenin:J, the positive Iiiage were :rerroved an::l stored in 1 ml SM buffer oontainin;J 1 drcp of dll.orofonn at 4.0C. '!his room of screenin:J was repeated if the cx:rcparison of Iiiage growth an::l anti.bcxly reactivity did not irxlicate that the :(ilage was IJU:re Preparation of gtll Rlage rNA an::l Isolation of crNA Insert Stocks of bacteriq:hage were propagated in bacteria grown on soft agar as follows. '1he plaque-positive :(ilage, whidl was ootained fran :iirm.moscreeni, was used to inoc:ul.ate a strain of i. coli Y1088. '!his strain of bacteria was employed because in contains the hsdRnsdW :necessacy to prevent restriction of foreign rNA prior to host m:xlification (Cales et al., 1983). In brief, 0.1 ml of Iiiage was incubated with 0.1 ml of a 5X i. coli sto::k (15 min at 37C). AWroxilnately 3 ml of soft agar was nelted, cooled to 47C, an::l mixed with the i. coli::(ilage suspension. A 150 X 15 nm petri dish containin;J 1. 5% agarose in YT medium suwlernented with o. 04 ng/ml 5-braco-4-dllo:ro-3-irrlolyl-B-galactoside (X-gal) an::l 0.078 ng/ml !PIG was wanned to 420c; the i. coli::(ilage:soft agar suspension was then poured onto the YT plate. '!he plate was incubated at R.T. (15 min) to allow the agarose to gel an::l then incubated at 37C tmtil confluent lysis of the i. coli ocnirred. To isolate the :(ilage, only those petri dishes whidl contained clear plaques initially (i.e., irxlicative of p,age containin;J crNA insert) were incubated with 2 ml SM buffer for 4 h at 4.0C; the buffer

PAGE 127

115 was then rem::,ved arrl centrifuged at 15,000 g for 2 min. 'lhe supernatant conta~ the Ji}age lysate was rem::,ved, suwlemented with 1 drq> of chlorofonn arrl stored at 4.0C. Preparation of Ji'}age Il'lA. was prefonned by a roodification of the method described by Maniatis et al. ( 1982) Briefly, 3 1 of the Ji'}age lysate suspension was incubated with 0.3 ml of the 5X i. roli stock Y1088 (15 min at 37C). Approxilllately 9 ml of a L.B. madium solution cont:ainin;J 0.4% agarose (SeaKem, ~) was melted, CX)Qled to 47C arrl then mixed with the i. roli:Ji}age suspension. '!his mixture was poured onto one 150 X 15 mm agar petri dish (1.5% agarose in L.B. madium), the petri dish was CX)Qled at R.T. (15 min) arrl then incubated at 37C until confluent lysis occurred (16 h). '!his step was repeated separately for an additional five petri dishes arrl the yield of Ji'}age Il'lA. was typically 0.15 to 0.2 ng. '!he isolation of piage Il'lA. was perfonned by incubatin;J the plates with 5 ml SM buffer for 4 h. '!he buffer was rem::,ved, pooled with the SM buffer fran the rema~ five petri dishes arrl centrifuged at 8,000 g for 10 min. '!his centrifugation step was interrled to rem::,ve any residual agarose. In order to rem::,ve bacterial RNA arrl Il'lA., the supematant was incubated with RNase A (0.01 ng/ml) arrl mase I (0.025 ng/ml) for 1 h at 37C. Ice-cold SM buffer conta~ 2 M NaCl arrl 20% polyethylene glyrol (MW 8,000) was then added to the solution arrl this mixture placed on ice for 1 h. 'lhe intact Ji'}age were then separated by centrifugation at 10,000 g for 20 min (4.0C). '!he supematant was decanted arrl the pellet conta~ the Ji}age was resusperrled in 4 ml of SM buffer. '!he piage suspension was then suwlemented with 0.04 ml of 0.5 M EIJl'A arrl

PAGE 128

116 0.04 ml of 10% soo, vortexed, am incubated with mase I-free Proteinase K ( O. 05 ng/ml) for 1 h ( 68 C) Protein was rercoved by extractin;J the suspension five times with ienol (distilled am equilibrated, see AWerrlix IV). '!he aqueoos piase was extracted an additional five times with a solution cx>ntainin;J ienol:dllorofonn:isoamyl alccnol (24:24:1) am then twice with dllorofonn:isoamyl alccnol (24:1). AWroximately 0.3 ml of 7.5 M anm:>nium acetate (NH40Ac) am an equal volume of ice-cold isopropanol was added to the rnA solution. '!he rnA was precipitated by incubatin;J this solution at -70C for 20 min, follOINed by centrifugation at 15, ooo g ( 10 min) '!he supernatant was decanted am the pellet containin;J the rnA dried by vacuum centrifugation. '!he :r;ra.ge rnA was then resusperrled in 2 ml TE ruffer (see AWerrlix IV) am dialyzed for 16 h against 2.0 1 of a ruffer cxmsistin;J of 10 nM Tris, pl 7.4 am 10 nM EIJl'A. '!he dialysis ruffer was dlan;Jed to 10 nM Tris, pr 7.4 am 0.1 nM ED1'A (2.0 1, 5 hat 4C) am the rnA precipitated as described above. '!his step was inten::led to rercove any contaminatin;J netal ions whidl may interfere with the subsequent en:lonuclease restriction digest. To analyze the crNA insert, the Ji')age rnA was resusperrled in o. 8 ml sterile distilled water am restricted with the bacterial errlonuclease F.coRl. in a reaction ruff er cxmsistin;J of O .1 ml of Bethesda Research laboratories reaction ruffer #3, 0.01 ml of 2-mercaptoethanol (0.5 M), 0.01 ml of BSA (fatty acid free at 15 ng/ml), 0.05 ml of distilled water, am 100 units of F.coRl. restriction en:lonuclease. After 1 h at 37C, an additional 100 units of F.coRl. errlonuclease was added. '!he reaction was terminated after an additional 1 ham the rnA precipitated by adclin;J o .1 ml of 7. 5 M NH40Ac am two volumes of 95% ethanol. '!his

PAGE 129

117 mixture was incubated at -1oc for 20 min arrl then centrifuged at 15,ooo g for 10 min. 'lhe pellet contai.nir] I:NA was dried by vaa.n.nn centrifugation arrl then resusperrled in 0.07 ml TE l:uffer suwleme.nted with 2 1 of I:NA gel loadin:J l:uffer. '!he er.NA insert was separated fran the Ibage I:NA by electrc:pio:resis on a 1% agarose gel with a TAE l:uffer system (20 volts, 16 h) as described by Davis et al. (1986). Followin:J electrc:plo:resis, the agarose gel was incubated with a solution contai.nir] o. 5 g/ml ethidium branide (30 min) '!he gel was destained for 30 min with nultiple dlanJes of water arrl the I:NA visualized with the aid of a UV light (15 sec) '!he cCNA fragment was then electroeluted fran the agarose gel as described by Davis et al. (1986). Briefly, the region of the agarose gel corresporning to the cCNA was rerocwed with the aid of a razor blade. 'lbe gel plug was place::l in dialysis tubin:J (MW cutoff 12,000 dalton.s) contai.nir] 2 ml of TAE l:uffer. 'lbe er.NA was then electroeluted at 60 volts for 16 h in a ''minigel" ~tus contai.nir] TAE l:uffer. '!he polarity was then reversed for 5 min to dislodge any I:NA fran the dialysis tubin:J wall. 'lbe dialysis bag was rinsed with an aatition 2 ml of TAE l:uffer, both solutions were then pooled arrl centrifuged at 10,000 g for 5 min to reroc,ve any contaminatin;J agarose. '!he cr:NA solution was concentrated to a volume of O. 3 ml by a series of sec-1:::Jutanol extractions. 'lhe cCNA was then ethanol precipitated arrl dried as described above. SUbclonim of the cr:NA Insert Identified by Inmmoscreenirq In order to sulx::lone the cCNA fragments ootained fran iJrm.moscreenin, plasmid I:NA was first :restricted for use in subsequent ligation reactions. 'lbe plasmid used in these tran.sfonnations was

PAGE 130

118 J:UC19. '!his plasmid was dlosen both for its anpicillin resistance gene arrl the presence of a unique cloninJ site within the lac z gene (Vieira arrl Messirg, 1982), thus allc,...rirg recanbinants containirg cloned CNA in the lac Z gene to be easily distin]uished fran non-recanbinants when 9rcM1 on X-gal plates. '!he plasmid was partially digested with F.caRl. by treatirg 0.01 ng of tvC19 with 5 units of F.caRl. for 30 min as described above. '!he CNA was then ethanol precipitated, dried arrl resuspemed in distilled water. AWroxiJnately 50 rg of J;VC19 was then incubated with a three-fold 11K>lar excess of cCNA. '!his reaction mixture consisted of o. 02 ml containirg tvC19 vector CNA, cCNA, 2 1 of 5 nN ATP, 2 1 of BRL lOX reaction ligase l:uffer, 1 1 of T4 CNA ligase. '!his mixture was incubated at R.T. for 4 h. Control reactions included vector CNA withalt cCNA in the presence or absence of T4 ligase. '!he follc,...rirg methcxlology is similar to the procedure used by Maniatis et al. (1982) to make bacterial strains c:x:rrpetent for tran.sfo:rmation with plasmid CNA. E coli strain 'IG-1 was used to inoculate a 50 ml culture flask containirg YT medhnn (16 h at 37"C) as described above. '!he suspension was centrifuged at 2,000 g (5 min) arrl the pellet containirg E coli was resuspemed in 10 ml of i~ld 50 nN cac12 '!he cells were incubated an additional 40 min on ice, centrifuged again, arrl then resusperrled in 5 ml i~ld cac12 '!he cells were stored on ice at 4.oc for no lorqer than 48 h. AWroxiJnately 1 1 of the ligation reaction was mixed with 0.1 ml of the E coli 'IG-1 c:x:rrpetent cells. Cells were also incubated separately with the tml.igated as well as the ligated vector. After a 1 h incubation (4.0"C), the cells were ''heat shocked" at 42"C for 3 min, transferred arrl spread unifo:nnly with a glass spreader on a petri dish

PAGE 131

119 containinJ 1. 5% agar in YT nwadia suwlenert:.ecI with o. 05 ng/ml of anpicillin, 0.04 ng/ml of X-gal, arrl 0.08 ng/ml of !PIG. 'lhe plates were maintained at R.T. for 15 min then inverted arrl incubated at 37C l.llltil white colonies awe,ared. 'lb.is incubation was stcg>ed as soon as white colonies awe,ared to avoid contamination with "feeder'' bacterial colonies. 'lhe in:tividual colonies were rem:::,ved, streaked onto an additional 1.5% agar:YT nwadium plate containinJ anp/X-gal/IPIG arrl the plate was incubated as described above. 'lhe in:lividual colonies fran this plate were used to inoculate a 5 ml YT nwadium culture for small scale plasmid I:NA isolation as described in~ V. Isolation of Total RNA fran Tissue CUlture Cells arrl Rat Liver Tissue 'lhe followin;J procedure describes the isolation of RNA fran both liver tissue arrl tissue culture hepatana cells (Chaoc:zynski arrl Sacx:ru., 1987; Chirgwin et al., 1979). 'lhe tissue culture cells were rinsed in PBS, scraped fran the petri dish with the aid of a rubber policeman arrl centrifuged at 2,000 g for 10 min. 'lhe cells were then suspenied in 1 ml of isolation b..rffer (4 M guanidinium thicx:yanate, 25 nM sodium citrate, i;:iI 7.5, 0.5% sarcosyl arrl 0.1 M 2-mercaptoethanol) arrl hcaoogenized usin;J a Potter-Elvehjern hcaoogenizer (20 strokes). '!his solution was then diluted with 0.1 ml of 2 M sodium acetate, i;:iI 4.0, 1 ml of distilled:equilibrated plenol (see~ IV), arrl 0.2 ml chlorofonn: isoamyl alcohol ( 49: 1) mixed vigorously arrl then cooled on ice for 15 min. 'Ihe sanples were centrifuged at 10,000 g for 20 min ( 4 o C) 'lhe uwer aqueous J;i1ase containinJ RNA was rem:::,ved arrl an equal voll..DOO of isopropanol was added. 'lb.is mixture was then incubated at -2oc (1 h) arrl centrifuged at 10,000 g for 20 min. 'lhe pellet containinJ RNA was dissolved in 0.3 ml of the isolation b..rffer arrl

PAGE 132

120 precipitated by the ao::tition of an equal volume of isoprq::,anol (-2oc for 1 h) '1he RNA was isolated by centrifugation at 10, 000 g for 10 min (4.0"C). '1he pellet was resusperxied in 75% ethanol, sedimented, dried, arxl resusperxied in 0.05 ml of 0.5% SIB by heatin;J at 65C for 10 min. For isolation of RNA fran liver tissue, the liver was rem:,ved, minced, arrl diluted with 5 volumes (v/w) of the isolation ruffer. '!he remainin;J steps were identical to those used for the isolation of RNA fran tissue culture hepatana cells, with the exception that reagent volumes were scaled up prqiortionally to the original haoogenization volume. Electra;noretic Separation of RNA arrl Northern Analysis RNA was separated by agarose gel elect:rq:i1o:resis as described by r:avis et al. (1986). In brief, ai:Proximately 15 g of total cellular RNA [or 2 g of poly(A+)RNA] was dried by vaCUllll\ centrifugation arxl resusperxied in 0.02 ml of a RNA gel loadin;J ruffer which consisted of 0.72 ml of deionized fo:nnamide, 0.16 ml of lOX M:>PS ruffer (0.2 M K>PS, pl 7.0, 0.05 M scxlium acetate, 0.01 M EIJI'A), 0.26 ml of fonnaldehyde (37% reagent grade stcx:k), 0.18 ml of H 2o, 0.1 ml of 80% glycerol, arxl 0.08 ml of bratqilenol blue. '!he RNA sanples were heated at 95C for 2 min arrl placed on ice (5 min). '1he RNA was then separated on a 1% agarose gel with a 1X M:>PS ruffer system containin;J 0.66 M fonnaldehyde (100 volts for 4 h). Followin;J electroplo:resis, the gel was incubated for 5 min each in two d"lanJes of distilled water arxl then 60 min in 20X SSC ( 3 M NaCl arrl o. 3 M scxlium citrate, pl 7. o) 'lhe gel was then placed upside down on top of two 2" spon:Je pads arxl four sheets of Wha'bnan #3 filter paper saturated with 20X SSC. Gene Screen nylon transfer membrane was placed on top of the gel arrl three layers of

PAGE 133

121 filter paper an:i a stack of folded paper t.c:Jwel.s was then placed on tq> of the membrane. A small weight was placed on tq> of the paper towels an:i the RNA was transferred overnight by capillary action. 'lhe followirg norni.nJ the membrane was rinsed in water, taut with Saran Wrap an:i with the RNA-side up irradiaterl at a distance of 35 en with seven gennicidal TN J:::w.bs, 15 wattsjrulb (Clmrc:h an:i Gilbert, 1984). Detection of specific mRNA species was perfonrm by hybridization with radiolabeled cIIU\ as described Wahl et al. (1979) an:i D:ivis et al. (1986). 'lhe Gene screen filter was first prehybridized at R.T. with B.lffer N (see~ IV). Awroximately 100 rg of cIIU\ fragment isolaterl by restriction of the :p.JC19 plasmid with F.coRl. was labeled with [32 PJ-dATP via the primer extension method an:i diluted with B.lffer N at a specific activity of 2 X 106 q:nVml. Followirg rerroval of the prehybridizirg solution, the prcbe was incubaterl with the nylon membrane an:i incubaterl 36 h at 42C. 'lhe nylon membrane was then washed as described by Maniatis et al. (1982). Briefly, the hybridization solution was renw::,ved an:i the filters washed fo.ir times, for 10 min each, (300 ml) with 2X SSC an:i 0.1% soo at roan tenperature. 'lhe nerbrane was then washed two times for 1 h each in 500 ml of a solution containirg 1X SSC an:i 0.1% soo at 65C. 'lhe nylon membrane was next washed for 0.5 h with 500 ml 0.2X SSC an:i 0.1% soo at 65C. Followirg this final wash, the membrane was air dried, cxwered with Saran Wrap, awlied to X-ray film an:i exposed at -70C.

PAGE 134

CHAPl'ER VI IDENl'IFICATION AND ClIARACTERIZATION OF AMINO ACID STARVATION INIXJCED HEPATIC MEMBRANE marEINS Introduction Eukacyotic cells respon:l to many different fonn.s of envirornrental changes in a dlaracteristic fashion. Exposure of cells to either acute elevations in ambient teJrperature, sulfhycb:yl-nooifyin:J reagents, or amino acid analogs causes a rapid in:iuction of a small graip of proteins known as "heat shock" proteins or hsp (craig, 1985). 'Ihe ll'Dlecular weights of these proteins range f:ran 28 kDi to 100 kDi (Weldl, 1983). At the cellular level, general RNA an:l protein biosynthesis is reduced dramatically, whereas the biosynthesis of a few niRNA's codin:J for the hsp proteins is enhanced (Bonner an:l Pardue, 1976; Tissieres et al., 1974). With the exception of a 68 kDi hsp protein, all of the hsp proteins are constitutively expressed at low levels. 'Ihe two 1l'Dst abtJrmnt hsp proteins (hsp 70 an:l hsp 90) are highly conserved am:nq organisms as diverse as Ecoli an:l man. 'Ihe ll'Dlecular medlanism responsible for the in:iuction at the protein level~ to be enhanced transcription of the respective structural genes. 'Ihe function of the major "heat shock" proteins is unknown; however, the mamnalian hsp 70, bi.ms an:l has the ability to hydrolyze, ATP (Zylic et al., 1983). Up::>n heat shock many nuclear proteins becx:lne insoluble, at the same time hsp 70 migrates to the nucleus, suggestin:J that one function might be to repair "heat" denatured proteins (Weldl an:l SUhan, 1987). 122

PAGE 135

123 Rotlnnan am Sctnnid (1986) denonstrated that the uncoatin;J of cx:>ated vesicles in vitro by the uncoatin;J ATPase is nmiated by hsp 70. '1hese authors cx:>Se.L"""Ved that in the presence of ATP hsp 70 birrls to clathrin cages am hydrolyzes ATP, resultin;J in disniption of the clathrin cages. Napolitano et al. (1987) denonstrated that the hsp 68 am 70 proteins partition into Triton X-100 soluble cellular fractions. Both proteins were co-isolated with an intennediate filament-rich cytoskeletal fraction. Coldlicine inhibited the interaction of hsp 68 with the cytoskeleton. '!he authors have sugJeSt that hsp 68 am hsp 70 may act as interfacin;J I1Dlecules between cytoskeletal elerents am other cytoplasmic stnictures urrler nonnal. con:litions. Another series of hepatic proteins awear to be expressed durin;J periods of glucose stai:vation arrljor calcil.nn iorqnore treatment (Lee, 1987). '!he I1Dlecular weights of the two major glucose-regulated proteins (grps) are 78 kIB am 94 kIB. Although originally detected in chick fibroolasts, the grps are very abumant in secreto:ry cells sud1 as hepatocytes (Pelham, 1986). 'lhese proteins are distinct fran the ''heat shock" proteins based on their ionic charge am electrcploretic nooility in two-dimensional polyac:rylamide gels. '!he function of the glucoseregulated proteins is unknown. Cellularly, glucose deprivation inhibits N-linked glycosylation of nascent protein in the emq:,lasmic reticulum, resultin;J in the acamulation of urrlerglycosylated protein aggregates. Munro am Pelham ( 1986) showed that grp 78 am grp 94 are very ab.Jnjant in the emq:,lasmic reticulum which has lead sate laboratories to postulate that they might function by birrlin;J abnonnal. or urrlerglycosylated proteins. It has also been~ that the 78 kIB protein may be involved in the stabilization of nsnbrane-bami am

PAGE 136

124 secretory protein (Wu, 1981). '!he 94 kDa protein may be the calcium trans!X)rt ATPase of the sarcoplasmic reticulum (Lee, 1987). Recently the availability of cr:NA clones as hybridization probes has alla.ved the study of the nolecular medlanism.s of regulation (Lin arrl Lee, 1984). 'lhese studies have revealed that glucose deprivation causes a 10-to 20-fold increase in the steady-state levels of mRNA for these proteins. '!he researdl described in this Oiapter fc:x:,.JS0d on the mntrol of hepatic nenbrane protein biosynthesis durin;;J amino acid sl:arrcition. Because of the i:nysiological irrportance of hepatic protein synthesis arrl gluconeogenesis, the cellular response to amino acid deprivation has been characterized in the liver. '!he liver is, after nuscle tissue, the organ with the highest rate of protein biosynthesis. Only 20% of the total protein made by the liver is used within the liver, whereas awroximately 80% of the protein synthesized is secreted into the blocxl. In vivo (i.e., liver perfusion studies with whole anbnal.s), the strin;;Jent anission of amino acids in the perfused rat liver results in a rapid decrease in the biosynthesis of r:NA, arrl RNA (Zem et al., 1982). Total liver RNA levels decrease due to both inhibition of de novo RNA biosynthesis as well as increased degradation of existin;;J RNA (Enwon arrl Munro, 1970). General protein biosynthesis also~ to decrease durin;;J amino acid starrcition. In liver, amino acids may be the primacy regulators of resident protein degradation. Hen.shaw et al. (1971) oosei:ved that within two days followin;;J amino acid sl:arrcition, total rat liver protein content was decreased as nudl as 40%. Inhibition of protein degradation can be elicited with cacplete amino acid mixtures at (X)ncentrations as low as half the nonnal plasma levels in the perfused rat liver (Poso et al., 1982; Poso arrl Mortirrore, 1984; Jeejeeliloy et

PAGE 137

125 al., 1973). Several studies with perfused rat liver (Woodside an::i MortinDre, 1972) an::i isolated rat hepatocytes (Seglen et al., 1980) have shown that sate amino acids can directly inhibit hepatic proteolysis. Perfusion studies with rat liver has denonstrated that Ir-leucine, Ir tyrosine, Ir-:r;:nenylalanine, Ir-glutamine, Ir-proline, Ir-histidine, Irtryptq::han, an::i Ir-nethionine are as a group as effective at inhibiting hepatic proteolysis as c::arplete plasma mixture (Paso et al., 1982). In addition to lowering total hepatic protein biosynthesis, amino acid deprivation results in degradation of both intracellular hepatic proteins an::i extracellular plasma proteins for degradation (a-istri.ta et al., 1986). '1he signals which initiate these events are unknown, hOiY'eVer, it is thought that a decrease in the steady state level of amino acyl-t.RNA's may be responsible (Oscar, 1983). other investigators have suggested that transcriptional an::i translational med1anisms nust be involved, since the intracellular ooncentration of amino acids does not chan:Je significantly. '1he response to amino acid starvation in vitro ai:pears to be quite distirct fran that of the whole anilllal.. '1he incoqx>ration of radiolabeled uridine into RNA, thymidine into mA, an::i Ir-leucine into protein only slightly affected follC1Ning amino acid starvation of cultured rat hepatocytes (Harrll.ogten an::i Kilberg, unpmlished observations). Irxieed, over a period of six hours, the rate an::i alOClUJ1t of radiolabeling is identical to cells cultured in amino acid suwlemented ne:li.1.ml. Similar results have also been observed with the rat hepatana cell line, Fao. Cellular processes other than protein nr.ibilization are also enhanced during amino acid deprivation. fttJst notably is the stinulation of neutral amino acid transport across the

PAGE 138

126 hepatic plasma membrane, a process referred to as adaptive regulation (Guidotti et al., 1978; Shotwell et al., 1983; Kilberg et al., 1986). System A is the plasma membrane carrier protein responsible for the enhanced transport. .AWroximately 60 to 90 min followinJ :rertJVal of amino acids fran the culture na:lium, System A-mediated transport begins to increase in primary cultures of rat hepatocytes (Kelly arrl It>tter, 1978; Kilberg et al., 1986). Imuction of System A activity is blocked by inhibitors for both de novo RNA (Kelly arrl It>tter, 1978) arrl protein biosynthesis (Kilberg et al., 1985) as well as the glycq:>rotein biosynthesis inhibitor 'l\micamycin (Barber et al., 1983). In contrast, the addition of a sinJle neutral amino acid to the culture na:lium causes a rapid decay in the stinulated transport activity (Hamlogten arrl Kilberg, 1984; Kilberg et al., 1986). Recent studies have in:licated that Irasparagine is the best repressive substrate, elicitinJ a decay in transport activity with a half tine of 1.5 h (Kilberg et al., 1986). '!here is little infonnation in the literature concemirg the imuction of proteins specific for amino acid deprivation. Ievinson et al. (1980) cilsel:ved that amino acid stru:vation of cultured chick ent>:ryo cells irrluces the biosynthesis of four proteins. Within 2 h after incubation of cells in amino acid free-na:lium, proteins correspon;ting to nolecular weights of 89, 73, 35, arrl 27 kDa were irrluoed. Biosynthesis reached steady state within a h arrl began to decay after 12 h. Kelly arrl SchlesinJer (1978) reported that exposure of chicken ent>cyo fibroolasts to hydrovaline imuce the biosynthesis of three proteins of nolecular weight 95, 76, arrl 22 kDa. 'lhe .irrluction of protein synthesis was blocked by actinanycin D suggestinJ transcription control. 'lhese proteins were detergent extractable arrl localized to a membrane-enriched

PAGE 139

127 fraction. '!he 22 kO:i protein was also fa.mi to bin:i to lentil lectin Sep'larose arrl contained mannose, suggestirg that it was a menbrane glycx:,protein. Further analysis revealed that these proteins :resporrled both to glucose deprivation arrl to elevations in 'ten'perature arrl are prd::>ably similar, if not identical, to the ''heat shock" or glucose regulated proteins. Although the 1IDlec:ular regulato:ry mechanisms whidl occur in mamnalian cells durirg amino acid stai:vation are not 'Well un::lerst:cxxi, in lower eukaryotes arrl bacteria a great detail of infonnation has been cbtained. In bacteria, depletion of a sirgle amino acid leads to increased transcriptional activity of those genes responsible for the enzymes in the rognate pathway (Ames arrl Garcy, 1959). For exanple, bacteria adapt to stai:vation of Ir-histidine by elevated levels of expression of all the enzymes for the biosynthesis of Ir-histidine. In the yeast Sacdlaranyces a:rrevisiae, the pathways of amino acid biosynthesis are regulated in response to both general arrl specific amino acid deprivation (Hi.nnel:xJsch, 1986). Specific control occurs 'When cells are deprived of a sirgle amino acid. 'As a result, the cells in::luce the biosynthesis of enzymes responsible for the production of the particular deficient amino acid. For exarrple, the activity of isopropylmalate isanerase, the SE!CX)OO enzyme in the pathway for leucine biosynthesis, is inversely proportional to the availability of Ir leucine. 'lhe stnictural gene for isopropylmalate isanerase, m.Jl, has been cloned arrl analysis of the cellular mRNA levels have revealed an increased expression of the m.Jl transcript 'When Ir-leucine is absent fran the culture medium (Hsu arrl Sdtlnmel., 1984) '!he response to total amino acid stai:vation results in increased

PAGE 140

128 biosynthesis of several enzynes which are capable of p:roducirq amino acids as the availability of extracellular amino acid d"laRJes. 'Ihe general cx:>ntrol systems coordinately :regulate the expression of stnictural. genes which encode the amino acid biosynthetic enzynes fran several different pathways. For exanple, '\lrhen yeast cells are placed in a medium deficient of Ir-histidine the expression of genes encod.in;J the enzynes for Ir-histidine biosynthesis are irrluced, however, the biosynthetic enzynes for Ir-arginine, I.r-leuci.ne, Ir-isoleuci.ne, I.rt.rypt:qi)an an::l Ir-lysine are also irrluced. Recently it has been shown that the general amino acid control response oocurs at the level of transcription. 'Ihe first step in the Ir-histidine biosynthetic pathway in yeast is the fonnation of J;Xl(JSinOribosyl-ATP. 'Ihe activity of this enzyme, ATP J;Xl(JSinOribosyl-transferase, is derepressed several-fold un:ier conlitions of Ir-histidine deprivation (Hinnebusch an::l Fink, 1983). Usirq a cloned HISl I:NA prcbe, Hinnebusch an::l Fink (1983) absei:ve::l a 2-to 10-fold increase in the HISl transcript durirq amino acid deprivation. 'Ihe general amino acid control response is mediate::l by a trans actirq protein, GNC4 an::l a cis-actirq I:NA sequence (5'~-3'). 'lb.is I:NA sequence is foum in the 5' -noncodin:J regions of all genes which respon:i to amino acid deprivation (Hinnebusch et al. 1985) Hq>e an::l Struhl (1985) suggeste::l that the activation of the IIDre than 25 genes involve::l in amino acid biosynthesis oocurs via a "global" IIDlecular signal. 'Ihe GNC4 protein was identifie::l an::l shown to bin:i at the cis actirq rNA sequerx::es. Yeast nutants which contained deletions within the GNC4 gene were unable to elevate transcription, yet these strains grew on amino acid suwlemented medium. 'lhese observations lead Anrlt

PAGE 141

129 et al. (1987) to identify t\oJO genes, BAS1 am BAS2, resp:,nsible for the control of basal level transcription of the HIS4 gene. In addition, the BAS2 gene was shown to encxxJe a protein that specifically boom to the 5 '-noncoclin;J region of the HIS4 gene. Materials am Methods See the materials am methods for protein dlemistry am nDlecular biology in Olapter V. Results In an effort to gain a better urrlerstan:lirq of the regulation of hepatic merrbrane protein biosynthesis by small nutrient nDlecules, a series of experiments were initiated to IlDllitor the effect of amino acid starvation on the synthesis of irrlividual merrbrane proteins usirg cultured rat hepatocytes. '!he goal of these initial studies was to identify any hepatic membrane proteins for wi.ch biosynthesis was controlled by amino acids, specifically those proteins that exhibit enhanced biosynthesis durirg amino acid deprivation. '!he basic experimental awroach was to incubate freshly isolated hepatocytes with L[4,5-3H]-leucine un:ler corxtitions of either amino acid deprivation (NaKRB), amino acid supplenentation (NaKRB containin;J 20 n'M L asparagine, NaKRB/ASN) or amino acid deprivation in the presence of 4 M actinanycin (NaKRB/ACT). A<;paragine was used because it was fa.m:i to repress the starvation-deperrlent transcriptional in:iuction of the hepatic System A transport activity to a greater extent than any other sirgle amino acid am as effectively as an amino acid-enriched culture medium such as MEM (Bracy et al., 1986). 'Ihose proteins wi.ch had incorporated L[4,5-3H]-leucine durirg this time were visualized by isolatirg a crude membrane fraction (10,000 g membrane pellet)

PAGE 142

Figure 14. Electrqiloretic Protein Pattern of a Ccx:lnassie Blue stained 'lwo-Dimensional Polyacrylamide Gel. .AWroximately 0.3 nq of protein fran a 10,000 g rat liver membrane fraction was separated by 2D-PAGE. '!he proteins were fixed in trichloroacetic acid am then stained with Ccx:lnassie Blue stain as described in Chapter V.

PAGE 143

131 pH 7.0 6.0 5.0 4.0 I') I 0 -)( 200 r .. .c a, G) 92 5 66 D 5 .. l 0 31 :::, "' Q) -+ 0 IE F ,. +

PAGE 144

132 separatin; the proteins by 2D-PAGE, arrl subjectin; the gels to fluo:rograply as described in Olapter V. '!he 10, ooo g membrane fraction represents a mixture of proteins derived fran the plasma membrane, mitochomrial membrane, am to a lesser extent erxlq:>lasmic retiall.rnn. Fig. 14 shows the typical protein patteni ooserved fran the 10,000 g membrane fraction after stainin; a 2D-polyac:cylamide gel with Ccmnassie Blue. 'As shown by the arrows in Fig. 15, a series of membrane proteins aweared by visual inspection of the fluorograms to exhibit increased synthesis when all. tured un:ler con:lition.s of amino acid deprivation. Although there are many c::han;Jes in the relative degree of L[4,5-3HJleuci.ne incorporation, five proteins of rrolecular weight arrl pl values: 73 k:Da, 7.0; 66 k:Da, 6.4; 66 k:Da, 6.2; 66 k:Da, 6.1; arrl 45 k:Da, 6.0 were readily detected arrl their synthesis was reproducibly elevated by amino acid stai:vation. '!he initial identification of these five membrane proteins was perfonned by Mary Harrllcxft:en of oor laboratocy. 'lb estimate the relative rate of de l10V'O protein biosynthesis for eadl of these five proteins, the ann.mt of L[4,5-3HJ-leuci.ne incorporated was quantitated. '!his was perfonned by excisin; the in:lividual proteins fran the gel arrl neasurin; the ann.mt of radiolabeled L-leuci.ne incorporation by scintillation spectranetcy. '!he data are presented in Fig. 16 arrl are expressed as a ratio of radiolabeled L-leuci.ne incorporated (i.e., c:pn NaKRB/c:pn NaKRB/ASN or cpn NaKRB/c:pn NaKRB/ACI'). '!he ratios for rrore than twenty additional proteins were also detennined arrl the stamard deviation of the average ratio is in:licated by the dash lines (0.8 0.20). In each case for the five proteins chosen, the inclusion of actinanycin D durin; the stai:vation period aweared to block lTl.ld1 of the increase in synthesis

PAGE 145

Figure 15. Fluorogram of the Synthesis of Irrlividual Hepatic Membrane Proteins in Response to Amino Acid st:ai:vation. Rat hepatocytes were isolated arxi placed into primary culture (25 x 106 cells :per 150 nun petri dish) in either Nal
PAGE 146

134 pH 8.0 7.0 6.0 s.o 4.0 M I 0 200 ,-)( .. 116 ..c tn 92 Q) s 66 D s I. 1 0 31 "' Q) + 0 22 I E F +

PAGE 147

C') 'o 200 >< .. 116 a, 92 Q) 3 66 .. 0 :::, 31 "' Q) o 22 135 8.0 7.0 pH 6.0 5.0 4.0 -----I EF ----#) + 5 D 5 l +

PAGE 148

136 8.0 s.o 4.0 C') I 0 200 )( .. 116 .c en 92 4) s 3 66 D s .. l g ::::, 31 "' 4) + -0 22 IEF---~) +

PAGE 149

Figure 16. Quantitation of the Rates of Inco:rporation of Radiolabeled IrI.eucine into Membrane Proteins. '!he proteins of interest -were localized by overlayirg the fluorograms shown in Fig. 15 on the dried gels. '!he proteins -were then excised as gel plugs am the radioactivity detennined by scintillation spectraooti:y. '!he results are expressed as the ratios of c:pn NaKRB/c:pn NaKRB + ASN or c:pn NaKRB/c:pn NaKRB + ACT inco:rporated into irrlividual proteins.

PAGE 150

'=' 3.0 u C .. 2.s 0 z "' 2.0 C + 1. s '1.0 ~o.s 0 .. 0 0 138 Control Protein Number [J ASH II ACT

PAGE 151

139 suggesti.rg that the effect was the result of transcriptional CXXItrol (Fig. 16). '!he protein designated as spot 1, corresporrlirg to an isoelectric point of 7. o am a nolecul.ar 1weight of 70-75 kDa was chosen for further stu:ly because: 1) the protein irrluction was blocked by actinanycin D; 2) the level of irrluction, although not large, was reproducible; 3) the protein was abJrrlant enaigh to be detected by Fast Green stain of nitrocellulose blots; am 4) the protein was 'Nell separated fran other protein spots. In order to characterize the protein, designated as MP-73, with respect to its sulx:ellular location am regulation by amino acids, nono specific polyclonal antibodies directed against the protein were raised in New Zealarn white rali:>its. Based on Fast Green staini.rg of a nitrocellulose blot containi.rg 300 g of the 10,000 g membrane preparation separated by 20-PAGE prior to electrd:>lotti.rg, the estimated content of MP-73 represents awroximately 0.4% of the membrane protein in the crude fraction studied. PUrification of the protein am production of antisera usi.rg conventional methodology was not possible because of the low al::ormnce am hydrq:'habic nature of MP-73. 'As a result, a nmification of the methcxi described by Knudsen (1986) was devised with advice fran Dr. T.W. O'Brien of this deparbtent. Knudsen separated a carplex mixture of chick embryo adhesion-related glycoproteins by ID-PAGE. Followi.rg transfer to nitrocellulose, awroximately 20 to 40 g of the nitrocellulose baJrrl protein was dissolved in diJnethylsu,lfoxide (I:J.1SO), diluted with an equal volume of Freun:i's adjuvant am injected into New Zealarn white ral::bits. After an additional four series of injections (spaced at two -week intervals), sennn antibody production was detected via conventional i.nnuncblotti.rg

PAGE 152

Figure 17. Anti.body Production Scheme for Prepari.rg :r.t:>nospecific Polyclonal Anti.bodies. Membrane proteins -were separated by 2D-PAGE. 'lhe proteins -were transferred to nitrocellulose paper am stained with Fast Green dye. 'lhe in:lividual proteins -were then renoved fran the nitrocellulose paper am i.nplant.ed into the back of a rali:>it as described in Cl1apter V.

PAGE 153

. SDSPAG 1 ... TWO-DIMENSIONAL GEL ELECTROPHORETIC ANALYSIS 141 ANTIBODY PRODUCTION METHODOLOGY PROTEINS ELECTROBLOTTED ,,, .... .. NITROCELLULOSE ... I ~ ... SERUM COLLECTED NITROCELLULOSE l SERUM SCREEN BY IIESTERN ANALYSIS AUTO RAD I OC RAM INDIVIDUAL PROTEIN SPOTS EXCISED .. .. PROTEIN SPOTS

PAGE 154

142 techniques. '!he antisennn :reco;ptlzed the glyooproteins bourrl to nitrocellulose an::l typically was used at dilution.5 of 1:5,000 in ELISA assays. By analogy we reasoned that, prcxiuction of antil:x:xlies against MP-73 followin;;J 2D-P1'.GE \vOUld provide, in effect, a one-step prrification. F\Jrt.hen'OC>re, thra.lgh transfer of the protein to nitrocellulose prior to inm.mization the :maxinum ann.m.t of protein possible can be presented to the ral:x>it. 'lb estimate the ann.m.t of protein used for the i.nmmizations, a stamard cw:ve was prepared by spottin;;J 1-10 g bovine sennn albnnin protein onto nitrocellulose paper an::l then stainin;;J the paper with 1% Fast Green as described in Oiapter V. 'lhe stainin;;J intensities of the excised MP-73 spots were cx:upared to this st:arrlard. cw:ve. Based on these estimates, one protein-bearin;;J spot oorrespon:led to awroximately 1 g of MP-73 protein. '!he protein spot oorrespon:lirg to MP-73 was then excised fran a series of six tvJo-dimensional polyacrylamide gels followin;;J elect.rqnoretic transfer of the proteins to nitrocellulose paper (Fig. 17). '!he piece of nitrocellulose to which MP-73 was i.nt'cd:>ilized was dried, dissolved in a minimal volume of mso (0.1 ml), an::l an equal volume of Freurrls's c:arplete adjuvant was added (Chiles et al. 1987) '!his mixture was used as the primacy inm.mogen by injectin;;J subcutaneously into the back of the ral:x>it. Four weeks after the primacy injection, the followin;;J i.nm.mization sdledule was followed: day one: 8 protein spots (10 g); day foorteen: 10 protein spots (10 g); day twenty-eight: 10 protein spots (10 g). 'lhese secomacy injection.5 were perfonood by sinply inplantin;;J the nitrocellulose paper subcutaneously followin;;J wettin;;J with Freurrl's incarplete adjuvant. '!he mso solubilization step used to prepare the

PAGE 155

Figure 18. Detection of MP-73 by Inm.lnd::>lottin;J of Rat Liver Membrane Proteins. A 10,000 g rat liver membrane fraction was isolated arrl analyzed (100 g protein) by ID-PAGE as described in Cllapter V. '!he proteins were then electrcploretically-transferred to nitrocellulose paper. Ral::bit sennn collected before (NI) or after (I) inm.mization was screen for reactivity with MP-73 by inm.Jncblot analysis (see Cllapter V).

PAGE 156

..., I 0 .. .c a, -' .. a : CD -0 144 NI I

PAGE 157

145 inmmogen in the first series of experiments, was not enploye.d durinJ the secorrlary injections. Instead, non-solubilize.d protein was used as antigen by sinply inplantinJ the nitrocellulose paper subcutanea.Jsly. 'lhis m:xtification resulted fran the ctiffia.ll.ty in han:llinJ am the loss of antigen whidl. occurred durinJ the solubilization am injection proce:iure. Recently, this laboratory as 'Well as those of Dr. C.M. Allen am T.W. O'brien laboratory have prepared antisera to a variety of proteins withalt any solubilization durinJ the primary or secorrlary inm.mizations, suggestinJ that the is not needed to elicit antibody production. Fig. 18 shows the specificity of the antisennn raised against MP-73 as jtxlged by lD-PAGE. AR;,roximately 0.1 ng of protein fran the 10,000 g membrane fraction was separated by lD-PAGE. Folla,.rinJ transfer of the proteins to nitrocellulose, MP-73 was detected with sennn obtained fran i.Imunize.d ral:i>its. A polypeptide of approximately 73 kD:l was recognized by the antisennn. A secom barrl of lesser intensity at approximately 55 kD:l awears to represent a proteolytic fragnent. Incubation of this membrane fraction at 37C results in the cx:mplete loss of the 73 kD:l fonn of MP-73 suggestinJ that the protein is sensitive to an ernogenous protease within the preparation. In contrast, the non-:inm.me (NI) sennn fran the same ral:i>i t collected prior to inm.mization showed no reactivity with MP-73. To dlaracterh: e the immme status of the ral:i>it with respect to antibody production, a time course of antibody production was perfonned usinJ nitrocellulose i.nm::t>ilize.d antigen (Fig. 19). 'lhe antigen preparation used to screen the sennn, diluted 1: 50, was obtained by separatinJ proteins fran the 10,000 g membrane fraction on lD-PAGE am

PAGE 158

Figure 19. T~ of Antil:xxly Production Against MP-73. A New Zealam White rali:>it was imrunized as described in the text. Four weeks folla.,rirg the primacy inplantation of nitrocellulose spots, the rali:>it was boosted at two week interrcus am bled 10 days after each l:x:>OSt. Sennn was assayed for antil:xxly prcduction (1:50 dilution) usirg an inm..mchlottirg procedure against a 10, 000 g crude membrane fraction separated by ID-PAGE am then transferred to nitrocel\~ paper (25 g protein/lane). BaJrrl antil:xxly was detected usirg [ !]-Protein A as described in Olapter V. Key: lane A represents total protein of the membrane fraction stained with Amide Black, whereas lanes B, c am Dare i.nm.moblots designed to test for the presence of antil:xxly at 6 (B), 8 (C), am 10 (D) weeks after the primacy injection. lanes E, F am G show the antil:xxly level at 4, 6 am 8 weeks folla.,rirg the last set of l:x:>OSt inplantations. lane H illustrates the lack of reactivity seen with pre-inm..me serum.

PAGE 159

c:> 0 ... )( .. .J: m Q) 0 ::, Q) -0 116 97 66 45 29 147 Antibody Production ABCDEFGH

PAGE 160

Figure 20. Sennn .Anti.body Titer Against MP-73. A 10,000 g mem:>rane fraction (50 g/lane) was separated by 1.IrPAGE, electrq:tloretically transferred to nitrocellulose paper and then subjected to .i.nm.mablottinJ. Sennn ootained after the third injection (secom l:x>ost at 8 after the primary injection) was assayed for anti.body titer by .i.nm.mablottinJ usinJ the followin:J dilutions: Key: lane A (1:50), lane B (1:75), lane C (l:100), lane D (l:300), lane E (l:500), lane F <:HsOOO), lane G (1:2000). Bourrl primary anti.body was detected with [ !]-Protein A as described in Olapter V.

PAGE 161

... 116 97 66 A 149 Antibody Titer B C D E F G

PAGE 162

150 then transferrin;J the proteins to nitrcx::ellulose. serum obtained at variCAJS times thra.lghaJt the innunization period was diluted 1:50 arrl use to screen the merrbrane proteins by l.llllll.lilCi>lottin;J. Ten days after the secorxi set of inplantations (first boost), anti1:xxly production was detected readily. Anti1:xxly levels -were maintained follCMin;J two additional :inplantations, each spaced at 2 'INeek i.ntei::vals. AR:>roximately 4 'INeeks follCMin;J the final injection of antigen, anti1:xxly was no le>n:Jer detectable. Initially, assays for anti1:xxly production -were not possible, because of the high degree of nonspecific birrlirg of antibodies to the nitrcx::ellulose paper. For reasons unknown, the rali)its awearecI to produoe antibodies to material within the nitrcx::ellulose paper. 'lhis resulted in an unusually high backgroon:l signal, which in turned masked the signal fran the anti1:xxly-MP-73 reaction. '1he backgroon:l calld, however, be substantially reduced if the sennn or p.rrified IgG, prior to use in inrrund:,lottin;J experiments, was incubated overnight with nitrcx::ellulose paper (blocked with 3% BSA arrl o. 5% Tween-20) '!he sennn anti1:xxly titer, tested after the third injection (seco:rn boost) of antigen, was sufficient to allCM inm..mcblottin;J to be routinely perfonned at dilutions rargin;J fran 1:250 to 1:500 (Fig. 20). '!he same anti1:xxly mixture, diluted 1:250, calld be used up to five times for i.Imund::>lottin;J without noticeable loss of :imnunoreactivity. '!he specificity of the antisennn prepared to MP-73 was examined by adaptin;J the ID-i.Imund::>lottin;J tedmique to analysis of merrbrane proteins separated by 2D-PAGE (Fig. 21). For these experiments, awroximately 0.3 ng of protein fran the 10,000 g merrbrane fraction was separated by 2D-PAGE arrl then transferred to nitrcx::ellulose. Antisennn

PAGE 163

Figure 21. Innunablot Detection of MP-73 FollOvlin:J 'Iwo-Di.mensional Polyacrylamide Gel Electrqiloresis. A 10,000 g rat liver membrane fraction was isolated am the membrane proteins (300 g) \tJeJ:'e separated by 2D-PAGE am then electrqnoretically transferra:l to nitrocellulose paper. Sennn ootained after the secon:i boost (8 weeks after the primary injection) was diluted 1:250 am used to detect the MP-73 protein by inm.md)lottin:J.

PAGE 164

152 pH 7.0 M I 0 >( .. .c 0) s 3 D s I. D :, \I G> -45 0 + -IE F +

PAGE 165

153 to MP-73 was then used to screen the nitrocellulose paper arrl the :(X)Sition of the inm.moreactive protein coirx::ided with that of the antigen previously ci:lServed by Coanassie Blue staining (i.e., pI 7.0, M.W. 73 k[a). 'lhe inm.moreactive polypeptide at lc:,wer nnlecular '#eight (47 kIB) seen in Fig. 21 was not present consistently arrl was not detected by pre-inm.me sennn. '!his polypeptide awarei,tly represents a proteolytic cleavage product contaminatin:J sare membrane preparations. It should be noted that while the 2D-inm.mablot analysis provided~ evidence that the antisennn was specific for MP-73, it is p::lSSible that antil:x:xlies may have been present to other "confonnational antigenic detenninants" which were not recognized by the antisennn after transfer to nitrocellulose (D.mbar, 1987). '!his inm.mization procedure was also erployed to prepare antil:x:xlies to an additional protein which aweared to be in:luced by amino acid stal'.Vation (i.e., M.W. 66 k[a, pI 6.4, see Fig. 15). In this case, both primary arx:l secorxJary inm.mizations used the protein i.nrcd:>ilized on nitrocellulose witha.rt dissolvin:J the paper in r.:MSO. After the third series of :mplantations, antibody production was ci:lServed via lD inm.md:>lot analysis (Fig. 22). 'lhe nnlecular '#eight of the inm.moreactive barn corresp::>rds with that of the protein used for inm.mization (i.e., M.W. 66 kIB). Interestin:Jly, 2D-inm.mablot analysis (Fig. 23) deJIDilS'trates that the antisennn also recognized two proteins with similar nnlecular '#eight yet differin:J isoelectric points (i.e., pI 6 .1 arx:l 6. 2) 'lhe reasons for the cross reactivity of the antisennn with these proteins is lll'lClear, however, the proteins may represent different isoelectric fo:nns of the same protein or the original antigen preparation localized on the 2D-nitrocellulose paper blot may have

PAGE 166

Figure 22. Inm.mablot of Imrcune or Noninm.me Sennn Against MP-66 Followi.rq One-Di.nensional Polyaccylamide Gel Elecb:q:.horesis. A 10,000 g nenbrane fraction was isolated arrl analyzed (100 g protein) by Ur PAGE as described in Chapter V. After separation, the proteins -were electrqiloretically transferred to nitrocellulose paper. Ral:i:>it sennn CX>llected before (NI) or aft.er (I) i.nm.mization was diluted 1:100 arrl used to screen for reactivity with MP-66 by inm.mcblot analysis.

PAGE 167

r, I 0 ... ... a :::, G) -155 NI I 29

PAGE 168

Figure 23. Imrm.lrloolot of MP-66 FollCM:mJ 'l\vo-Di.men.sional Polyacrylamide Gel Electrq:horesis. A 10,000 g rat liver :membrane fraction (300 g protein) was separated by 2D-PAGE arrl electrc:ploretically transferred to nitrocellulose paper. Sennn obtained after the third series of i.nplantations (6 weeks after the primary injection) was diluted 1:50 arrl used to detect the MP-66 protein by the .i.nmmd::>lottin:J.

PAGE 169

M I 0 >< 116 97 .c m j 66 :::, "' G) o 45 157 ------I E F -----+ s D s +

PAGE 170

158 contained both of these proteins. It is possible that this series of protein spots represent the same protein with different isoelectric species, because many membrane glycq:>roteins contain sialic acid. In reference to the later sug;Jestion, MP-66 arxi the t\\10 proteins of similar nolecular "tNeight migrate to p:::isitions very close to one another in the isoelectric focusing gel. As a result, the Coanassie Blue stained pattem.s of these proteins usually shOW' sane degree of streaking, sug:JeSting cross cxmtamination. Fig. 23 also shows an .inm.trx>reactive protein of awroximate nolecular "tNeight of 85 kDl. Analysis of sennn obtained prior to ilmunization of the ral::bit reveals the same protein (Fig. 22). Detailed analysis of MP-66 will be the subject of further research in Dr. Kil.berg's laborato:ry. '!he antisennn prepared against MP-73 was enployed to detennine the nolecular "tNeight of the i.mnunoreactive protein (MP-73). '!his was ascertained by first separating 100 g of hepatic nerbrane protein (10,000 g nerbrane fraction) by l.lrPAGE, electroolotting the proteins to nitrocellulose arxi then screening with antisennn to detect the protein as in Fig. 18. As seen in Fig. 24, the calculated nolecular "tNeight based on nd:>ility in a 7. 5% reducing (2-mercaptoethanol) polyac:rylamide gel is 73 kDl. It shoold be noted that membrane proteins have been reported to bi.rrl markedly different annmts of Sll5 due to their hydrcplooic nature arxi cartx:nydrate stnicture (segrest et al., 1971). '!his bi.ming may therefore affect the nd:>ility of the protein in the polyac:rylamide gel. As a result, the calculated nolecular "tNeight, is at best, an awroximation. '!he nd:>ility of MP-73 was also ascertained un:ler nonreducing (lacking 2-mercaptoethanol) corrlitions. Membrane proteins fran the 10,000 g fraction "tNere separated on a 5% nonreducing

PAGE 171

Figure 24. Estimation of the M:>lecular Weight of MP-73 FollowinJ OneDimensional Sodimn-Dod.ecyl-SUlfate Polyacrylamide Gel Elect:rc.ploresis. AJ;:proximately 50 g of protein fran the 10,000 g nenbrane fraction was separated on a 7.5% sodium-dodecyl-sulfate polyacrylamide gel (shown in Fig. 18). 'Ihe nenbrane proteins were then electrd::>lotted to nitrocellulose paper am screened with antisennn (diluted 1:250) to MP73. 'Ihe protein nolecular 'INeight stamards (Sigma SIE-6D) were ral:i:)it nuscle myosin, 205 kl)l; F.sdleridtla ex>li B-gal.actosidase, 116 kl)l; ral:i:)it nuscle ~cylase b, 97 .4 kl)l; bovine aJ..b.nnin, 66 kl)l; ec;R aJ..b.nnin, 45 kl)l; am bovine eryt:hra::yte carbonic anhydrase, 29 kl)l. 'Ihe protein nolecular \eight staooards were visualized by Fast Green stain am are represented as the log of the nolecular 'INeight versus the relative nd:>ility.

PAGE 172

5.4 5.2 5.0 c:n 0 4.8 -._ 4.6 4.4 4.2 4.0 160 116kDa 97.4kDa 0.1 0.3 o.s 0.7 0.9 Rf (cm)

PAGE 173

Figure 25. Inm.mablot of MP-73 Followin;J One-Dimansional Non-Reducin;J Sodium-Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis. Approximately 50 g of protein fran the 10,000 g membrane fraction was separated on a nonreducin;J sodium-dcx:1ecyl-sulfate polyacrylamide gel. '!he membrane proteins were then electrcblotted to nitrocellulose :paper am screened with antisennn (diluted 1:250) to MP-73.

PAGE 174

t;> 0 ... >< .. 205 m -::, u G) -0 162 -BME

PAGE 175

Figure 26. Estimation of the Molecular Weight of MP-73 Followirg one Dimensional Nonreducirg Sodium-D:xiecyl-SUlfate Polyacrylamide Gel Elect.rop:ioresis. Jg>roximately 50 g of protein fran the 10,000 g membrane fraction was separated on a 5. 0% nonreducirg soctium-dodecylsulfate polyacrylamide gel (Fig. 25). '!he membrane proteins -were then electrcblotted to nitrocellulose paper am. screened with antisennn to MP-73. 'lbe protein nnlecular ,;..ieight stamards (Sigma Sr:6-6D) are described in Fig. 24. '!he protein nnlecular ,;..ieight stamards -were visualized by Fast Green stain am. are represented as the log of the nnlecular ,;..ieight versus the relative :nd:>ility.

PAGE 176

5.4 5.2 5.0 0, 0 4.8 -._ 4.6 4.4 4.2 4.0 164 116kDa 0.1 0.3 0.5 o. 7 0.9 Rf (cm)

PAGE 177

165 polyacrylamide gel (Fig. 25) '!he estimated nolecular weight, as detennined by inm.mablottin;J, was 78 kDa which is not significantly different fran 73 kDa given the separation capabilities of PAGE (Fig. 26). 'Ihese results irrlicate that within the fractionation rarge of the 5% polyacrylamide gel (i.e., 75 kDa to 300 kDa) MP-73 not to be associated with other larger nolecular weight membrane ccnponents via disulfide borrlmJ. Given that the membrane preparation used for the initial identification of MP-73 contained plasma meni:>rane, mitoc::homrial membrane arrl to a lesser extent lysosanal arrl errloplasmic reticular membranes, the antisennn was used to detennine the sul:x::el.lular location. 'Ibis was achieved by sul:x::el.lular fractionation of rat liver usin;J three distinct fractionation nethoos, each designed for q,timal yield of particular hepatic sul:x::el.lular carpartments as outlined in Crapter v. Followin;J isolation, the proteins fran each of the sul:x::el.lular fractions were separated by lD-PAGE, electrqnoretically transferred to nitrocellulose, arrl then assayed for the presence of MP-73 by inm.mablottin;J analysis. Figs. 27 arrl 28, show representative Ox:lnassie Blue arrl silver-stained gel patterns of the suboellular fractions. '!he results of inm.mablottin;J experilllents of the isolated meni:>rane fractions suggested that a fraction enriched for mitochorrlrial proteins contained the irrrrunoreactive MP-73 protein, whereas fractions corresporrlin:J to the nuclei, cytosol, golgi, errloplasmic reticulum, arrl plasma membrane were devoid of the protein at the sensitivity level of the assay (Fig. 29). Enzyme analysis of the mitochorrlrial-enriched membrane fraction revealed minimal contamination with either glucose-6-~tase, a marker enzyme for errloplasmic reticulum (swanson, 1955), acid

PAGE 178

Figure 27. Coanassie Blue stain of Proteins fran the SUbcel.lular Fractionation of Rat Liver. Rat liver was subjected to subcellular fractionation as described in Olapter V. Fadl of the subcellular fractions (25 g protein/lane) were subjected to lD-PAGE, the proteins were then fixed in trichloracetic acid arrl stained with Coanassie Blue stain as described in Chapter v. Key: lane A (mitocnorrlria), lane B (nucleus), lane C (cytosol), lane D (golgi), lane E (microsanes), arrl lane F (plasma nembrane).

PAGE 179

167

PAGE 180

Figure 28. Silver stain of Proteins fran the SUbcel.lular Fractionation of Rat Liver. Rat liver was subjected to subcellular fractionation as described in Olapter V. F.adl of the subcellular fractions (25 g protein/lane) were subjected to lD-PAGE, the proteins were fixed in tridlloracetic acid an:l develcp:!d by silver stainirg (see AWen:lix III). Key: lane A (mitochorrlria), lane B (nucleus), lane C (cytosol), lane D (Golgi), lane E (microsanes), an:l lane F (plasma nenbrane).

PAGE 181

169 205 116 .., 97 I 0 ,c 66 .. .21 I "" C 'S 45 "' Q) 0 29

PAGE 182

Figure 29. SUbcellular Localization of MP-73 by Imnund::>lot Analysis. Rat liver was subjected to suocel.lular fractionation as described in Cl1apter V. F.adl of the suocel.lular fractions (25 g protein/lane) were subjected to lD-PAGE am transferred to nitrcx:lellulose. '!he fractions were screened by i.nm.mcblotti.rg with anti-MP-73. Key: lane A (mitochorrlria), lane B (nucleus), lane C (cytosol), lane D (Golgi), lane E (microsanes), am lane F (plasma irenbrane).

PAGE 183

171 Su bcel lula r Fractionation A B C D E F r:> 0 ... 116 >< .. ..c 97 a, Q) 3 66 I. a ::, u Q) -0 45

PAGE 184

172 plOSii}atase, a marker enzyme for lysosanes, (Sdllosnagle et al., 1974), or S'-rn.1Cleotidase activity, an enzyme located primarily in the plasma membrane (M:>ore, 1971). In contrast, suocinate:cytodu:'ane c reductase, an enzyme present in the inner mitochorrlrial membrane (Fleischer am Kervina, 1974), was enridled 10-fold over the haoogenate (Table 12). It is also clear fran the data in Fig. 29 (lanes A am B) that the antibody recognizes a unique protein of similar nolecular \eight in subfractionated bovine liver mitochorrlria as \ell, sug:JestinJ the presence, am at least partial consei:vation, of MP-73 in species other than rat. '!he bovine mitochorrlrial fractions \ere the genera.JS gift of Ors. T.W. O'Brien am N. DenslCJlrl of this departltelt. '!he manmalian mitochorrlrion is m1if.06d of four distinct carpartments, an a.rt:er membrane, i.ntentenbrane space, inner membrane, am a matrix (I.ehn.in;Jer, 1982). To ascertain the sul::rn.i.tochon:lrial localization of MP-73, mitochomria \ere separated into mitq>lasts ( inner membrane an.1 matrix) an.1 a fraction containinJ the proteins of the a.rt:er membranes an.1 the intennernbrane space. '!he mitq>lasts \ere subjected to further fractionation by sonication an.1 subsequent centrifugation to separate the matrix proteins fran those associated with the inner membrane. F.adl of the sul::rn.i.tochorrlrial fractions \ere separated by 1D-PAGE, transferred to nitrocellulose, an.1 screened with MP-73 antiserum. Figs. 30 an.1 31 shCJlrl representative Coanassie Blue an.1 silver-stained gels of the sul::rn.i.tochorrlrial fractions, respectively. As depicted in Fig. 32, the major i.rmunoreactive protein was detected in the lanes containinJ intact mitochomria (lane C) mitoplasts (lane E) or inner membrane (lanes A an.1 G). '!he fractions correspon:lirg to solubilized a.rt:er meJTbrane proteins an.1 released intennernbrane space

PAGE 185

173 Table 12. Enzyme Marker Analysis of Isolated Rat Liver Mitochondria Fractioo Total Protein 5'-NUc:leo-Gluoose-6-Succinate:cyt.c Acid-(mg) tidase P'ase Reductase P'ase Harogenate 1772 8,1 0.8 14,2 0.4 841 28,1 56,2 ...2,9 Mi todlordria 14 o.8 1.8b 2,4 l.lC 8071 105,oc 5.7 ._0,2C RSA 0,1 0.2 9.6 0.1 Mitochcn:iria were isolated fran rat liver as described in Chapter v. 'lhe freshly isolated mitodlordria were stored at -7o0c; enzyme activities were assaye;i within 24 h followi.rq isolation. 'lhe enzymes 5'-nucleotidase (Moore, 1971), gluoose-6-~tase (8wanson, 1955), suc:x::inate:cytcx::hrane c roouctase (Fleischer an:i Ke.Ivina, 1974), an:i acid i::nosphatase (Schlosnagle Al,., 1974) were assaye;i as marker enzymes for the plasma meri:>rane, emoplasmic reticulum, mitochcn:iz:~, arxl 1~, :i:espectively. Enzyrre activities are expressed as n:,l Pi releasedng proteinmini for both ~'i-nucleotidase arxl glua:ise-6-fi1~tase arxi nrrol cytochrane C reduoedng proteinmin for sucx:inate:cytoc:hrane _I reductase. _1 Acid ~tase activity is expressed as nrrol p-nit.rq:i"lerol prooucedng proteinmin 'lhe relative specific activity was calculated as the ratio: specific activity in the mitodlcn:lria/specific activity in the hcm:qenate. brhese values are significantly different top values< 0.025. Ihese values are significantly different top values< 0.005.

PAGE 186

Figure 30. Coanassie Blue stain of Proteins fran the Fractionation of Rat Ll.ver Mitochomria. Mitochomria were fractionated as described in Olapter V. F.ach fraction (25 g protein/lane) was then subjected to IDPAGE. '!he gel was fixed in tridtl.oracetic acid am stained with Coanassie Blue dye. 'Ihe protein nolecular "tNeight starnards (Sigma SIS6D) were ral:'bit nuscle myosin, 205 kD:i; Escheridri.a coli B galactosidase, 116 kD:i; ral:'bit nuscle Jilosp10i::ylase b, 97.4 kD:i; bovine alb.nnin, 66 kD:i; egg alb.nnin, 45 kD:i; am bovine ecythrocyte cartx:mic anhydrase, 29 kD:i. Key: lane A (protein nolecular "tNeight markers), lane B (liver haoogenate), lane c (rat mitochomria), lane D (rat outer nenbrane am intentert>rane space proteins), lane E (rat mitc:plasts), lane F (rat matrix proteins), am lane G (rat inner nenbrane).

PAGE 187

175 A B C D E F G

PAGE 188

Figure 31. Silver stain of Proteins fran the Fractionation of Rat Liver Mitochomria. Mitochomria were fractionated as described in Olapter V. '!he proteins of eadl. fraction (25 g protein/lane) were separated by 1Ir PAGE. '!he gel was then fixed in tridl.loraoetic acid arrl silver stained as described in III. '!he protein m:>lecular ,;.,.ieight starrlards (Sigma MW-SIS-200) were ra1::bit nuscle myosin, 205 k:D:l; F.scheridl.ia coli B-galactosidase, 116 k:D:l; ra1::bit nuscle }ilosI:ho:rylase b, 97 .4 k:D:l; bovine album.in, 66 kD:l; eg;J album.in, 45 kD:l; arrl bovine e:rythrocyte cartx:mic anhydrase, 29 k:D:l. Key: lane A (protein m:>lecular ,;.,.ieight markers) lane B (liver haoogenate) lane C (rat mitodlorrlria) lane D (rat art:er nstt,rane arrl intenneni)rane space proteins), lane E (rat mitcplasts), lane F (rat matrix proteins), arrl lane G (rat inner nenbrane).

PAGE 189

'? 0 116 97 ... 66 )( .. D 45 "'5 ... Q) 0 :e 29 A 177 B C D E F G

PAGE 190

Figure 32. Inm.md::>lot Analysis of Fractionated Rat Liver Mitoc::hon:lria. Mitoc:hon:tria were fractionated into proteins fran mitq:>lasts, outer neti:)rane am. :intentenbrane space, inner membrane am. matrix as described in Chapter V. Fadl fraction (25 g protein/lane) was then subjected to lD-PAGE am. transferred to nitrocellulose paper.. 'lhe fractions were screened by inm.mablottin;J usin;J antisera rai.ssd again.st MP-73. Key: lane A (bovine inner membrane), lane B (bovine lt!t.'lt.rix), lane C (rat mitoc::hon:lria), lane D (rat outer membrane ari~ idr..ennent>rane space proteins), lane E (rat mitq:>lasts), lane F (rat. ;;.,;c:::.~'ix proteins), am. lane G (rat inner membrane)

PAGE 191

179 Mitochondrial Fractionation A B C D E F c:, 116 0 .. 97 >< .. .c 66 m .:.-.~ f loc:: 4) L : : : f:I[ !),_,; mi: ms :.-:r.Jsrri :J' .. 0 :::, 4) -45 0

PAGE 192

180 proteins (lane D), or that containin;J the nBtrix proteins (lanes B am F) -were essentially devoid of any detectable levels of MP-73. To confinn the separation of the mitq>lasts into nBtrix am inner membrane fractions, the distrib.Ition of glutamate dehydrogenase, an enzyme localized in the mitodlon:lrial nBtrix (Beaufay et al., 1959), am suocinate:cytochrane c reductase, an enzyme localized in the inner me.rrbrane (Fleischer am Kenrina, 1974), -were detennined in eadl of the mitq>last fractions. As illustrated in Table 13, the sonicated mitq>last supernatant (nBtrix proteins) contains high levels of glutamate dehydrogenase activity relative to the inner me.rrbrane pellet, but was essentially devoid of suocinate:cytochrane c reductase activity. In contrast, the membrane pellet contained m::>St of the suocinate:cytochrane c activity am a small aioount of glutamate dehydrogenase, presumably due to traw.IDJ. Further evidence that MP-73 is a integral membrane protein was provided by Triton X-114 :(ilase separation of intact rat liver mitq>lasts. In the method developed by Boldier (1981), integral membrane proteins bi.rxi the non-ionic detergent Triton X-114 dur.IDJ solubilization in 1% detergent at 40c. If the tenperature of the mixture is then shifted to 37c am the mixture centrifuged at 300 g, hydrq:ru.lic proteins preferentially partition into the aqueous :(ilase, whereas the integral membrane proteins are foorxi to partition into the lower detergent :(ilase. In his initial studies, Boldier (1981) foun:i that the anpriJ;itllic integral membrane proteins, acetyldlolinesterase, bacterio:rhodopsin, am cytochrane c oxidase partitioned into the detergent :(ilase, whereas oval..b.nnin, catalase, helooglabin, am cytochrane c, all hydrq:ru.lic proteins partitioned into the aqueous :(ilase. Many

PAGE 193

181 Table 13. Enzyme Marker Analysis of Rat Liver Submitochondrial Fractions Mitochcn:Jrial Glutamate SUOCi.nate:cytoc:hrane c Fractia, Dehydrogenase Reductase Mitq:>lasts 22 1 0.40 0.02 Matrix 39 le 0.01 0.0lC Inner l'IIBDmcU'l8 9 2C 0.30 0.01b SUl:m:i. tochcn:Jrial fractionation was perf onned as described in Olapter V. '!he enzyme activities for glutamate dehydrogenase, a mitodlon:irial matrix protein, am sucx:inate: cytoc:hrane c reductase, a mi tochorrlrial inner I!V:!Il'brane protein, were assayed as described by Hogeboon et gJ,., (1953) am Kilberg~ al., (12?9), -~ively. '!he data are expressed as nm:>!_1NADP_~ng protein min am poc,l cytoc:hrane c reduoedng protein min for the respective enzyme activity am are the averages stan:iard deviations for three assays. values are significantly different top values< 0.01. tlleS8 values are significantly different top values< 0.005.

PAGE 194

Figure 33. MP-73 Inm.moreactivity Followin:J Triton X-114 Riase Separation of Rat Liver Mitcplasts. Mitcplasts -were adjusted to 1 ng protein/ml with 10 nM Tris-HCl, pl 7. 4, 150 nM NaCl, am 1% Triton x-114. 'Ihe mixture was incubated for 30 min at 40c. 'Ihe nerbranes -were then shifted to 370c for 5 min am centrifuged at 300 g for 2 min. 'Ihe aqueous Ji}ase was renoved am saved for analysis, whereas the lower detergent Ji}ase was mixed with 1 ml of 10 nM Tris-HCl, pl 7. 4, 150 nM NaCl, am 1% Triton x-114 am incubated am centrifuged as described above. 'Ihe aqueais Ji}ase fran the secx::ni extraction was discarded, whereas the lower detergent Ji}ase was renoved am save for analysis. 'Ihe proteins in eadl fraction -were then separated by lDPAGE am transferred to nitrocellulose paper. 'Ihe nitrocellulose paper was screened by .i.nmund:>lottin:J with antisennn raised against MP-73. Key: lane A (intact mitcplasts), lane B (~ aqueous Ji}ase), lane C (lower detergent piase).

PAGE 195

er, 0 ... 116 >< 97 i, 66 Q) .. .i 45 :::, Q) -0 183 Triton X-114 A B C

PAGE 196

Figure 34. Fast Green stain of the Proteins Followin;J Triton X-114 Fhase Separation of Rat Liver Mitq>lasts. 'Ihe proteins in rat liver mitq>lasts were separated into aqueous arrl detergent fractions as described in Figure 33. 'Ihe proteins in eadl fraction were then separated by lD-PAGE, transferred to nitrocellulose, arrl stained with Fast Green stain. Key: lane A (intact mitq>lasts), lane B ('UR)er aqueous p,ase), lane c (lower detergent p,ase).

PAGE 197

185 c:, 0 116 .97 >< .. 66 .c a, Cl) 3 "' 45 a ::, Cl) 29 -0

PAGE 198

186 laboratories have used this procedure to study a variety of merri:>rane systems. Alcaraz et al. ( 1984) dem::>nstrated that upon bim:irg IgE, the IgE receptor CX1Tplex partitions into the Triton X-114 p,ase. Maher an:l Si.n:}er (1985) used Triton X-114 i;ilase separation to pirify the acetyldloli.ne receptor fran californica elect:.rq>lax. When isolated rat liver mitq,lasts were subjected to Triton X-114 i;ilase separation an:l the resultin;J aqueoos an:l detergent }.i1ases analyzed by lll111.l1100lottin;J with antisennn against MP-73, the inmmoreactivity was detected only in the lower detergent }.i1ase (Fig. 33). Fig. 34 shows a representative Fast Green stain of the proteins transferred to nitrocellulose followin;J }.i1ase separation, clearly the majority of proteins partitioned into the soluble p,ase. 'lhese results dem::>nstrate that MP-73 is Weed an integral nenbrane protein, an:l are consistent with its assigmnent to the mitochon:iria inner merri:>rane. In order to study the biosynthetic regulation of MP-73 durin;J amino acid starvation of hepatocytes, I initially prq:,osed to use i.nm.mc:precipitation as the major tool to quantitate the relative changes in the biosynthesis of MP-73 durin;J amino acid starvation. Unfortunately, after tcyin;J many different con:titions (i.e., vai:yin;J the solubilization, bim:irg, an:l washir)3s procedures) the i.nm.mc:precipitations were not consistent ena.igh to allow any conclusions to be drawn. Fig. 35 shows a typical i.nm.mc:precipitation usin;J antiserum raised to MP-73. Clearly, the preiirm.me antiserum isolates many of the same 100lecular -weight size proteins as does the iirm.me serum (Fig. 35). In many experiments, the preiirm.me serum actually i.nm.mc:precipitated 100re proteins than the iirm.me serum, whereas no proteins in the rcm:Je of 70-75 were detected with the iirm.me serum.

PAGE 199

Figure 35. Inm.lnq>re.cipitation of MP-73 Fran (3HJ-Ir-I.eucine labeled Cells. Freshly isolated hepatocytes were placed into primai:y culture in 150 nm culture dishes in NaKRB (25 x 106 cells per dish) 'lhe nmium was suwlemented with 1 nei of ( 3HJ-leucine for awroxilnately 3 h (1 h at 4C). '!he cells were then lysed by incubation (1 hat 4C) with 10 rrM Tris, 150 rrM NaCl, pl 7 .4, 1% Triton X-100, 1 rrM :EMSF arrl 5 rrM benzamidine. '!he lysate was then centrifuged at 15,000 g for 15 min, arrl 0.15 ml of Protein A-Sep'larose (10% stock solution) was added. 'Ibis mixture was incubated overnight at 4C with erxi-over-errl mi.xi.rg. '!he followirq ncmirq, the Protein A-Sep'larose cx:ntainirq bam:l antigen was isolated by centrifugation, washed 3 ti.m:!s with 10 rrM Tris-base, pl 7 .4, 150 rrM NaCl, 1 ng/ml BSA arrl 0.5% Triton x-100. '!he antigen was then separated fran the Protein A-Sep'larose by boilirq in Sanple Dilution aiffer for 2 min. '!he labeled proteins were analyzed by l.lrPAGE arrl fluorograiily. '1he Protein A-Sep'larose stock was prepared by incubatirq with 1 ng/ml BSA in PBS for 1 h. Key: lane NI (nonimmune sennn), lane I ( inm.me sennn)

PAGE 200

188 NI I 0 .->< .. .c a, -45 Cl) a 29 :, "' Cl) -0 i

PAGE 201

Figure 36. Biosynthesis of MP-73 D.lrirg Amino Acid Deprivation. Rat hepatocytes -were isolated am placed into primary culture (25 x 106 cells per dish) containi.rg NaKRB suwlemented with 20 nM ASN. After 2 h, one-half of the cultured cells -were rinsed three times with am then incubated in NaKRB. '!he remai.nin;J cells -were rinsed am cultured in NaKRB containi.rg 20 nM ASN. At varioos times (O, 2, 4, 6, 8, h) the cells -were rinsed several times in Pm am a 10,000 g ment>rane fraction prepared as described in Oiapter V. '!he ment>ranes proteins -were then separated by l.lrPAGE, electroolotted to nitrocellulose paper am screened with antisennn (diluted 1:250) to MP-73.

PAGE 202

190 0 2 4 6 8 + + ... ... kDa A B C D E F G H I J 97 66 HOUR AA DAYl DAY2

PAGE 203

191 'Ihese irxx>nsistencies in the inm.mcprecipitation made localization of MP-73 on the polyacrylamide gel ani>iguous. On the other bani, these corx:lition.s 'Nere sufficient to allow inm.mcprecipitation of the cacplement protein C9 usin:J antiserum raised to C9 ( a gift of Ron Iaine am. Dr. A. Esser, Dept. of Expt. PatllOlcqy). 'As mentioned above, a variety of corx:lition.s 'Nere enployed in an effort to ootain consistent inm.mcprecipitations. 'lhese include an assortroont of detergents for the initial solubilization: soo (0.1%, 0.5%, am. 1.0%), Triton X-100 (0.5%, 1.0%, am. 1.5%), am. Triton X-100 in the presence of 2 M urea am. 1.0% dlolate. Further attenpts to reduce the nonspecific co-isolation by increasin:J the salt concentration am. addin:J nonionic detergents durin:J the wash corx:litions of the final Protein A-Seplarose-antigen carplex 'Nere un.sucx:::essful. It is tmClear why the antiserum did not recognize MP-73 in these eJq:>eriments. 'lhe antilxxlies 'Nere generated to MP-73 i.mocbilized on nitrocellulose; it is :possible that the above corx:litions do not pennit antilxxly recognition of MP-73. other laboratories whidl have enployed similar nwathods for antilxxly production, i.e., antigen i.mocbilized to nitrocellulose, have also failed to ootain consistent inm.mcprecipitation of the antigen (Dr. C.M. Allen, Dept. Biochemistry am. M:>lecular Biolcqy, personal camunication) Whole serum am. :p.irified IgG also failed to detect MP-73 in l10IlCClll)etitive solid-i;:tlase radio:hmunoassays. I:nm:lbilization of either antigen or IgG on the solid-i;:tlase of a 96 'Well mic:rotiter plate (Falcon 3911), followed by bridgin:J with radiolabeled Protein A or antigen, respectively, resulted in a failure to detect MP-73. In:ieed, in control eJq:>eriments I was able to show that these corx:lition.s 'Nere sufficient to allow detection of either C9 or rat serum albumin (data not shown).

PAGE 204

Figure 37. Adaptive Regulation of System A nrrirq Amino Acid starvation of Rat Hepatocytes. Freshly isolatai rat hepatocytes were suspen:ied in NaKRB suwlementoo with 20 nM ASN arrl placed into 24 well cluster trays. After 2 h, one-half of the wells were rinsed three times arrl incuba.tai in NaKRB. '!he :remainin:J cells were rinsed arrl a.ll.tured in NaKRB containin:J 20 nM ASN. At various times (O, 2, 4, 6, 9, 12 h) the cells were rinsed with CllolKRP arrl the activi% of System A assayed by ~in:J the Na+-depen:lent uptake of 50 M [ HJ-AIB for 1 min (370c) as described in Olapter II. '!he results are the averages of 4 detenninations arrl the starnard deviations were less than 10%.

PAGE 205

-. C E C Q) 0 a. ,rm E IQ ca: -0 E a. 150 120 90 60 30 0 2 193 NaKRB+A .SN 4 6 Time, ( h) 9 12

PAGE 206

194 An alternative awroadl was taken to quantitate dlanges in the biosynthesis of MP-73. Rat hepatocytes -were isolated arrl placed into primacy culture contai.nirq NaKRB supplemented with 20 nM ASN for 2 h. '!his initial incubation was interned to allow all of the cells exposure to the same con::litions (i.e., amino acid supplemented meditnn) prior to amino acid sta:rvation. After 2 h, one-half of the cultured cells -were rinsed in NaKRB arrl incubated in NaKRB. '!he remai.nirq cells -were rinsed arrl cultured in fresh NaKRB contai.nirq 20 nM ASN. At varioos times (O, 2, 4, 6, 9, 12 h) the cells -were rinsed in PIE arrl a 10,000 g membrane fraction prepared as described in Olapter V. '!he nenbranes proteins -were then separated by 10-PAGE, elect.rd:>lotted to nitrocellulose, arrl screened with antisennn to MP-73. As seen in Fig. 36, the net level of biosynthesis for MP-73 awea,red not to d1anJe as a ftmction of ti.me in amino acid-free meditnn. In order to ensure that the cells -were e>
PAGE 207

195 Despite the inability to detect dlan;Jes in the concentration of the MP-73 protein by inm.molCXJical methods, the results presenta:i in Fig. 15 am Fig. 16 suggested that the in::luction of MP-73 may occur via de :nerve mRNA synthesis. '!he followin;J series of experiments were designed to study the mechanisms responsible for the in::luction of MP-73 at the nnlecular level arrl to detennine the ex>ntrib.Ition, if any, of de novo mRNA biosynthesis. 'lb this eni, two liver-derived gtll cJ:NA expression libraries were screened with antisennn raised again.st MP-73. '!he cJ:NA clones obtained were then used as prooes to nonitor mRNA levels durin;J of amino acid deprivation of both nonnal hepatocytes arrl a rat am human hepatana cell line. '!he gtll cJ:NA expression system was originally described by Yoorg am r.avis (1983). '!he Iilage gtll is a 43.7 kb linear da.lble-straooed mA bacteriqilage clonin;J vector designed for clonin;J small F.coR1 cJ:NA fragnents. '!his vector system can aCXX1111odate up to 8. 3 kb of cJ:NA insert, asstnni.n;J a maxim.nu packageable Iilage mA length of 52 kb. '!he / da.lble-strcm:led cmAs are produced fran a i;x:,lyadenylata:i RNA pcpllation am are then inserted into the gtll genane at a unique F.coR1 enionuclease restriction site. '!his site is locata:i within the lac z gene near the carboxyl tenninus (Fig 38). Because the site of foreign mA insertion is within the B-galactosidase gene, cmA sequences can be expressed as B-galactosidase fusion proteins. '!his allows the cJ:NA libraries to be screened with anti.body specific for a given protein (Yoorg arrl I.avis, 1983b). Given that m::ist eukaryotic proteins are rapidly degraded in bacterial systems, the fusion protein increases the st.ability of the i;x:,lypeptide in the bacteria (Itakura, 1977). Furt:henoore, durin;J screenin;J, the fusion protein is synthesized by a

PAGE 208

Figure 38. Map of the I.ambda gtll Genane Shc:Min:J Major Restriction Sites. '!he pi.age gtll is a 43. 7 kb linear dooble stramed mA bacteriq:tlage clonin:J vector designed for clonin:J small EcaR1 mA fragments. '!his vector system can acx:x::xc,colate a maxinum cmA insert of 8. 3 kb. cmAs are inserted into the gtll genare at a tmique EcaR1 errlonuclease restriction site. '!his site is located within the lac Z gene (boxed area) near the cartx:,xyl tenninus. Because the site of foreign mA insertion is within the B-gal.actosidase gene, cmA sequences can be expressed as B-gal.actosidase fusion proteins (Yoon;J an::l Il3.vis, 1983b) '!his figure was taken fran Il3.vis et al. ( 1986)

PAGE 209

197 0 0 ,, Q 0 n :; N :-19.~ f ... ~LEFT BamHI Pvul Kpnl Kpnl EcoRI Sstl Pvul Xbol Sstl Hindm BamHl Xhol BamHl Pvul Hindm Hindm 43.7kb RIGHT Diagram of Agtll, showing major RE sites in mult i ple cloning region

PAGE 210

198 protease deficient~-cx,li, strain Y1090, (Bukhari arrl Zipser, 1973). An additional advantage of insertirq the cmA into the lac Z gene is that it ren:lers the bacteriqilage gal whereas nonrecarbi.nants are ga1+. 'lherefore, in the a,wrcpriate strain of~-cx,li (i.e., Y1090, gal-) recanbi.nants fonn clear plaques wnen grown on agarose medil.nn containirq the c::hrc:ltqnoric substrate, 5-braro-4-dll.oro-3-irnolyl-B-D galactoside (Xgal), whereas nonrecarbinants fonn blue plaques. '!his then allows the presence or abserx::e of cmA inserts to be ascertained. In a typical inm.moscreenirg (Fig. 39), 105 to 106 in:lepemeilt recanbi.nants are screened in the fonn of :Ei1,age plaques on a lawn of ~. cx,li Y1090. 'lhe ~-cx,li are prq:,agated at 42C, this inactivates the tenperature-se:n.sitive repressor (cI857) of lytic grcMth, allowirq for lytic grcMth of the piage (Neubauer arrl Calef, 1970; Goldberg arrl Howe, 1969) When the rnnnber of infected bacterial cells within the plaque is sufficiently large, lac Z-directed expression of the fusion protein is irrluced by inactivatirq the lac operon repressor (the product of the host cells lac I gene) with B-D-thiogalactcpyranoside (!Pm). Proteins released durirq lysis of the cells within the plaques are ilmd:>ilized on nitrocellulose paper placed over the lawn of plaques. Protein balrrl to the nitrocellulose paper is then screened with antisennn usirq cx,nventional .inm.mc.blottirq tedmiques. 'lhe :piage pop.llation responsible for the expression of the fusion protein is then lcx::al.ized by overlayirq the nitrocellulose paper on the original plate. 'lhe two cmA libraries screened in our studies 'iNere ootained fran Clontec:h (Palo Alto, ca.). 'lhe mRNA for the first librai:y was isolated fran a nonnal adult rat liver (Sprague-I:awley, female) arrl contained 6.8 X 105 in:leperrlent clones (lot # 0186). 'lhe average er.NA insert size was

PAGE 211

Figure 39. Inmmoscreeni.rg of a Human Fetal Liver cCNA Expression Library with Antisennn Against MP-73. ~coli Yl090 was infected with I,i1age gtll arx:l grown on 150 mn X 15 mn agar petri dishes (1.5% agarose in L.B. medium). Followirg incubation at 42C (3.5-5 h), IPffi saturated nitrocellulose filters were overlaid onto the lawn of I,tlage. After 3 h at 37C, the filters were rerroved arx:l inm.moscreened with antisennn prepared against MP-73 as described in Oiapter V. Key: Filter A, a representative filter fran the initial screenirg of 200,000 i.mepen:lent I,i1age plaques. Filter B, secorrl :room of screenirg with a~ plaque positive obtained fran the first screenirg.

PAGE 212

200 lmmunoscreening of Human >, gtll cDNA library Filter A Filter B I /./ -, : : . ,. .. . .. .. .. . ... . : .. .. I ., t -~. :.....-

PAGE 213

201 1.1 kb arrl the cmAs rarged fran 0.34 to 3.4 kb. '1he secorrl ci:NA library was produced usinJ mRNA cbtained fran a human fetal liver (first trimester, male) '!he average ci:NA insert size was 1.1 kb arrl the cmAs rarged fran 0.14 to 2.3 kb (lot# 0186). '!he rnnnber of irrleperrlent clones was 2.3 X 105. Both libraries 'tt1ere screened with antiserum raised to MP-73. Initial screenirg of 2 X 105 imepernent clones fran the human fetal library resulted in the detection of 10 positive piage plaques. '!he area includin:J arrl i.nrnediately surroun:ti.n:J the plaque was rencived arrl the ?'}age rescreened rmtil awroximately 90% of the piage plaques on a given lawn of bacteria 'tt1ere antibody positive. Fig. 39, shows a nitrocellulose filter lift of the first arrl seconi rourrl of inm..moscreenin with the human liver library. Of the initial 10 positive clones identified, only four remained antibody reactive through the subsequent rourrls of screenirg. Initial screenin;J of the adult rat liver library (2 X 105 inieperrlent plaques) was perfonood by Neil F. Shay of our laboratocy arrl resulted in the detection of five antibody reactive plaques. 'l\.1o of whidl remained antibody positive through subsequent screenin;J. '!he size of the ci:NA inserts was then estimated by restriction enionuclease treat.Ioont of isolated gtll rnA fran eadl clone. '!he rnA was separated on a 1% agarose gel arrl the rrolecular size of the ci:NA inserts was detennined by carparison with size markers. '!he size of the ci:NA fragneit cbtained fran the human fetal library was awroximately 1,100 bp (Fig. 40), while the two cmAs cbtained fran the adult rat liver are aw:roximately 2,000 bp am 1,000 bp (Fig. 41a). Both of the rat cmAs arrl the one human ci:NA 'tt1ere subcloned into the bacterial plasmid p]C19 (Fig. 42). 'Ihis was perfonood by first

PAGE 214

Figure 40. Molecular Size of the Human Fetal Liver cCNA Insert fran Bacteriqilage lambda gtll an:l p]C19. .AW:roxilllately O. 07 ng of Ii}age gtll I:NA was restricted with F.coR1 en:lonuclease. 'Ihe I:NA was then separated on a 1% agarose gel. 'Ihe cCNA insert was lcx:::alized by ethidium branide stai.ninJ of the gel, excised an:l subcloned into J;VC19 as described in Chapter v. At:Proximately o. 04 ng of plasmid J:NA contai.ninJ the cCNA insert was then restricted with F.coR1 an:l analyzed by agarose gel electrcpioresis. Key: Lane A, Him III restricted lambda I:NA size markers: Lane B, p]C19 plasmid: Lane C, p]C19 restricted with F.coRl.: Lane D, J:{JC19 contai.ninJ the cCNA fragment restricted with F.coRl; Lane E, plaque positive :(ilage I:NA restricted with F.coRl. 'Ihe 100lecular size of the lambda markers are: 23,130: 9,416: 6,557: 4,361: 2,322: 2027: 564: 125 bp.

PAGE 215

0. ..Q .. 23.1 71, 2.3 f 2.0 _, Std 203 EcoR 1 pUC .,,, .........._ pUC pTCHl gt 11

PAGE 216

Figure 41. Molecular Size of the Adult Rat Liver CCNA Insert f:ran Bacteriqtiage lambda gt11 am pJC19. ~roximately o. 04 irg plaque positive J:i}age J:NA was restricted with F.coRl. as described in Olapter V am then analyzed by agarose gel electrcploresis. 'lhe CCNA fragments v.1ere localize1 by Ethictium Branide stainirg of the gel, electroeluted am then subcloned into pJC19. 'lhe size markers v.1ere generated f:ran restriction with HiroIII am F.caRl. of laniJda J:NA. Key: Figure A: lane 3.2, plaque positive J:i}age J:NA restricted with F.coRl.; lane 6.2, plaque positive J:i}age J:NA restricted with F.caRl.. Figure B: lane pJC, 0.01 irg pJC; lane pJC, F.caRl. restricted p]Cl9 (0.01 irg); lane 2, CCNA fragment (2089 :t:p) subcloned into pJC19 am restricted with F.coRl.; lane 1, CCNA fragment (1047 :t:p) subcloned into pJC19 am restricted with F.coRl.. 'lhis analysis was perfonned by Neil F. Shay of this laboratory.

PAGE 217

A. 205 Eco R 1 2 1 7 9 10 M "gt 11 phage DNA 5.1 5.0 4.3 20 1.9 1.6 13 0.9 QS a. ..a ... .:::: D) C (1) ...

PAGE 218

206 B Eco R 1 Std pUC / pUC p NSRl 2 1 5.1 Q. 4.9 4.3 .a .. .r; 0, C: 2.0 cu 1.9 1.5 1.3 0.9 0.8

PAGE 219

207 rrifyin:;J the cCNAs by agarose gel elect:rqiloresis arrl then ligatin:;J with EcoR1 -restricted plasmid as described in Olapt.er V. Followin:;J ligation, the plasmid CNA was used to transfonn E. coli bacterial strain 'IG-1. Preparative isolation of plasmid CNA was :perfo:rmed as described in AWerrlix V arrl was used as the source for all cCNA fragments. Figs. 40 arrl 41.b show the rrified plasmid CNA arrl the F.coRl-treated plasmid CNA fran both the hl.nllail arrl the rat, respectively. In both cases, the sizes of the cCNAs ootained fran EcoRl-digestion of plasmid CNA corresporrl to those ootained fran EcoR1 digestion of gtll JX}age CNA. In order to confinn that the cCNA initially identified fran the hl.nllail fetal gtll library was sul:x::loned into i:vc19, restriction errlorruclease digestion was :perfo:rmed on both of the rrified cCNAs. In both cases, HaeIII restriction analysis yields foor major CNA fragments of m::>lecular weights 500, 200, 170, arrl 110 q> (Fig. 43). Ethidium branide stainin:;J of the agarose gel also revealed two additional CNA fragnw:mts of lower abumance corresporrling to awroximately 330 arrl 310 q>. '1hese results de.nonstrate that the same cCNA ootained fran the imnunoscreeni.r of the hl.nllail liver library was sul:x::loned into i:vc19. Because the same anti~ was errployed to screen both the rat arrl hl.nllail cCNA libraries, it was of interest to detennine if any of the rat liver cCNAs were haoologous to the cCNA isolated fran the hl.nllail fetal library. As mentioned above, the rat cCNAs were identified arrl sul:x::loned by Neil F. Shay. Plasmid CNA containin:;J the rat liver cr:NAs (pNSR1 arrl p{SR2) hl.nllail cCNA (p'l'Oll) arrl a IOOUSe cCNA (pWWMl, contains a 900 q> CNA sequence fran the IOOUSe lipocortin gene) was restricted with EcoR1. 'lhe cCNAs were separated fran the plasmid CNA on a 1%

PAGE 220

Figure 42. Restriction Map of IiJC19 Genane. '!he IiJC19 is a 2686 bp plasmid that CX>Iltains the Pvu IIjF.caR]. fragment of pBR322 (Vieira am Messirg, 1982). '!his fragmant contains an anpicillin gene, am an origin of replication. '!he plasmid also contains the sequences of the lac z gene. cr:NA fragments obtained fran i.nmmoscreenirg 'INere inserted into the mrique F.coRl. restriction site located within the lac Z gene. 'lhis results in loss of ~actosidase activity upon transfo:rmation with the bacterial host am allows for selection of recart>inants as described in Oiapter v. '!his figure was taken fran r:avis et al. (1986).

PAGE 221

209 puc Multiple Cloning Sites pUC-8 (2678 bp) Sal I Smal Xmal Sall pUC-9 (2768 bp) Hind Ill ONA SyntheSlS pUC 12 ACGAA 11 CGAGC I CGCCCGr.<,r.A I cc IC I A(;A(; I CGAC:C: I GC. Alif". r:r : AA( ;c I 11 ,liC:AC I G (M13mp10) Sstl 8amHI Xba I Sa/I Ace I Hine II Hind Ill ONA Synthesis pUC 13 ACOCCAAOC TTOGGCTOCAGG TCOACTC T AGAGGA TCCCCGGOCGAGC T CGAA n CACTO (M13mp11) pUC -18 (M13mp19) pUC-19 (M13mp18) Sa/I Ace I Xba I Hine II SamH I EcoR I ONA Synthesis ACGCCAAOCTTOCATGCCTOCAGO TCOACTCT AGAGGA TCCCCGGG T ACCO.AOC TCGAA TTCACTG s;:r::Ja Hind Ill Pst I Ace I Xba I SamH I Sma I Kpn I Sst I Hine II Xmal ONA SynthestS A(;GAA llCGAG<.; ICOG T ACCCOGGOATCCTC r AGAG TCGACC IGCAGGCA !<.CAAGC noGCACTG Sst I Kpn I Sma I SamH I Xba I Ace I Pst I Hind 111 Xmal Hine II

PAGE 222

Figure 43. Restriction Errlonuclease Analysis of the Bacteriqnage lambda gtll am. the SUbcloned Hlnnan Fetal Liver crnA Insert. Rlage mA am. J;i]C19 both conta~ the crnA fragment -were restricted with F.caRl.. 'lhe mA was then separated on a 1% agarose gel am. the mA inserts -were then electroeluted. AR:>roxilnately 1 g of each crnA fragment was restricted with Hae III am. analyzed on a 6.0% polyacrylamide gel conta~ 50 nM Tris, pl 7 .4, 50 nM boric acid, am. 1 nM EIJI'A. 'lhe m::>lea.ll.ar 1weight markers -were ootained by restrictin:J J;i]C19 with M5pI which generates mA fragments of: 501, 489, 404, 331, 242, 190, 147, 111, 110 q>.

PAGE 223

211 gtll pTCHl Std

PAGE 224

Figure 44. Hybridization Analysis of Rat and Hmnan Liver crNA's. AI:Proxilnately 0.01 nq of plasmid CNA containin:J the rat liver crnAs (J;NSRJ. and ~), human crNA (pTCHl) and a nntSe crNA (pWWMl., lipocortin gene) was restricted with F.coRl. en::lonuclea.se as described in Chapter V. '1he rNA was then analyzed on a 1% agarose gel. Followin;J electrq:horesis, the gel was incubated twice for 10 min in 0.25 M HCl, rinsed with distilled water, and then incubated twice for 25 min eadl with 0.5 M NaaI. '1he gel was neutralized with 1 M TBE (twice for 25 min eadl), then with 50 ICM TBE (twice for 25 min eadl). '1he agarose gel was then incubated for 30 min with 20X SSC and transferred to Gene Screen nylon membrane as described for the Northern analysis (Chapter V). '1he nylon membrane was incubated with a [32p]-labeled sin;Jle strarxied crNA pre.be (J;NSRJ.) cbtained fran the rat library (Fig.a). '1he nylon membrane was incubated with TE 1:uffer containin:J 1% sr:s for 30 min at 950c to reroc,ve the radiolabeled crNA pre.be and then incubated with a [32p]labeled crNA (pTCHl) ootained fran the human library (b). '1he hybridization corrlition.s and washinJs were identical to those described for the Northern analysis (Chapter V)

PAGE 225

213 A. pUC19 [3 2p]pNS R 1

PAGE 226

214 B pUC19 [32p]pTCH 1

PAGE 227

Figure 45. Northern Analysis of Polyadenylated RNA Cctrplementary to the cr:NA Isolated fran the Adult Rat Liver cCNA Expression Library .Awroximately 20 g of total cellular RNA d::>tained fran nonnal rat liver tissue was prepared by the nethcxi of Maniatis et al. ( 1982) am then separated on an agarose: fonnaldehyde gel. Followin:J transfer to Gene Screen nylon meni::>rane, the RNA was hybridized with a [32p]-labeled 1,000 q> cCNA probe, prepared by raman primer extension (Feinberg am Vogelstein, 1983), that was ccnplementary to the cCNA insert isolated fran the adult rat liver library.

PAGE 228

.. .c .. a, C G) ... 0.6 216 I. G) > ... 2.4 1.9

PAGE 229

Figure 46. Northern Analysis of Polyadenylated RNA carplementai:y to the cr:NA Isolated fran the Human Fetal Liver er.NA Expression Library .AW:roximately 20 g of total cellular RNA tained fran human ~2 hepatana cells was analyzed on an agarose: fonnaldehyde gel. Followin;J transfer to Gene Screen nylon merrbrane, the RNA was hybridized with a [ 32PJ-labeled 1,100 tp cr:NA prd.Je isolated fran the human fetal liver er.NA expression library.

PAGE 230

.a 5.1 ~ ... j (ii' .. en C a, ...a 2.0 218 HepG2 RNA 2.4kb 1.9kb

PAGE 231

219 agarose gel am then transferred to Gene Screen nylon membrane. '!he nylon membrane was incubated with a [32p]-labeled sirgle strarrled cCNA probe ootained fran either the rat librai:y (i;:tiSIU) or the human liver librai:y (plOil). '!he crnAs were labeled with [32P]-dATP by the rarrlan primer extension net.hod of Feinberg am Vogelstein (1983). 'As shown in Fig. 44a, the rat [32P]-cI:NA hybridizes to the 1.1 kq> human cr:NA, yet does not hybridize to the 2. o kq> rat cCNA or the m::iuse lipocortin cINA. '!he 1. O kq> human [ 32P]-cI:NA hybridizes to the 1. o kq> rat cCNA, with no detectable hybridization to the rat 2.0 kl:p cCNA or the m::iuse lipocortin 0.9 kq> cCNA (Fig. 44b). '!he DDlecular size of the polyadenylated mRNA catplementary to the crnAs isolated fran inmmoscreening was also investigated. 'lhis was perfonned by isolatirg total cellular RNA fran either nonnal hepatocytes or Hep;2 cells. '!he RNA was then separated on a dena:turirg 1% agarose:fonnaldehyde, transferred to Gene Screen nylon membrane, crosslinked with UV irradiation, am incubated with the [32p]-labeled rat cCNA (i;:tiSR].). FollOlrlirg hybridization (36 hat 420c), all of the blots were washed umer strirgent corrlitions (twice with 1X SSC am 0.1% soo at 650c for lh; 0.2X SSC am 0.1% soo at 650c for 30 min) to rem:::r.re any probe balm nonspecifically to the nylon membrane or to nonhcm:>logous mRNA sequences. 'As shown in Fig. 45, the rat 1.0 kq> CINA fragment detects two rat liver mRNA species of DDlecular size 2.4 kb am 1.9 kb, whereas the human 1.1 kq> human cCNA (plOil) detects an ab.lrrlant ttep:;2 mRNA species of DDlecular size 2. 4 kb am a lower ab.lrrlant mRNA of 1. 9 kb (Fig. 46). Discussion '!he major ct>jective of this i:nase of my research was to identify

PAGE 232

220 am characterize a hepatic membrane protein for which biosynthesis is transcriptionally oontrolled by amino acids. UsiDJ primacy cultures of freshly isolated hepatocytes, five membrane proteins have been identified for which synthesis awean; to be enhaJx=ed duriDJ amino acid deprivation. In particular, one membrane protein, MP-73, oorresp::nlin;J to an isoelectric point of 7. o am a nolecular -weight of 73 kIB was chosen for further study because: 1) the protein irrluction was reduced by actinanycin D; 2) the level of irrluction, although not large, was reproducible; 3) the protein was arurrlant enaigh to be detected by Fast Green stainiDJ of the nitrocellulose blots; am 4) the protein was 'Well separated fran other oontaminatiDJ protein spots. In an order to study MP-73 at the cellular am nolecular level, -we chose to develop nonospecific polyclonal antil:xxlies. Antil:xxlies have becane a valuable tool in studies ainai at elucidatiDJ membrane protein topology, stnicture, am function. In addition, nonospecific antil:xxlies can provide the selectivity needed for further isolation am characterization of the membrane antigen. Often, a limitation in generatiDJ a nonospecific antilxxly is the necessity to ootain a sufficient annmt of the p.irified protein to use as inm.mogen. Many membrane proteins are of low aburrlance relative to the total protein content of the cell, so p.irification requires large am::,unts of startiDJ material am is cx:nplicated by the insolubility of membrane proteins in aqueous solutions. 'lb circumvent these problems, I have described a methcxlology that allows the production of nonospecific polyclonal antil:xxlies followiDJ separation of ccnplex mixtures of membrane proteins by b.u-dimensional polyacrylamide gel electrqiloresis am electrablottiDJ. Previous reports have derconstrated the utility of

PAGE 233

221 generatin] antil:xxlies fran polyacrylamide gel plugs contai.ninJ a specific protein either by direct injection of the gel plug or extractin] the protein fran the polyacrylamide prior to injection (Bravo et al., 1983; Tracy et al., 1983; Bou.lard an:l I.ecroisey, 1982). Either of these prooedures results in sane loss of protein sanple because of the han:llin} prooedures required. Develqm with the kirrl assistance of Dr. T.W. O'brien, the methcxiology described in Olapter V of this thesis utilized electrcpioresed proteins that were initially transferred to nitrocellulose prior to immunization in order to concentrate the protein sanple an:l to maximize the delivecy of a low-al::,unjance antigen (Chiles et al., 1987). '1he nitrocellulose-inm:::t>ilized proteins were identified by Fast Green stai.ninJ an:l the area of paper contai.ninJ the specific protein of interest was excised fran several replicate transfers. '1hese pieces of nitrocellulose were then used as a solid-base i.nrrunogen. Usin] this methodology, antisennn has been raised to ttNO different hepatic ment>rane proteins, MP-66 an:l MP-73. Based on ONO-dimensional i.nm.mct>lot analysis of the ment>rane antigen, the antisera to both was m::>nospecific. '!he antisennn against MP-73 was used to further study the synthesis of the protein. Less than 20 g of protein antigen was required to elicit significant anti.body production. '1he sennn anti.body titer was sufficient to allow detection of MP-73 at a sennn dilution of 1:2000. Collectively, these results in:licate that nonospecific anti.body production can be elicited through the use of antigen that is concentrated an:l inm:::t>ilized on nitrocellulose. '1he procedure can be readily awlied to sanples that are cbtainable only in small quantities ( <20 g) such as proteins present in cells maintained in tissue culture. An additional feature is the ability to prepare antil:xxlies

PAGE 234

222 again.st membrane-boom proteins that are difficult to solubilize an:l ?,Irify. Irrleed, this methodology does not require protein ?,Irification beyon:i identification of the protein in a two-dimensional gel electrqiloretic pattern. Separation of proteins by two-dimensional rather than one-dimensional gel electrqiloresis prior to :innunization greatly enhances the prooability of ootainirg a nonospecific antibody an:l the use of nitrocellulose-l:x:AJ.nj protein as a solid-p,ase inmmogen results in nearly a 100% delivecy of the antigen. 'lhe methodology shcw.d allow production of anti.bodies again.st proteins that can be identified on a two-dimensional gel or blot by procedures such as enzymatic activity, ligam bi.rxlin;J, or selective covalent m::xtification. other laboratories have reported similar methods for antibody production (Abou-Zeid et al., 1987; Jacxi:> et al., 1986). For exanple, Diano et al. (1987) described a similar procedure for prepari.rg anti.bodies to paramyosin an:l trqx:myosin frcm C>wenia fusifonnis. In contrast to oor procedure, these authors rut the nitrocellulose pa:per into fine strips in PBS an:l then sonicated the pa:per l.ll'ltil the nitrocellulose was reduced to a fine~-AWroximately 20 to 30 g of protein was then injected subcutaneously on the back of a ratbit. Antibody production was detected by i.ntnund:)lot analysis after the secorrl series of injections. 'lhe serum titer was sufficient to allow .i.mrra.mcblotti.rg to be perfo:rmed at dilutions of 1:10,000. M::>nospecific polyclonal anti.bodies were used to localize MP-73 to a membrane fraction enridled for mitochorrlrial inner membranes. Based on Triton X-114 solubilization studies, MP-73 to be an ext.renely hydrqilobic integral membrane protein. It does not to be associated via disulfide borrli.rg with other mitoc:horrlrial membrane

PAGE 235

223 carponents or itself (i.e., 1IDilCl'OOJ:"S am dimers). 'lhe identification of the i:nysiological or biochemical function of MP-73 at this time is unknown, however, several p::,ssibilities exist. Cleclercq et al. (1987) am Woeltje et al. (1987) have recently published a series of reports describin:;J the dlaracterization of the rat liver mitochorrlrial cai:nitine palmitoyltran.sferase I. 'Ibis transport protein is located within the inner membrane of the mitodl.omrial am is irreversibly inactivated by tetradecylglycidyl-caA ('m-CaA). 'lhe concentration of 'm-CaA needed to reduce activity by 50% was 60 rj.f. When radiolabeled 'm-CaA was bourrl to mitocnomrial membranes am then the proteins analyzed by ~PAGE, two major proteins of nolecular ,;,.,,eight 75 kD3. am 86 kD3. were labeled. 'lhe migration of both proteins was uncharged when analyzed by nonreducin:;J PAGE. 'lhe proteins were not soluble in either cligitonin or octylglucoside, however, the authors noted that the 'm-CaA-insensitive cai:nitine palmitoyltransferase II was solubilized with 'l\t.1een-20 am Triton x-100. 'lhe nolecular ,;,.,,eight of this protein was fourrl to be awroximately 80 kl:B. Ohnishi et al. (1986) am Hatefi (1978) have isolated the submits of the bovine heart mitochomrial NAIE:ubiquinone oxidoreductase cx:nplex I. 'lhe nultimeric carplex can be separated into three catp:>nents by treatin:;J mitodl.omrial inner membranes with chaot:rq:>ic agents sud1 as sodium perchlorate. 'lhe carponents are: 1) a soluble flavo-iron sulfur protein fraction with NAIE dehydrogenase activity; 2) a soluble iron -sulfur protein fraction; am 3) an insoluble hydrcpld:>ic protein fraction. '1he NAIE dehydrogenase fraction consists of three proteins of nolecular ,;,.,,eight 51, 24, am 10 kl:B. TreatJnent of the soluble ironsulfur protein fraction with deoxydlolate am urea solubilizes a 75 kD3.

PAGE 236

224 protein, whereas octylglucoside solubilizes three proteins of m::>lecular weights 49, 30, am 13 kDa. 'Ihe 75 kDa protein does not react with antisennn prepared against MP-73 on lllllllilOOlots (the proteins of catplex I were the generous gift of Dr. Hatefi). Recent stulles with the suocinate:ubiquinone oxidoreductase (catplex II) has revealed that this catplex is cx:.ttPJSE!d of foor polypeptides of m::>lecular weight 70, 27, 15.5, am 13.5 kDa (Hatefi am Galante, 1980). Treatment of catplex II with sodimn perdllorate results in the selective release of suocinate dehydrogenase, an enzyme wni.dl is cc.rnposed of two proteins of m::>lecular weight 70 am 27 kDa (Hatefi am Hanstein, 1974). It is unknown at the present ti.me whether MP-73 antisennn reacts with suocinate dehydrogenase. Very feM proteins have been identified for wni.dl nutrient m::>lecules directly regulate gene transcription. Expression of 3-hydroxyl-3methylglutaryl ex>eneyme reductase (HM:; CoA reductase) is, holNever, regulated at the gene level by cholesterol. Cloned cr:NAs for the HM:; CoA reductase have been isolated am errployed to m::>nitor mRNA levels. In rat liver, the level of HM:; CoA reductase mRNA is increased by cholestyramine, the administration of mevalonolactone, dlolesterol, or hydroxysterols decreases the mRNA (Goodridge, 1987). 'Ihe effects of cholesterol is due in part to a decrease in the rate transcription of the HM:; CoA reductase mRNA (Clarke et al. 1985) Sequences responsible for prcm:rl:er activity am for mediatin} the inhibition of the transcription have been identified (Osborn et al., 1985). 'Ihe sequences are distribrted over 500 tp 5'upstream fran the initiation codon. Recently, Osborn et al. (1987) dem:>nstrated that at least two upstream elements are absolutely required for transcription of the HM:; CoA gene.

PAGE 237

225 'Ihe first area is located 85 bp upstream fran the initiation codon am is awroximately 18 bp in 1~; the secorrl area is located at a distance awroximately 30 bp upstream fran the initiation codon. Amino acid sta?:vation irrluces the biosynthesis of MP-73; this response may be specific for amino acid sta?:vation or it may be a result of a m:>re general stress response. A preliminary experiment designed to test for dlarges in MP-73 biosynthesis followinJ heat-shock proved negative even though the usual family of heat-shock proteins were irrluced (Harrll.ogten am Kilberg, llnp.lblished results). 'Ihe availability of the cINA clones as hybridization probes will allow detailed studies on the kinetics of irrluction, the levels of acctmtl.ation am the rate of transcription of the MP-73 mRNA. '1hese crnA probes will also allow us to ascertain 'Whether MP-73 is umer general or specific amino acid control by m:>nitorinJ the repressor activity of imividual amino acids.

PAGE 238

CHAPTER VI I SUMMARY '!he general outline of these studies has been to describe sare of the processes regulated in hepatic tissue durin;J amino acid deprivation. Because the System A carrier is regulated by amino acids, it contrirutes significantly in the transport of nutrient nnlecules durin;J amino acid starvation. 'lhese studies are the first atte.rrpt to characterize the carrier protein at the chemical level. Usin;J a variety of protein m:xlifyin;J reagents, -we have abser:ved that the System A carrier in hepatic tissue requires a free sulfhydl:yl groop(s) for transport activity. '!he results presented suggests that the sulfhydl:yl groop(s) is located within the amino acid birrli.rg site of the carrier protein an::l is d::>ligato:ry for active transport. Al tha.lgh rn.nneroos enzymatic an::l transport activities have been shown to be sensitive to sulfhydl:yl m:xlification, few investigations have provided details at the nnlecular level (X)ncei:nin;J the relation between the m:xlified cysteine residue an::l the actual catalytic events. An exception is the recent work by Profy an::l Schilmel (1986). '!he It-glycine aminoacyl-tRNA synthetase fran ~(X)li has been shown to be inactivated by NEM via direct dlemical m:xlification of the B-subunit (Ostrem an::l Berg, 1974). To confinn these studies, Profy an::l Schi.nme1 (1986) used oligorrucleotide-directed mutagenesis on the gly s gene. In this stlXly eadl B --submit I.r-cysteine ccx:lon was replaced .irrlividually by a I.ralanine ccx:lon. '!he resultin;J proteins -were active in vivo an::l interestin;Jly their in vitro aminoacylation activities -were catparable to the native protein. 226

PAGE 239

227 F\lrthenIDre, a :rcutant protein incorporatin:J all of the amino acid substitutions was also active, suggestin:J that the B -sub.mit cysteine thiol is not required for the catalysis of aminoacylation. studies by Kaback am co-workers on the lactose transporter (lac y gene) in F..scheridria coli. Beyreuther et al. (1981) showed that eys148 of the lactose carrier was nmified by NEM. '!his nmification am the resultant inactivation was blocked by substrate (Truni)le et al. 1984) However, when a lllltant lac y gene product was generated by oligonucleoti~ site-specific nutagenesis so that the eys148 residue was replaced by a glycine, the nutant cells exhibited slower initial rates of uptake, yet aca.mulated lactose to steady-state levels equal to those seen in wild type cells. 'Ihese am other results irrlicate that although eys148 is inp:>rtant for substrate protection against sulfhycb:yl inactivation, it is not obligatory for lactose transport (Viitanen et al., 1985). In fact, a series of experiments were recently reported in whidl eadl of the eight cysteine groups in the lac pernease were systematically replaced with either glycine or serine (Merrick et al., 1987). '1he results suggested that only eys154 is obligatory for transport. Until similar sbrlies are perfonood with the System A carrier, one cannot rule out the possibility that the amino acid-protected sulfhycb:yl group is not necessary for transport, but is involved in the PCMBS-deperrlent inactivation. We have also identified am characterized a series of hepatic nenbrane proteins for which biosynthesis awears to be regulated by the concentration of amino acids in the extracellular medium. '1he production of anti.1:x:xlies has allowed the identification am isolation of potential crnA clones fonn both human am rat origins. '1he availability

PAGE 240

228 of the cCNA clones as hybridization prd:>es will allow detailed studies on the kinetics of imuction, the levels of aoa.mulation arrl the rate of transcription of the MP-73 :mRNA. 'lhese cI:NA prd:>es will also allow us to ascertain whether MP-73 is urrler general or specific amino acid control by nonitorinJ the repressor activity of in:lividual amino acids. Future experinents in the laboratocy will fcx:::us on the regulatocy sequences 5'-to the MP-73 gene in order to detennine how amino acids regulate :mRNA synthesis. 'lhese studies will be deperrlent on the isolation of a full len:fth cCNA clone, in the event that a full-len:fth clone has not been obtained fran the initial screening of the libraries. An oligonucleotide ('X)~ to the sequences flankinJ the initiation a:xlon of the full lenJtll cl:NA will then be enployed to screen a genanic libracy in order to identify the sequences 5'-to the MP-73 gene. To study the effects of amino acid starvation on pramter activity, the I:NA fragments containinJ the 5'-sequences of the MP-73 gene will be introduced into a plasmid containinJ the reporter coc:lirg sequences for the dll.oranpieni_('X)l acetyltransferase gene. To detennine which regions 5'-to the gene are inp::>rtant for amino acid regulation, deletions generated by cleavinJ with the awropriate restriction enzynes or by treat:nent with the exonuclease III or Bal 31 rruclease will be perfo:nned. In addition to the deletion studies, in vivo protein-I:NA interactions will be examined usinJ genanic sequencinJ nethodology (Church am Gilbert, 1984; Nick am Gilbert, 1985). A tremerrlous am::,unt of .info:nnation concerning gene regulation in bacterial systems durinJ amino acid deprivation is known. studies with genetic nutants have revealed a cx:rrplex series of activator am repressor proteins, which resporrled to external dl.an:Jes in amino acid

PAGE 241

229 levels arx:l then regulate gene expression of the respective amino acid biosynthetic enzymes. Recent studies with yeast nutants have revealed an even ll)re oc:rrplex system of gene regulation durin;J amino acid starvation (Hq:>e arx:l struhl, 1985). Hov.1':!ver, very little infonnation is known abait how higher eukacyotic cells respon:ied to d1an;Jes in extracellular amino acid concentrations at the level of the gene. It is hope that by identifyin;J the genes for proteins, sud1 as MP-73, which are in:luced durin;J amino acid deprivation, the ll)lecular :medlanisms may be ascertained.

PAGE 242

APPENDIX I TISSUE ClJIJIURE MEDIUM AND TRANS:EORI' IlJFFERS Preparation of Tissue CUl ture Meditnn: Choll
PAGE 243

NaKRB: 231 25 nM scxlium bicart>onate, 119 nM NaCl, 5.6 nM glucose, 5.9 nM KCl, 1.2 nM magnesium sulfate, 1 g penicillin. 100 ng streptanycin, 75 ng i;:nerx:,l red (scxlium salt), 2.3 ng n-rutylp-hydroxybenzoate, 2.5 nM calcium dlloride, 10 g BSA ( q;,tional) 1. Dissolve first ten chemicals in 8. O 1 of water. 2. Dissolve calcium dll.oride in 0.5 1 of water, then stir it into the above solution. 3. Bul:ble this solution with 5% cart>on dioxide for at least 1 h. 4. Add dissolved BSA (0.5 1) or 0.5 1 water if BSA is not usa:l. 5. Adjust the volume to 10. o 1, pl to 7. 35, filter arrl store at 4 Oc. MEM: A 5.0 1 package of MEM is 5tWlemented with 1.46 g Irqlutamine, 10 g scxlium bicarbonate, 50 ng streptanycin, 500 ng penicillin, 142 ng gentamicin, 1.2 ng n-rutyl-p-hydroxybenzoate, 10 g BSA. 1. Dissolve the MEM arrl first six chemicals in 4. 5 1 of water. 2. Bul:ble with 5% cart>on dioxide for 1 to 2 h. 3. Dissolve the BSA in 0.5 1 water, add it to the above solution, pl to 7.33, filter, arrl store at 40c.

PAGE 244

232 Preparation of Radiolabeled Amino Acid Uptake Buffers: 1. For the majority of transport measurements the final concentration of amino acid was 50 M. 2. 'lhe annmt of radiolabeled amino acid used in transport ~ts was usually 6 1/ml, the final concentration of the amino acid was then prepared by usin;J the awi:q,riate tmlabeled amino acid stock solutions prepared in either NaKRP or CllolKRP buffers. All trititnn labeled amino acid carpcmrls were evaporated before use in order to re.nJV'e trace annmts of radiolabeled water. 'lhe specific activity of the amino acids is shown below am were ootained at a concentration of 1 nCi/ml. To prepare a 21 ml NaKRP uptake buffer CX>11tainin;J 50 M AIB, 1050 moles of AIB is needed .AWroximately 126 1 of radiolabeled AIB is evaporated in a small glass beaker; this CX>11tains 12.6 moles of radiolabeled AIB .AWroximately 104 1 of a 10 IrM unlabeled AIB stock solution (in NaKRP) is then added to yield 1050 moles of AIB total. Finally, 20.8 ml of NaKRP is added to yield a 50 M solution. All uptake buffers were stored -700c. IrAlanine L-Cysteine IrGlutamine IrProline L-'Ihreonine lr'l'1:yptqilan Irl'yrosine AIB 30 Ci/mnol 0.8 Ci/mnol 46 Ci/mnol 41 Ci/mnol 0.2 Ci/mnol 6.1 Ci/mnol 51 Ci/nt00l 10 Ci/nt00l IrHistidine IrI.eucine I.rMethionine IrSerine Ir-Glycine Ir:Rlenylalanine IrAsparagine MeAIB 56 Ci/nt00l 136 Ci/111001 11 Ci/111001 28 Ci/111001 0.1 Ci/mool 50 Ci/111001 14 Ci/111001 0.5 Ci/nt00l

PAGE 245

APPENDIX II SOI.lJI'IONS FOR FOLYACRYIAMIDE GEL EIECIR)RDRESIS (Ref: RdJerts et al. 1984) Acrylamide stock Soln 1 31.96 g a~lamide (Bio-Rad) 5.64 g diallyltartardiamide (DATA: Sigma D-2391) Bri.n;J this soln to 100 ml with distilled water. Store in brown bottle at 4C (1-2 m:>nths). Riboflavin--TEMED Soln 2 2.0 ng rilx>flavin 50.0 ml distilled water Inmediately before use, add 8 1 N,N,N,N'tetraethylmethylenediamine (TEMED) to 1 ml of this solution. Anpl. Soln 3 5.5 g urea (Pierce No 29700) 5o.o ng orr (0.5%) Adjust final volume to 10 ml with 2% NP-40 (Partical ll:lta laboratories) in 5 nM potassium bicamonate. Aliquot in to 0.2 ml arx:l store at -70C. NP-40 orr Soln 4 5.5 g urea (9.3 M) 5o.o ng orr (0.5%) Dilute to 10 ml with 2% NP-40 in 5 nM potassium bicamonate. Wom F.quilibration Buffer Soln 5 3.9 g Tris base (65 nM) 5.0 g SIB (1.0%) Dilute to 500 ml with distilled water arx:l Iii to 6.9. Add 1% 2mercaptoetllanol ilmaliately before use. Rurmin:J Gel Acrylamide Soln 6 75.0 g a~lamide 2.0 g N,N'-nethylene-bis-a~lamide Dilute to a final volume of 250 ml with distilled water arx:l filter. store in brown bottle at 4 c. Rurmin:J Gel Buffer B Soln 7 90.5 g Tris base 1.6 ml TEMED Dissolve Tris arx:l TEMED in 450 ml distilled water arx:l add about 9 ml 12 N HCl. Dilute to a final concentration of 500 ml arx:l Iii to 233

PAGE 246

8.8-9.0. store at 4C. stackin;J Gel Blffer E Soln 8 5.98 g Tris base 0.46 ml TEMED 234 Dissolve the two dlernicals into 90 ml distilled water, add 4 ml 12 N HCl, am J:iI to 6. 6-6. 8. BrinJ to a final voltnne of 10 ml am store at 4C. Top Gel Sealer Soln 9 1. 0 g agarose 100 ml Worm equilibration 1:uffer Divide into 10 ml portions am freeze. Illlnediately before use, nelt in a boilinJ water bath, make 1% with 2-mercaptoethanol am add 3-4 drops of 0.15% bratqhenol blue. Electrqnoresis Tank Blffer 12.2 g Tris base 57.6 g glycine 4.0 g soo 4.0 1 deionized water 0.04 M NaaI 3.2 gin 2.0 1 distilled water, degas before use. 5 nM Potassium Bicarbonate 69. O ng in 100 ml distilled water 8 M Urea 24.02 gin 50 ml distilled water. Anunonium Persulfate 28. o ng in 10 ml distilled water 20% soo 10.0 gin 50 ml distilled water 0.06 M SUlfuric Acid 4 .125 ml in 2. 5 1 distilled water Fast Green Trackin;J Dye 0.1 ml NP-4o-urr Soln 4. 0.1 ml distilled water o .1 ml 1% fast green dye stock Gel Fixative 5% 'IC.A,/10% acetic acid/30% methanol coanassie Blue Dye staininJ Solution

PAGE 247

235 1. 25 g ooanassie blue in 500 ml of a solution ex:>ntai.nin;J 50% methanol/10% acetic acid, ani filter. Destai.nin;J Soln 20% ethanol/10% acetic acid/70% deionized water Sanple Dilution Buffer for lD-PAGE: 5.0 ml stackirg gel 1::uffer E 1. 0 ml 20% soo 0.4 ml bratqilenol blue stock (1.5 nq/ml) 4.0 ml 60% glycerol 1. o ml II ani 8. 6 ml distilled water

PAGE 248

APPENDIX III GENERAL MEllDil3 .AND ENZYME ASSAYS Purification of IgG fran Ral:i:>it Serum: (Ref: stei.nru.cn am Audran, 1969; Tijssen, 1986) 1. Blood is ex>llected in 50 ml sterile Cornirg plastic test tubes am incubated at roan terrperature for 1 h. 2. 'lhe clot is rinmed fran the walls of the tube with the aid of a wooden awlicator stick am the blood incubated overnight at 4C. 3. 'Ihe tube is then centrifuged at 10, ooo g for 20 min to obtain a sen.nu enriched supeznatant. 4. 'lhe sen.nu is rem::,ved am the EiJ adjusted to 5.0 with 3 M acetic acid ( 1 drq>/ml) 5. One part n-octanoic acid (Sigma C-2875) is then added to 20 parts sen.nu. 'lhe acid is added while mixin:J the sen.nu vigo:rously. 'Ihe solution is then stirred an additional 30 min. 6. 'lhe mixture is centrifuged at 20,000 g for 30 min. 7. 'lhe supeznatant is then rem::,ved am an equal volume of saturated anm:>rliurn sulfate is added to the supeznatant (stir at roan terrperature for 1-2 h or overnight at 4C). 8. 'lhe solution is then centrifuged at 20, 000 g for 30 min. 9. 'lhe pellet enriched for IgG, is washed with PBS ex>ntainiD3' an equal volume of saturated amoonium sulfate. 10. 'lhe pellet is then resuspened in 2-3 ml PBS ex>ntainiD3' 0.02% scxlium azide am dialyzed 12 h with two changes (LO 1 per~ at 4C) against PBS ex>ntainiD3' 0.1% scxliurn azide. 11. 'lb quantitate the IgG a 1/100 dilution is made of the final IgG fraction am the abso:rbance maasured at 280 nm (E=l.4 ~1 x an-1). Solutions: Saturated Anm:>niurn SUlfate; Add 900 g amrionium sulfate to 1.0 1 distilled water. Heat until dissolved am quickly filter through a 236

PAGE 249

237 Whabnan #1 filter. After CXX)lllXJ, the solution is adjusted to pl 7.4 with amronium hydroxide. 3M Acetic Acid; 19 ml acetic acid in 81 ml distilled water. RadiolabelirXJ Proteins with [125!]-Iodine: (Ref: Ht.mter am Greenwood, 1962) 1. 'lhe protein is diluted into PBS at 0.2 ng/0.5 ml. 2. 1.0 nCi radioactive c125r]iodine is added to the protein mixture. 3. 0.01 ml Chloramine T (25 ng/10 ml in PBS) is then added. 4. 'Ibis solution is mixed errl-over-errl for 30-45 sec. 5. 'lhe reaction is stcg:>ed by addin:J O. 02 ml sodium metabisulfide ( 60 ng/10 ml in PBS). 6. 'lhe radiolabeled protein is then separated frcm the free iodine by gel filtration on a 8-12 ml 5eiiladex G-100 coll.mm equilibrated with PBS containirXJ 0.01% sodium azide. 7. o. 5 ml fractions are collected am the radiolabeled protein solution is stored at 4"C.

PAGE 250

238 Silver sta~ of Proteins in Folyaccylamide Gels: (Ref: Wray et al. 1981) 1. 'lhe gel is soaked in 50% methanol overnight with several dlanges. 2. 'lhe silver stain is prepared by addirg 0.8 g of silver nitrate to 4 ml distilled water. 21 ml of a 36% NaaI solution is then added to 1.4 ml of 14.8 M NH4oo. 'lhe silver nitrate solution is then added dropwise with constant stirrirxJ to the Naaf/NH4 CH. '!his solution is the taken to 100 ml with distilled water arrl used within 15 min. 3. 'lhe gel is incubated with the silver stain solution for 15 to 30 min. 4. 'lhe silver stain solution is then decanted arrl the gel washed with 3 dlanges of distilled water (200 ml) for 5 min eadl. 5. 'lhe gel is developed with a solution conta~ 2.5 ml of 1% citric acid arrl o. 25 ml of 38% fo:rmaldehyde in 500 ml distilled water. If the developirxJ solution turns dark, rinse the gel as described in step 4 arrl then add ll'Dre developer. 6. 'lhe reaction is stop by incubatirxJ the gel in a 45% methanol/10% acetic acid solution. 7. 'lhe gel can be destained with film stren:Jth Kodak Rapid Fix, followed by rin.sirxJ with water as described in step 4 arrl then washml in hypo-clear (25 g/1 distilled water. 'lhe gel is then placed in 50% methanol to stabilize the protein stain.

PAGE 251

5'-Nucleotidase Assay: (Ref: ~rre, 1971) 239 1. Me.rri:>ranes are diluted to 1 ng protein/ml am wanned to 37C. 2. 'Ib initiate the assay, o .1 ml of the membranes is added to o. 9 ml substrate mix, vortexed am place in water bath at 37C for 15 min. 3. '!he reaction is tenninated by addinJ 1 ml ice-cold 10% '!CA. 4. '!he sanples are centrifuged at 10,000 g for 20 min. 5. One-half ml of the supernatant is re.roved for inorganic ~te detennination as describoo. below. 6. '!he b.u control sanples are: 0.1 ml water (in place of the membranes) am 0.9 ml substrate mix; 0.9 ml water (in place of the substrate mix) am o .1 ml nenbranes. SUbstrate Mix; 5.5 :rrM magD?Sil.nn dll.oride, 55 :rrM Tris base, 11 :rrM 5'-AMP (Sigma Type III), 10 :rrM Na+~-tartrate, pl 8.4.

PAGE 252

Glucose-6-Fhosp1.ate 'Assay: (Ref: swanson, 1955) 240 1. Membranes are adjusted to a concentration of 1 ng protein/ml am wanooa to 37c. 2. '!he reaction is initiated by addin3 0.1 ml of the membranes to 0.3 ml maleic acid ruffer am 0.1 ml substrate mix. 3. '!his mixture is incubated for 15 min at 37C, then tenninated by a.ddin:J 1 ml of ice-cold 10% TCA, 2. 5 ml water, am vortexed. 4. '!he solution is centrifuged at 10, ooo g for 10 min am o. 5 ml is I."e.llOV'ed for inorganic piosplate detennination. 5. '!he t\oJO controls sanples are: O .1 ml substrate mix, O. 3 ml maleic acid ruffer, am 0.1 ml water (in place of membranes); 0.1 ml membranes, o. 3 ml maleic acid ruff er am O .1 ml water ( in place of substrate mix) Maleic acid ruff er; 1.16 g maleic acid in 60 ml distilled water, NaOH pellets are added \llltil the pl is 6.3, the solution is then diluted to 100 ml am pl to 6.5. SUbstrate mix; 0.1 M glucose-6-piosr,hate in distilled water.

PAGE 253

241 SUccinate: cytochrane C Reductase Assay: (Ref: Kilberg arrl. Christensen, 1979) 1. Reference cuvette contains the followi.rg solution: 0.1 ml buffer, 0 .1 ml KCN, 0 .1 ml cytochrane C arrl. 0. 7 ml water. 2. Two sanple tubes are prepared. 'lhe first tube neasures the nonenzymatic rate of the reaction. 'lhe seconj tube measured the enzymatic rate of the reaction. Both tubes contain the followi.rg solution: 0.1 ml buffer, 0.1 ml cytochrane c, 0.1 ml KCN, 0.1 ml sucx:::inate, arrl. o. 5 ml water. 3. 'lhe first tube is diluted with 0.1 ml of distilled water arrl. nonitored for a ~e in abso:rbance at 550 nm (non-enzymatic rate). 4. 'lhe secorrl tube is mixed with 0.1 ml of nenbranes (1 ng protein/ml) arrl. the abso:rbance nonitored at 550 nm. 5. Steps 3. arrl. 4. are repeated, rut the KCN is replaced with o .1 ml of distilled water. 6. 'lhe results were expressed as micraiole of cytochrane C reduced per ng protein per min. (E=18,500 ~1 c:m-1) Buffer consisted of 250 nM Tris, 5 nM IDI'A, arrl. 50 nM magnesil..Ill\ chloride, pH 7.4. other solutions; 10 nM KCN, 0.4 nM cytochrane c, 10 nM sucx:::inate (pH 7 .5)

PAGE 254

Glutamate Dehydrogenase Assay: (Ref: Beaufay et al., 1959) 242 1. Meltt>ranes are adjusted to 1 nq protein/ml. 2. A 0.1 ml aliquot of neri:>ranes is added to solution A (0. 7 ml), the mixture is vortexed an::l incubated at rcx::m terrperature for 15 min. 3. A 0.2 ml aliquot of substrate mix is then added, the solution is vortexed an::l the absomanoe m::>nitored at 340 run. 4. '!he reference cell a::>ntain.s O. 7 ml solution A an::l o. 2 ml substrate mix. Solution A; 37.5 nM Nicotinamide c, 0.5 M Nam, 0.125% Triton X-100, 1. 75 nM Nicotinamide adenine dinucleotide, NAO, (Si gma N-3886), an::l 1.25 nM EDI'A. SUbstrate mix; 65 nM Irglutamate, :pl 7 .4 E(NAD)= 18 X 103 ~l X an-1.

PAGE 255

lactate Dehydrogenase 'Assay: (ref: Hem:y et al. 1960) 243 1. PBS ( 2. 5 ml) is added to a 5 ml plastic test tube. 2. A o. 2 ml aliquot of NAIE am a o .1 ml aliquot of membranes ( 1 ng protein/ml) are added, the solution vortexed am i.nmadiately placed in a 3 ml cuvette. 3. '!he mixture is incubated for 20 min at roan terrperature. 4. '!he reaction is initiated by aaiin:] 0.2 ml of substrate am the absort>ance was then llDTlitored at 340 nm. 5. Controls sarrples contain: 2. 5 ml PBS, o .1 ml membranes, o. 2 ml NAIE, am 0.2 ml water (in place of substrate); 2.5 ml PBS, NAIE, o. 2 ml substrate am o. 3 ml water ( in place of membranes) NAIE (Nicx::,tinamide adenine dinucleotide, reduced fonn); 2.5 ng NAIE/ml in PBS SUbstrate; 1 ng soditnn Irpyruvate/ml in PBS. calculation of Enzyne Specific Activity: units/ng protein= c::haDJe in absort>ance at 340 rntV6.2(min) (ng protein)

PAGE 256

244 Inorganic ~te Detennination 'Assay: (Ref: Ieloir am cantini, 1957) 1. A inorganic ~te starrlard cw:ve is prepared rargin;J fran O to 1500 rnooles of inorganic ~te, the volmne is adjusted to 1 ml with water am then diluted with 1 ml 10% '!CA. 2. 'lhe sanples (0.5 ml) am stamard cw:ve (0.5 ml) are adjusted to 3.5 ml with distilled water. 3. M:>lybdate reagent (1 ml) am reducin;J reagent (0.5 ml) are then added to eadl tube, am vortex. 4. 'lhe tubes are incubated at roan terrperature for 30 min, centrifuged at 10,000 g for 10 min am then the abso:rbance at 660 rnn is measured. Molybdate reagent; 25 g aimonium nolybdate is added to 500 ml distilled water. SUlfuric acid (139 ml) is then added am the solution diluted to a final volmne of 1.0 1. Ihosp1ate reducin;J :reagent; 250 ng 1-amino-2-napithol-4-sulfonic acid, 14. 6 g sodium bisulfite, am 500 ng sodium sulfite is added to 100 ml boilin;J water. 'lhe solution is then filtered (Whatman #1), am stored in a brown bottle. Inorganic ~te stock; 17. 4 ng dipotassium Jilosp1ate in 10 ml distilled water.

PAGE 257

245 M:xlified Il:Mry Protein Assay: (Ref: Ben.sadOllll am Weinstein, 1976) 1. '!he protein unknowns am the BSA stan::Jard an:ve (10 to 60 ug protein) are plaO:d in 15 ml plastic test tubes. '!he final volune is adjusted to 1 ml with distilled water. 2. A 0.01 ml aliquot of 10% soo is added, the tubes vortexed, am incubated at roan tenperature for 15 min. 3. A O. 75 ml aliquot of ice-cold 24% '!CA is added to each tube, the solution is vortexed am then centrifuged at 10,000 g for 20 min. 4. '!he supernatant is decanted am 0.1 ml of 0.2% S00/0.2 N NaOH is added. 5. A 0.6 ml aliquot of the Il:Mry cq:per reagent is added, tubes incubated for 10 min at roan tenperature, am then 0.06 ml of I,ilenol reagent is added. 6. After incubating the sanples for 30 min at roan tercperature, the absort:>ance is measured at 750 nm. Il:Mry cq:per reagent; 0.58 nM EIJI'A (cq:per disodiurn salt), 189 nM sodium cartx:>nate, am 100 nM NaOH in 1% soo. Ihenol reagent; Folin-Cicx::alteu reagent (Sigma F-9252) diluted with equal volune water. BSA stock; 1 nq BSA/ml (Sigma Fraction V, A-9647) in distilled water, store at -1oc.

PAGE 258

APPENDIX IV SOIIJI'IONS FOR K>I..EClJI.AR CI.DNING 40X TAE b.lffer 1.6 M Tris-base 0.8 M Na acetate:3H20 40 nM Na2EIJI'A:2H20 Adjust the pl to 7.2 with acetic acid. Buffer N 40 ml deionized fonnamide 16 ml 20X SSC 1. 6 ml Tris-base, 1 M, :pl 7. 4 0.8 ml lOOX Denhardts solution 0. 8 ml Sal.non spei:m INA ( 2 ng/ml) 20.8 ml distilled water store at -20c TBS b.lffer 50 nM Tris-base 150 nM NaCl Adjust the pl to 7. 4. SM b.lffer 5.8 g NaCl 2.0 g M:JS04:7H20 50.0 ml 1 M Tris-base, :pl 7 .5 5.0 ml 2% gelatin Adjust the voll.Ilne to 1. o 1 am autoclave. YT meditnn 8.0 g Bacto-tryptone 5.0 g yeast extract 5.0 g NaCl Adjust the voll.Ilne to LO 1, pl to 7.4 am autoclave. For agar plates the solution is suwle.mented with 1.5% with Bactoagar. IB meditnn 10.0 g Bacto-tryptone 10.0 g NaCl 5.0 g Ba.etc-yeast extract Adjust the voll.Ilne to 1. o 1, pl to 7. 4, am autoclave. For agar plates the solution is suwle.mented with either 0.4% or 1.5% with Bactoagar. 246

PAGE 259

rNA gel loadi.rg ruffer 30 nM EDI'A 25% fiex>ll 0.4 ml 2% Xylene cyaool 0.4 ml 2% Bratq;ilenol blue 1.1 ml distilled water 500X Anpicillin stock 247 25. o Irg anpicillin/ml distilled water, store wrawed in foil at -2oc RNase A stock 10.0 Irg RNase A in 10 ml of a ruffer CX)lltaining 10 nM Tris-base, pi 7. 5, arrl 15 nM NaCl. Heat this solution to 100 C for 15 min to inactivate any contaminatin:J rnase activity: store at -2oc. Ethidium branide solution (10,000 X) 10.0 Irg ethidium branide in 1 ml distilled water, store in dark at 4.oc. lOOX Clenhardts solution 10.0 g polyvinylpyrrolidone 10.0 g BSA 10.0 g fiex>ll Adjust the volume to 500 ml with distilled water, filter arrl store at 4.oc. 100 nM B-D-thiogalactq,yranoside (!PIG) 23.8 Irg !PIG in 1 ml water, store at -2oc. 'Ihe workin:J concentration is 3 1/ml. 10% 5-dtloro-3-brato-2-irrlolyl-B-galactoside (X-gal) 100 Irg X-gal in 1 ml dimethylfonnamide, store at -2oc. '!he workin:J concentration is 3 1/ml. TE ruffer 10 nM Tris-base 0.1 nM EDI'A Adjust the pi to 8.0 arrl autoclave. Saturated J;ilerK)l Fhenol is distilled at 160"C arrl stored at -2oc. As needed, the Jnenol is melted at 68"C arrl made 0.1% with hydroxyquinoline. 'Ihe melted J;ilerK)l is then extracted several times with an equal volume of 1. O M Tris-base, pi 8, followed by 0.1 M Tris-base, pi 8 until the pi of the aqueous Jnase is >7.6.

PAGE 260

248 Fonnami.de Adi 5.0 g of Bio-Rad AG 501-XS ion exdlan:Je resin to 50 ml fonnami.de, stir at 4.0C for 30 min am. then filter. store at -20c.

PAGE 261

APPENDIX V GENERAL MEIHOC6 FOR M::>I.EaJIAR CTDNING Small Scale Bacterial Plasmid Preparation: (Ref: Ish-Horowicz arrl :&irke, 1981) 1. 'lhe evenirg before use the white colonies obtained fran the tran.sfonnations to inoculate a 5 ml YT natium culture containing 0.05 ng/ml anpicillin. Grow this culture overnight at 370c. '!he foll0v1ing IIDming streak a portion of this culture onto a new YT natium plate containing 1.5% agar/anp/X-gal/IPIG. 2. Centrifuge this solution at 2,000 g for 5 min arrl then resusperrl the cells in o .1 ml Gl'E buffer. 3. Transfer the cells to an ~rf test tube arrl incubate for 5 min at roan t.errperature. 4. Add 0.2 ml of the alkali-SOO solution, tap gently, arxl incubate on ice for 10 min. Add 0.15 ml of the acetate solution, vortex arrl incubate on ice for an additional 5 min. '!here should be large white precipitates visible at this point. 5. Centrifuge the tubes in a microfuJe for 10 min (4.00c). :Rem:Jve the supernatant, which should be totally devoid of any insoluble material, arxl add 0.9 ml 100% ethanol. Incubate the test tubes at -700c for 15 min. 6. 'lhe mA is then isolated by centrifugation in a microfuge ( 4. oOc) for 10 min. 'lhe supernatant is discarded, the pellet containing plasmid mA is dried (speed-vac), arxl then resusperrled in 0.02 ml TE buffer. 7. .AWroxiinately 1-2 1 of the plasmid mA arrl the original ?JC19 (2 g) is then electrcploretically separated on a 1.5% agarose:TAE ''minigel" (40 volts for 2 h) to determine if the plasmid CNA contains insert cmA. 8. Before a large scale plasmid preparation is perfo:rmed, 5 1 of the plasmid mA should be rut with F.coRl. to confinn that the ?)'C19 contains the cCNA insert. sterile Solutions: Gl'E buffer; 50 nM Glucose, 25 nM Tris-base, 10 nM EIJI'A, pi 8.0 249

PAGE 262

250 Alkali-SOO; 0.2 N NaOH in 1% SOO (make fresh) Acetate Solution; 60 ml of 5 M ROAc, 11.5 ml HOAc, 28.5 ml H 2o large Scale Preparation of Plasmid mA: (Maniatis et al. 1982) 1. ~coli strain '1G 1 containin;J ?}C19 with the cmA insert is used to inoculate a 5 ml culture of YT meditnn suwlemented with 0.05 ng/ml arrpicillin. '!his culture is irK::ubated overnight at 37C with constant aeration. 2. A 1.0 liter flask containin;J YT meditnn suwlemented with arrpicillin (0.05 ng/ml) at 37C is then inoculated with the 5 ml culture am allor.,,ied. to grow several hours l.ll1til the strain is in late log Ji)ase growth (usually 6 h at an O.D. of 1.5 to 3.0, 550 rnn). 3. When the~coli readies late log piase growth, 0.17 ng of dll.oranpienicol is added am the cells are allor.,,ied. to shake overnight at 37CX:.. 4. '!he followirg 11Drnirg, the cells are transferred to 1. O 1 bottles am centrifuged at 5,ooo g for 15 min. 5. '!he cell pellet is then resusperxled in 14 ml GrE buffer, the solution is decanted into two So:rvall SS-34 centrifugation tubes am 5 ng/ml lysozyme is added. 6. After 5 min, 14 ml of the alkali-soo solution is added to each tube, the tubes are mixed, am placed on ice for 10 min. 7. '!he tubes are then centrifuged at 20,000 rpn for 20 min (4.0C) to renove protein am dlraoosanal mA. 8. '!he supeniatant is then divided into foor So:rvall SS-34 centrifugation tubes am 0.6 voltmeS of isq:>rq,anol (9 ml) is added, mix am irK::ubate at roan tenperature for 15 min. '!he RNA am plasmid mA will fonn a precipitate. 9. '!he RNA am plasmid mA is isolated by centrifugation at 10, ooo g for 10 min. '!he supeniatants are decanted am the pellets resuspen:ied with 100% ethanol, pooled, am dried (speed-vac). 'Ihe final pellet is resuspen:ied in 6.5 ml TE buffer. 10. AR;)roxiinately 6.5 g of CsCl (IBI, ultra p.ire 11Dlecular biology grade) is added to the 6.5 ml TE:mA solution (i.e., 1 g CsCl/ml TE:mA solution). Add 0.8 ml of ethiditnn b:ranide per 10 ml of the CsCl solution. '!he ethiditnn branide is made fresh at a concentration of 10 ng/ml in water.

PAGE 263

251 11. If there is any precipitate, centrifuge the solution at 10,000 g for 10 min. (R. T.) '!he :refractive i.n:lex of this solution is checked arrl shool.d be 1. 386. '!he J:NA solutions are then transferred to Beckman Type 65 centrifuge tubes, balaoced (USl.DJ 1 g <;sel/ml TE ruffer) arrl centrifuged at 40,000 :rpn for 20-30 h at 20 c, brake off. 12. RNA shool.d pellet, while drraoosanal arrl nicked circular J:NA are present in the uwer barrl. '!he plasmid J:NA is located in the lower barrl. 13. '!he plasmid I:NA is then :rerocr.red with the aid of a 18 gauge needle arrl transferred to a 15 ml Comirg tube. Five voltnneS of N-butanol saturated with TE ruffer is added arrl the suspension rocked for 15 min to extract the ethidil.nn branide. '!his extraction is repeated l.llltil all of the ethiditnn branide is :rerocr.red. 14. '!he J:NA is then dialyzed against 4.0 1 TE rutfer at 4.oc (16 h). '!he TE ruffer is replaced with fresh ruffer (4.0 1) arrl the dialysis continued overnight. 15. '!he J:NA is then concentrated by reducl.DJ the aqueous voltnne with a series of sec-rutanol extractions. '!his is perfo:nood by addi.rg an equal voltnne sec-rutanol, mi.xin;J, arrl then centrifugation. '!he uwer sec-rutanol p,ase is discarded, whereas the lower aqueous p,ase is extracted lllltil the aw:r.-q>riate voltnne is readied. A few microliters of the J:NA is analyzed on a minigel for the presence of plasmid J:NA arrl RNA contamination. If the plasmid J:NA is oontaminated with RNA, the solution shool.d be treated first with 100 ng/ml of boiled RNase A (30 min, R. T.) follO'Ned by extraction with plen()l:chlorofonn:isoamyl alcdlol extractions. 16. '!he J:NA is then ethanol precipitated arrl spooled ait of solution with the aid of a glass rod. '!he plasmid J:NA is stored at 4.oc in TE ruffer (1 ml) sterile solutions: Gl'E ruf fer; 50 Il'M Glucose, 25 Il'M Tris-base, 10 Il'M EIJI'A, :pl 8. o Alkali-SOO; 0.2 N NaOH in 1% SOO (make fresh) Acetate Solution; 60 ml of 5 M KOAc, 11.5 ml HOAc, 28.5 ml H 2o

PAGE 264

2sa Radiolabelin':J of mA by the Priloor Extension Method: (Ref: Feinberg am Vogelstein, 1983) 1. In a sterile ei;:perrlorf test tube, mix 100 to 200 n;J mA am add water to 9 1. 2. Boil the mA sarrple for 5 min am then exx>l the solution on ice for 5 min. 3. Add 10 1 of the 2. 5X reaction ruff er, mix am then add 5 1 [ 32p]dATP (10 mCijml, 800 Ci/imole). 4. Add 5 mtlts of Klenow fragmant of~coli mA polyioorase I, mix am then centrifuge briefly. 5. Incubate the mixture at roan tenperature for 16 h. 6. '!he prooe is then separated fran 'lllUI1COrporated nucleotides by an etharx>l precipitation. 2.5X Reaction ruffer: 500 ITM Hepes, pl 6. 6 12 5 ITM M:JCl 2 25 ITM 2-nercaptoetanol 125 ITM Tris-HCl, pl 8.0 o. 05 ITM eadl of dCI'P, dITP, am dGTP (Ihannacia) 1.0 ng/ml BSA (Sigma# A-7511, essentially fatty acid-free) 7.5 ng/ml oligodeoxyhexamer (Rlannacia 27-2166-01) '!he ruffer is prepared withcut the oligodeoxyhexamer in a final volume of o. 3 ml. '!he ruff er is then added to the bottle containin:J the oligodeoxyhexamars, mixed, aliquoted into 10 1 portions, am stored at -2oc.

PAGE 265

APPENDIX VI RF.AGENI'S FOR 'IHE OIEMICAL K>DIFICATION OF PRJl'EINS (Ref: Means am Feeney, 1971) Protein M:xtifyinJ Reagent: Acetic anhydride SUccinic anhydride Chloroacetate Iodoacetate Iodoacetamide N-ethyl.maleimide P-chloranercuribenzene sulfonate P-chloranercuribenzoate Fluorescein isothiocyanate Fhenyl isothiocyanate N-acetylimidazole N-braoosuccinimide Trinitrobenzene sulfonate Fhenylglyoxal Ethoxyfonnic anhydride HgCl2 Amirx:> Acid Reactivity: NH2 = = CysfJ = Hish NH2 = CysfJ > Hish = Tyi:b Me~-8.5, HiSp1>5.5, ~7, NH2?>8.5 Me~-8. 5, HiSp1>5. 5, ~7, NH2?>8. 5 Me~-8.51 ~>5.5, ~7, NH2?>8.5 ~5, NH2?>7 cys cys cys = 'fip > Tyr > His NH2 > CysfJ aF.asily reversible, regeneratinJ original group. ~ly reversible urrler the reaction con::litions or upon dilution, regeneratinJ the original group. 253

PAGE 266

BIBLicx;RAFHY Aden, D.P., Fogel, A., Plotkin, S., O:unjanov, I. an:l Knowles, B.B. ( 1979) Controlled synthesis of HBsAg in a differentiated hl..Illlan liver carcinana-derived cell line. Nature 282, 615-616. Alcarz, G., Ki.net, J-P., Wank, S. an:l Metzger, H. (1984) Rlase separation of the receptor for .i.nmmoglcbllin E an:l its subunits in Triton X-114. J. Biol. Chem. 259, 14922-14927. Aledort, L.M., Troup, S.B. an:l Weed, R.I. (1968) Inhibition of sulfhydryl-deperrlent platelet functions by penetrating an:l nonpenetrating analogues of parachlorarercuribezene. Blood 31, 471-479. Ames, B.N. an:l Gary, B (1959) Coordinate repression of the synthesis of four histidine biosynthetic enzyires by histidine. Proc. Natl. Acad. Sci. 45, 1453-1461. Al::n:lt, K.T., Styles, C. an:l Fink, G.R. (1987) Multiple global regulators control HIS4 transcription in yeast. Science 237, 874-880. Ballatori, N. l>k>seley, R.H. an:l Boyer, J. L. ( 1986) Sodium gradient-deperrlent I.rglutamate transport is localized to the canalicular darrain of liver plasm membranes. J. Biol. Cllem. 261, 6216-6221. Barber, E.F., Han:llogten, M.E. an:l Kilberg, M.S. (1983) Irrluction of amino acid transport System A is blocked by tunicamycin. J. Biol. Cllem. 258, 11851-11855. Batt, E.R., Abbott, R.E. an:l Schachter, D. (1976) Transport of m::>nOSaocharides. J. Biol. Cllem. 251, 7184-7190. Beaufay, H., Berxlal.l, D.S., Baudhuin, P. an:l deDeve, C. (1959) Intracellular distribution of sate dehydrogenases, alkaline deoxyribonuclease an:l iron in rat liver tissue. Biochem. J. 73, 623-628. Beckwith, J Davies, J. an:l Gallant, J. (1983) Gene function in procaryotes. Cold Sprin:Js Hart>or PUblishin;J, New York, N.Y. Bensada.m, A. an:l Weinstein, D. (1976) Assay of proteins in the presence of interfering materials. Anal. Biochem. 70, 241-250. Beyreuther, K. Bieseleler, B. Ehring, R. an:l Muller-Hill, B. (1981) In Methods in Protein Sequence Analysis (Elzina, M., ed.) :pp. 139, Ht.nnana Press, Clifton, N.J. 254

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PAGE 268

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271 BICGRAm!CAL SKEI'CH 'Ihanas Crane Ori.les was born on May 11, 1960, in Jacksonville, Florida. He received his Badlelor of Scierx:ie degree in 1983 fran the University of Florida in Gainesville. In Septent,er, 1983 he began his graduate education in the Department of Biochemistcy am ltk>lecular Biology at the University of Florida, vJOrkirg unier the direction of Dr. Midlael S. Kilberg. After recei vi.DJ his doctoral degree in June, 1988, he plans to ootain a postdoctoral position in the field of marine biology.

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I certify that I have read this study arrl that in my opinion it conforms to acceptable stamards of scholarly presentation arrl is fully adequate, in scope arrl quality, as a dissertation for the degree of IX>ctor of Rrllosophy. ID~~ Associate Professor of Biochemistry arrl Molecular Biology I certify that I have read this study arrl that in my opinion it conforms to acceptable starrlards of scholarly presentation arrl is fully adequate, in scope arrl quality, as a dissertation for the degree of IX>ctor of Rrllosophy. 'Susan C. Frost Assistant Professor of Biochemistry arrl Molecular Biology I certify that I have read this study arrl that in my opinion it conforms to acceptable stamards of scholarly presentation arrl is fully adequate, in scope arrl quality, as a dissertation for the degree of =tor of Rlila,qny. ~~rLL Ri P. Boyce Professor of Biochemistry arrl Molecular Biology I certify that I have read this study arrl that in my opinion it conforms to acceptable starrlards of scholarly presentation arrl is fully adequate, in scope arrl quality, as a dissertation for the degree of IX>ctor of Rrllosophy. 'lhctnaS W. O'Brien Professor of Biochemistry arrl Molecular Biology

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I certify that I have read this study am that in my opinion it confonns to acx::eptable starrlards of scholarly presentation am is fully adequate, in scope am quality, as a dissertation for the degree of Doctor of Rlilosophy. Wo.K~-Paul A. Klein Associate Professor of Pathology '!his dissertation was sul:tnitted to the Graduate Faculty of the College of Medicine am to the Graduate School am was accepted as partial fulfillment of the requinnents for the degree of Doctor of Rlilosophy. ~. Dean, College of Medicl.Ile Dean, Graduate Sdlool April, 1988