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Long-term alcohol intake enhances ADP-ribosylation of the multifunctional enzyme, phosphoglucomutase, in rat liver
Long-Term Alcohol Intake Enhances ADP-Ribosylation of the Multifunctional Enzyme, Phosphoglucomutase, in Rat Liver FUMIO NOMURA,1 MASATOSHI NODA,2 MASAMI MIYAKE,2
Adenosine diphosphate (ADP)-ribosylation is a posttranslational protein modification that, in turn, alters several regulatory proteins in mammalian cells. We demonstrated that long-term alcohol intake enhanced the ADP-ribosylation of a 58-kd protein in rat liver plasma membranes. To assess the biological significance of this phenomenon, we partially purified the 58-kd acceptor protein from solubilized rat liver homogenates by two sequential preparative high-pressure liquid chromatographies. Microsequencing revealed that it was phosphoglucomutase (PGM) (EC 5,4,2,2). This enzyme underwent negligible auto ADP-ribosylation, but the ADP-ribosylation was remarkably increased by adding rat liver plasma membranes. The extent of the increase was greater in alcohol-fed rats than in pair-fed controls, suggesting enhanced enzyme activities toward ADP-ribosylation of PGM after chronic alcohol consumption. Several important enzymes are ADP-ribosylated, after which their activities are modified. The results of this study showed that PGM is a novel substrate for ADPribosylation in the liver and that the ADP-ribosylation is increased after chronic alcohol consumption. In view of the variety of roles of PGM in the liver (carbohydrate metabolism and Ca2/ homeostasis), specific roles of this modification in terms of the effects of alcohol on hepatocytes may deserve further investigation. (HEPATOLOGY 1996;24:1246-1249.) Enzyme activities are often regulated by posttranslational covalent modifications at critical points of metabolic pathways. One example is adenosine diphosphate (ADP)-ribosylation, in which the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) is transferred to an acceptor protein.1,2 Modification of protein functions by mono-ADP-ribosylation is best known as the mechanism of action of bacterial toxins such as cholera toxin3 and pertussis toxin.4 They exert biological effects on vertebrate cells by catalyzing the mono-ADPribosylation of the stimulatory and inhibitory guanosine triphosphate binding proteins of the adenylate cyclase system. These toxins have been widely used as probes to investigate the role of G-proteins in the transduction of hormonal and sensory signals and their intracellular processes.5,6 Endogenous enzymes that mimic these bacterial toxins have been
Abbreviations: ADP, adenosine diphosphate; NAD, nicotinamide adenine dinucleotide; PGM, phosphoglucomutase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CM, carboxymethyl. From the 1Department of Clinical Pathology, Institute of Clinical Medicine, Tsukuba University, Ibaraki, and 2Second Department of Microbiology, Chiba University School of Medicine, Chiba, Japan. Received January 3, 1996; accepted July 2, 1996. Supported by Japan Educational Ministry Grant and by Kurozumi Medical Foundation. Address reprint requests to: Fumio Nomura, M.D., Department of Clinical Pathology, Institute of Clinical Medicine, Tsukuba University, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2405-0060$3.00/0
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identified in various eukaryotic cells, and mounting evidence suggests that endogenous ADP-ribosylation plays significant biological roles in mammalian cells.7-11 These enzymes may be involved in the regulation of adenylate cyclase,12,13 Ca2/ release from mitochondria,14 and the phosphorylation of nuclear proteins.15 In view of the diverse roles of ADP-ribosylation in cells, the effects of alcohol on this reaction are of interest. We demonstrated that long-term alcohol intake remarkably enhanced the endogenous ADP-ribosylation of a 58-kd hepatic protein in the rat.16 To further clarify the biological roles of this phenomenon, the 58-kd protein needed to be identified. We describe here that the 58-kd protein is the multifunctional enzyme, phosphoglucomutase (PGM) (EC 5,4,2,2). The modification of this enzyme by ADP-ribose was increased by chronic alcohol consumption. MATERIALS AND METHODS Animal Procedures. A total of 18 male Sprague-Dawley rats (Charles River Japan, Kanagawa, Japan) weighing 140 to 150 g were used. Ten of the rats were pair-fed with a nutritionally adequate liquid diet containing ethanol as 36% of the total energy, as well as an isocaloric control diet for 4 weeks as described.17 Animal care was in accordance with our institutional guidelines. To equalize the rate of consumption before plasma membrane isolation, one third of the daily ration was given at 9 AM, and two thirds was given at 5 PM on the day before killing. In addition, one third of the daily intake was administered 2 hours before the procedures. The animals were decapitated and their livers were perfused through the portal vein with ice-cold buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L NaN3 , and 250 mmol/L sucrose (TENS buffer). The livers were minced and homogenized by 10 passes with a loose-fitting Dounce homogenizer (Wheaton, Millville, NJ) to prepare 20% homogenates in the same buffer. Liver plasma membrane fractions were prepared as described by Prpic et al.18 ADP-Ribosylation Assay. Mono-ADP-ribosylation was performed as previously described.16,19 Briefly, the reaction mixture (final volume, 200 mL) contained 100 mmol/L Tris-HCl (pH 7.5 at 377C), 10 mmol/L thymidine, 0.5 mmol/L guanosine triphosphate, 1 mmol/L ethylenediaminetetraacetic acid, 5 mmol/L MgCl2 , 1 mmol/L [32P] NAD (10 mCi), and 100 mg protein of samples (homogenates, membranes, column eluates) containing both ADP-ribosyltransferases and their acceptor proteins. After incubation at 377C for 30 minutes, 2 mL ice-cold 7.5% trichloroacetic acid was added, and the samples were kept at 47C overnight. Precipitated proteins were pelleted and dissolved in 1% sodium dodecyl sulfate (SDS)/5% mercaptoethanol (607C; 10 minutes), then resolved by electrophoresis in 10% polyacrylamide gels according to Laemmli.20 The gels were autoradiographed on Kodak X-Omat film (Eastman Kodak Co., Rochester NY) or were evaluated using a Bio-image analyzer (Fujix BAS 2000, Fujicolor, Tokyo, Japan). Identification of a 58-kd Protein. One rat liver (10 g) was homogenized in 40 mL of the TENS buffer, and the homogenates were solubilized in the TENS buffer containing Triton X-100 (final concentration, 0.1%) for 1 hour. The solubilized samples were concentrated using Centricon-100 (molecular-weight cutoff, 100,000) (Grace Japan, Amicon, Tokyo, Japan). The filtrate was then subjected to Centricon 30 (molecular-weight cutoff, 30,000). The residue was fractionated by two rounds of preparative high-pressure liquid chromatographies using a Waters ALC/GPC208 system with a type 440 absorbance detector (Millipore Waters Chromatograph, Tokyo, Japan).
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The first high-pressure liquid chromatography separation was accomplished on a Protein-pak DEAE (10 1 100 mm) (Millipore Waters Chromatograph). The residue from the Centricon 30 centrifugal concentrator was placed on this column, which was equilibrated with buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L NaN3 , and 0.1% (vol/vol) Triton X-100 (Triton-Tris-EDTA-NaN3 buffer), and eluted with a stepwise gradient of 0.2 to 1.0 mol/L NaCl. The flow rate was 1.0 mL/min, and fractions collected every minute were monitored at 280 nm. Fractions containing high levels of endogenous ADP-ribosylation of a 58-kd protein, which appeared in the flow-through, were pooled and injected onto a protein-Pak carboxymethyl (CM) (10 1 100 mm) (Millipore Waters Chromatograph) equilibrated with the Triton-TrisEDTA-NaN3 buffer, pH 6.0, and then eluted with a stepwise 0.2 to 1.0 mmol/L NaCl gradient. Column fractions collected every 0.5 minute were monitored at 280 nm with a flow rate of 1 mL/min, and those containing high levels of endogenous ADP-ribosylation of a 58kd protein eluted at 0.5 mol/L NaCl were pooled, concentrated, and resolved by SDS–polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and stained with Coomassie brilliant blue R250. A band equivalent to the 32P-incorporated 58-kd protein was excised, and then the amino-terminal of the 58-kd protein was determined using a protein sequencer (model PPSQ-10, Shimadzu Inc., Kyoto, Japan). Sequence homology was determined by searching PIR-international protein sequence database. The final preparation (CM eluate at 0.5 mmol/L NaCl) was subjected to ADP-ribosylation as described above, and a portion of the reaction mixture (30 mg protein) was analyzed by two-dimensional electrophoresis according to O’Farrell.21 The first-dimension isoelectric focusing was performed in the presence of 2% Nonidet P-40 in capillary glass tubing (1 1 70 mm). The second dimension was a 12% SDS-PAGE according to Laemmli.20 Following electrophoresis, the gel was fixed, silver-stained, and subjected to autoradiography as described above. Other Methods. Protein concentrations were determined by means of the Bio-Rad protein assay dye reagent kit (Bio-Rad, Richmond, CA) using bovine serum albumin as the standard. [Adenylate-32P]NAD di(triethylammonium) salt was obtained from Du Pont NEN Research Products (Boston, MA). PGM from rabbit skeletal muscle was obtained from Boehringer Mannheim (Mannheim, Germany). Other reagents were obtained from standard chemical sources. RESULTS
Incubation of rat liver homogenates in the reaction mixture as described above resulted in endogenous 32P incorporation in five protein bands (Fig. 1). It was notable that ADP-ribosylation of the 58-kd form was greater in alcohol-fed rats than in pair-fed controls. Thus, our finding in the plasma membrane fractions were reproducible in homogenates. Therefore, to isolate the 58-kd protein, solubilized rat liver homogenates were partially purified by two sequential rounds of preparative high-pressure liquid chromatographies. Figure 2 shows the protein profiles (Fig. 2A) and 32P-autoradiographs (Fig. 2B) at each step of the purification procedure. The final CM eluate was transferred to a polyvinylidene difluoride membrane following SDS-PAGE. The N-terminal amino acids of the 58-kd protein were sequenced as described in Materials and Methods. The analysis yielded the sequence of up to 15 amino acids (VKIVTVKTQAYPDQK), which was identical to that of residues in rat liver PGM.22 Two-dimensional electrophoresis of the final preparation (CM eluate at 0.5 mol/L NaCl) resulted in one major spot accompanied by two minor ones with pIs close to that of the major spot at 58 kd (Fig. 3A), and selective 32P-labeling was seen in the major spot following ADP-ribosylation (Fig. 3B). Incubating PGM obtained from rabbit skeletal muscle (95% homologous to the rat liver form in terms of amino acid sequences) in the reaction mixture described in Experimental Procedures led to little 32P incorporation of the 58-kd band (Fig. 4), indicating that PGM undergoes negligible auto-ADPribosylation. By contrast, the addition of rat liver plasma membranes obtained from naive rats to the ADP-ribosylation medium together with exogenous PGM strikingly enhanced
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FIG. 1. Endogenous ADP-ribosylation of liver homogenates from control rats and those given alcohol chronically. Rat liver homogenates (120 mg of protein) were ADP-ribosylated with 1 mmol/L [32P]NAD (10m Ci) in the reaction mixture detailed in the text. After an incubation for 30 minutes at 377C, trichloroacetic acid–precipitated proteins were separated on 10% SDS-PAGE and were visualized by autoradiography. Similar results were obtained in three other experiments. C, control; A, alcohol-fed.
the radiolabeling of the 58-kd protein (Fig. 4), suggesting the presence of substantial ADP-ribosyltransferase activities in the membrane fractions. The addition of the cytosol fraction failed to stimulate ADP-ribosylation of the protein (Fig. 5). Furthermore, the enhanced radiolabeling by membrane fractions obtained from alcohol-fed rats was remarkably greater than that by membranes obtained from pair-fed controls (Fig. 5). Incubation of the plasma membranes alone without exogenously added PGM resulted in minimal ADP-ribosylation of the 58-kd protein (Fig. 5). These findings suggested an increase of enzyme activities toward ADP-ribosylation of the 58-kd protein after long-term alcohol intake. DISCUSSION
ADP-ribosylation is a posttranslational protein modification by which living organisms modify the functions of many important regulatory proteins, including guanosine triphosphate-binding protein.2-6 In view of the range of effects of ethanol on cellular metabolism, those on this posttranslational modification deserve attention. We demonstrated that long-term alcohol intake enhanced the endogenous ADP-ribosylation of a 58-kd protein in rat liver plasma membranes.16 To assess the biological significance of this phenomenon, it appeared essential to identify the acceptor protein. In this study, a 58-kd protein was purified by successive column chromatography and microsequenced. N-terminal amino
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FIG. 2. Protein profiles and autoradiographs of a 58-kd protein in liver homogenates and during purification. (A) SDS-PAGE gels stained with Coomassie brilliant blue. Lane 1, liver homogenates (80 mg); lane 2, solubilized liver homogenates (80 mg); lane 3, DEAE flow-through (40 mg); lane 4, CM eluate at 0.5 mol/L NaCl (40 mg). (B) Autoradiographs of preparations during purification, which were ADP-ribosylated as detailed in the text. Lane 1, liver homogenates (80 mg); lane 2, solubilized liver homogenates (80 mg); lane 3, DEAE flow-through (40 mg); lane 4, CM eluate at 0.5 mol/L NaCl (40 mg).
acids sequence analysis revealed that it was the multifunctional enzyme, PGM (EC 5.4.2.2). It was confirmed by twodimensional electrophoresis that a major protein at 58-kd was selectively 32P-labeled following ADP-ribosylation. Furthermore, ADP-ribosylation of a 58-kd protein was strikingly enhanced when PGM was added as an exogenous acceptor to the incubation mixture. Taken together, these observations indicate that the ADP-ribosylated 58-kd protein is PGM. The results of experiments using PGM obtained from rabbit skeletal muscle (95% identical to the rat liver form in terms of amino acid sequences) showed that this enzyme undergoes negligible auto-ADP-ribosylation, but that the protein labeling was remarkably increased by the addition of rat liver plasma membranes. Furthermore, the extent of the increase caused by membrane fractions obtained from alcohol-fed rats was greater than that by those obtained from pair-fed controls, suggesting enhanced ADP-ribosyltransferase activity toward PGM after long-term alcohol intake. Alternatively, it is also possible that PGM was modified nonenzymatically by free ADP-ribose that was generated by NAD glycohydrolase. This is unlikely, because 32P–ADP-ribose failed to bind to the 58-kd protein (data not shown). Also, the ADP-ribosylation of the 58-kd protein was stimulated by GTP,16 which inhibits NAD glycohydrolase.23 It is still possible, however, that a novel linkage of NAD to a protein, as recently clarified,24 can partially account for the protein modification. Several key enzymes are ADP-ribosylated, which, in turn,
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FIG. 3. Two-dimensional electrophoresis of a 58-kd protein in the partially purified preparation. The final preparation (CM eluate at 0.5 mol/L NaCl) was subjected to ADP-ribosylation, and a portion of the reaction mixture (30 mg protein) was analyzed by two-dimensional electrophoresis as described in Materials and Methods. (A) Silver-stained gel. (B) An autoradiogram of the gel shown in (A). A major 58-kd spot in (A) and selective radiolabeling of the 58kd spot in (B) are indicated by arrows. The positions of the molecular mass standards in kilodaltons are shown.
modifies their activities, as reviewed recently.25 Substrates modified by arginine-specific ADP-ribosyltransferase include Ha-ras p21,26 tubulin,27 phosphorylase kinase,28 and Ca2/dependent adenosine triphosphatase.29 Enzymatic and nonenzymatic ADP-ribosylation of cysteine residue of proteins is also known.30 The results of this study showed that PGM is a novel substrate for ADP-ribosylation in the liver and that the enzyme activity of ADP-ribosyltransferase toward PGM is increased after chronic alcohol consumption. Among hepatic membrane-bound enzymes, Na-K adenosine triphosphatase and g-glutamyl transferase were tested for their capacity to be exogenous acceptor proteins. They were not found
FIG. 4. Auto-ADP-ribosylation and enhanced ADP-ribosylation of the 58kd protein by rat liver plasma membranes. PGM obtained from rabbit skeletal muscle (95% homologous to rat liver PGM in terms of amino acid sequences) was ADP-ribosylated as detailed in the text at 377C for 30 minutes in the absence (lanes 1-3) or presence (lanes 4-6) of rat liver plasma membranes (40 mg of protein) obtained from control rats. Lanes 1 and 4, 20 mg PGM; lanes 2 and 5, 40 mg PGM; lanes 3 and 6, 80 mg PGM.
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FIG. 5. Enhanced ADP-ribosylation of PGM by liver plasma membranes obtained from alcohol-fed rats and from pair-fed controls. PGM (40 mg) obtained from rabbit skeletal muscle (95% homologous to rat liver PGM in terms of amino acids sequences) was ADP-ribosylated as detailed in the text. Lane 1, PGM alone; lane 2, PGM / cytosol fraction from control rats (40 mg); lane 3, PGM / membrane fraction from control rats (40 mg); lane 4, PGM / membrane fraction from alcohol-fed rats (40 mg); lane 5, plasma membranes alone from control rats; lane 6, plasma membranes alone from alcohol-fed rats. Similar results were obtained in three other experiments.
to be substrates for ADP-ribosylation (data not shown). The reasons for this apparent specificity for PGM are not currently clear. Identification of the binding site for ADP-ribose, which is currently underway, will provide some explanations for the specificity. PGM is a widely distributed multifunctional enzyme. The classical function of PGM is to link glycolysis and glycogen metabolism by interconverting glucose-1phosphate and glucose-6-phosphate. The activity of PGM is under hormonal control and may play a physiological role in glucose metabolism.31 Alterations of this enzyme may have to be considered in alcohol effects on carbohydrate metabolism. In this context, it is notable that PGM activity was reportedly decreased after an acute ethanol load in the rat.32 Long-term alcohol intake, however, resulted in an increase of PGM activities (Nomura et al., Unpublished observation, June 1996). Mechanisms for the regulation of PGM activities appear to be complex, because this protein is also a substrate for Ca2//calmodulin–dependent protein kinase. The contribution of ADP-ribosylation to the regulation of PGM activity remains to be detailed. In addition to its classical role in glucose metabolism, PGM has another important role. Lee et al.33 have indicated that PGM plays a crucial role in Ca2/ regulation. It is tempting to speculate that hepatic Ca2/ homeostasis can be altered by the modification of PGM by ADP-ribosylation. Thus, our finding that long-term alcohol intake enhanced endogenous ADP-ribosylation of a 58-kd protein in the rat liver16 has been substantiated. We are further clarifying the pathophysiological roles of the posttranslational modification of this multifunctional enzyme in terms of the effects of alcohol on hepatocytes. Acknowledgment: We wish to thank Drs. Motoo Yamasaki and Keiichi Yano, Tokyo Research Laboratory of Kyowa Hakko Kogyo Co., Ltd., for their amino acids sequence analysis. We also thank Yukiko Miyokawa and Sachiko Takegami for their excellent technical assistance. REFERENCES 1. Honjo T, Nishizuka Y, Hayaishi O, Kato I. Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem 1968;243:3553-3555. 2. Ueda K, Hayaishi O. ADP-ribosylation. Annu Rev Biochem 1985;54:73100. 3. Moss J, Vaughan M. Mechanism of choleragen: evidence for ADP-ribosyltransferase activity with arginine as an acceptor. J Biol Chem 1977;252: 2455-2457. 4. Ui M. Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol Sci 1984;5:277-279.
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5. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 1987;56:615-649. 6. Moss J, Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol 1988;61:303-379. 7. Moss J, Stanley SJ, Watkins PA. Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes. J Biol Chem 1980;255:5838-5840. 8. Tanuma S, Kawashima K, Endo H. Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein. J Biol Chem 1988;263:5485-5489. 9. Maehama T, Takahashi K, Ohoka Y, Ohtsuka T, Ui M, Katada T. Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADPribosylation of GTP-binding proteins. J Biol Chem 1991;266:10062-10065. 10. Seki K, Hirai A, Noda M, Tamura Y, Kato I, Yoshida S. Epoxyeicosatrienoic acid stimulates ADP-ribosylation of a 52kDa protein in rat liver cytosol. Biochem J 1992;281:185-190. 11. McDonald LJ, Moss J. Nitric oxide-independent, thiol-associated ADPribosylation inactivates aldehyde dehydrogenase. J Biol Chem 1993;268: 17878-17882. 12. Molina y Vedia L, Nolan RD, Lapetina EG. The effect of iloprost on the ADP-ribosylation of Gsa (the a-subunit of Gs). Biochem J 1989;261:841845. 13. Tanuma S, Endo H. Mono(ADP-ribosyl) ation of Gi by eukaryotic cysteinespecific mono(ADP-ribosyl)transferase attenuates inhibition of adenylate cyclase by epinephrine. Biochim Biophys Acta 1989;1010:246-249. 14. Richter C, Winterhalter KH, Baumhuter S, Lotscher HR, Moser B. ADPribosylation in inner membrane of rat liver mitochondria. Proc Natl Acad Sci U S A 1983;80:3188-3192. 15. Tanigawa Y, Tsuchiya M, Imai Y, Shimoyama M. ADP-ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP-dependent protein kinase. FEBS Lett 1983;160:217-220. 16. Nomura F, Noda M. Stimulation of mono-ADP-ribosylation in rat liver plasma membranes after long-term alcohol intake. HEPATOLOGY 1993;18: 870-873. 17. Lieber CS, DeCarli LM. The feeding of ethanol in liquid diets: 1986 update. Alcohol Clin Exp Res 1986;10:550-553. 18. Prpic V, Green KC, Blakemore PF, Exton JH. Vasopressin-, angiotensin II- and a1-adrenergic-induced inhibition of Ca2/ transport by rat liver plasma membrane vesicles. J Biol Chem 1984;259:1382-1385. 19. Kato I, Noda M. ADP-ribosylation of cell membrane proteins by staphylococcal a-toxin and leukocidin in rabbit erythrocytes and polymorphonuclear leukocytes. FEBS Lett 1989;281:185-190. 20. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4 . Nature 1970;227:680-685. 21. O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007-4021. 22. Revera AA, Elton TS, Dey NB, Bounelis P, Marchase RB. Isolation and expression of a rat liver cDNA encoding phosphoglucomutase. Gene 1993; 133:261-266. 23. Watkins P, Moss J. Effects of nucleotides on activity of a purified ADPribosyltransferase from turkey erythrocytes. Arch Biochem Biophys 1982; 216:74-80. 24. McDonald LJ, Moss J. Stimulation by nitric oxide of a novel linkage of NAD to glyceraldehyde 3-phosphate dehydrogenase. Proc Natl Acad Sci U S A 1993;90:6238-6241. 25. Tsuchiya M, Shimoyama M. Target protein for eukaryotic arginine-specific ADP-ribosyltransferase. Mol Cell Biochem 1994;138:113-118. 26. Tsai SC, Adamic R, Moss J, Vaughan M, Manne V, Kung HF. Effects of phospholipid and ADP-ribosylation on GTP hydrolysis by Escherichia colisynthesized Ha-ras–encoded p21. Proc Natl Acad Sci U S A 1985;82:83108314. 27. Scaife RM, Wilson L, Purich DL. Microtuble protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization. Biochemistry 1992;31:310-316. 28. Tsuchiya M, Tanigawa Y, Ushiroyama T, Matsuura R, Shimoyama M. ADP-ribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme. Eur J Biochem 1985;147:33-40. 29. Hara N, Tsuchiya M, Mishima K, Tanigawa Y, Shimoyama M. ADP-ribosylation of Ca2/-dependent ATPase in vitro suppress the enzyme activity. Biochem Biophys Res Commun 1987;148:989-994. 30. McDonald LJ, Moss J. Enzymatic and nonenzymatic ADP-ribosylation of cysteine. Mol Cell Biochem 1994;138:221-226. 31. Hashimoto T, Sasaki H, Yoshikawa H. Hormonal control of phosphoglucomutase activity. Biochem Biophys Res Commun 1967;27:368-371. 32. Kanazawa K, Ashida H. Relationship between oxidative stress and hepatic phosphoglucomutase activity in rats. Int J Tissue React 1991;13:225-231. 33. Lee YS, Marks AR, Gureckas N, Lacro R, Nadal-Ginard B, Kim DH. Purification, characterization, and molecular cloning of a 60kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase. J Biol Chem 1992;267:21080-21088.
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Adenosine diphosphate (ADP)-ribosylation is a posttranslational protein modification that, in turn, alters several regulatory proteins in mammalian cells. We demonstrated that long-term alcohol intake enhanced the ADP-ribosylation of a 58-kd protein in rat liver plasma membranes. To assess the biological significance of this phenomenon, we partially purified the 58-kd acceptor protein from solubilized rat liver homogenates by two sequential preparative high-pressure liquid chromatographies. Microsequencing revealed that it was phosphoglucomutase (PGM) (EC 5,4,2,2). This enzyme underwent negligible auto ADP-ribosylation, but the ADP-ribosylation was remarkably increased by adding rat liver plasma membranes. The extent of the increase was greater in alcohol-fed rats than in pair-fed controls, suggesting enhanced enzyme activities toward ADP-ribosylation of PGM after chronic alcohol consumption. Several important enzymes are ADP-ribosylated, after which their activities are modified. The results of this study showed that PGM is a novel substrate for ADPribosylation in the liver and that the ADP-ribosylation is increased after chronic alcohol consumption. In view of the variety of roles of PGM in the liver (carbohydrate metabolism and Ca2/ homeostasis), specific roles of this modification in terms of the effects of alcohol on hepatocytes may deserve further investigation. (HEPATOLOGY 1996;24:1246-1249.) Enzyme activities are often regulated by posttranslational covalent modifications at critical points of metabolic pathways. One example is adenosine diphosphate (ADP)-ribosylation, in which the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) is transferred to an acceptor protein.1,2 Modification of protein functions by mono-ADP-ribosylation is best known as the mechanism of action of bacterial toxins such as cholera toxin3 and pertussis toxin.4 They exert biological effects on vertebrate cells by catalyzing the mono-ADPribosylation of the stimulatory and inhibitory guanosine triphosphate binding proteins of the adenylate cyclase system. These toxins have been widely used as probes to investigate the role of G-proteins in the transduction of hormonal and sensory signals and their intracellular processes.5,6 Endogenous enzymes that mimic these bacterial toxins have been
Abbreviations: ADP, adenosine diphosphate; NAD, nicotinamide adenine dinucleotide; PGM, phosphoglucomutase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CM, carboxymethyl. From the 1Department of Clinical Pathology, Institute of Clinical Medicine, Tsukuba University, Ibaraki, and 2Second Department of Microbiology, Chiba University School of Medicine, Chiba, Japan. Received January 3, 1996; accepted July 2, 1996. Supported by Japan Educational Ministry Grant and by Kurozumi Medical Foundation. Address reprint requests to: Fumio Nomura, M.D., Department of Clinical Pathology, Institute of Clinical Medicine, Tsukuba University, 1-1-1 Tennoudai, Tsukuba City, Ibaraki 305, Japan. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2405-0060$3.00/0
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identified in various eukaryotic cells, and mounting evidence suggests that endogenous ADP-ribosylation plays significant biological roles in mammalian cells.7-11 These enzymes may be involved in the regulation of adenylate cyclase,12,13 Ca2/ release from mitochondria,14 and the phosphorylation of nuclear proteins.15 In view of the diverse roles of ADP-ribosylation in cells, the effects of alcohol on this reaction are of interest. We demonstrated that long-term alcohol intake remarkably enhanced the endogenous ADP-ribosylation of a 58-kd hepatic protein in the rat.16 To further clarify the biological roles of this phenomenon, the 58-kd protein needed to be identified. We describe here that the 58-kd protein is the multifunctional enzyme, phosphoglucomutase (PGM) (EC 5,4,2,2). The modification of this enzyme by ADP-ribose was increased by chronic alcohol consumption. MATERIALS AND METHODS Animal Procedures. A total of 18 male Sprague-Dawley rats (Charles River Japan, Kanagawa, Japan) weighing 140 to 150 g were used. Ten of the rats were pair-fed with a nutritionally adequate liquid diet containing ethanol as 36% of the total energy, as well as an isocaloric control diet for 4 weeks as described.17 Animal care was in accordance with our institutional guidelines. To equalize the rate of consumption before plasma membrane isolation, one third of the daily ration was given at 9 AM, and two thirds was given at 5 PM on the day before killing. In addition, one third of the daily intake was administered 2 hours before the procedures. The animals were decapitated and their livers were perfused through the portal vein with ice-cold buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L NaN3 , and 250 mmol/L sucrose (TENS buffer). The livers were minced and homogenized by 10 passes with a loose-fitting Dounce homogenizer (Wheaton, Millville, NJ) to prepare 20% homogenates in the same buffer. Liver plasma membrane fractions were prepared as described by Prpic et al.18 ADP-Ribosylation Assay. Mono-ADP-ribosylation was performed as previously described.16,19 Briefly, the reaction mixture (final volume, 200 mL) contained 100 mmol/L Tris-HCl (pH 7.5 at 377C), 10 mmol/L thymidine, 0.5 mmol/L guanosine triphosphate, 1 mmol/L ethylenediaminetetraacetic acid, 5 mmol/L MgCl2 , 1 mmol/L [32P] NAD (10 mCi), and 100 mg protein of samples (homogenates, membranes, column eluates) containing both ADP-ribosyltransferases and their acceptor proteins. After incubation at 377C for 30 minutes, 2 mL ice-cold 7.5% trichloroacetic acid was added, and the samples were kept at 47C overnight. Precipitated proteins were pelleted and dissolved in 1% sodium dodecyl sulfate (SDS)/5% mercaptoethanol (607C; 10 minutes), then resolved by electrophoresis in 10% polyacrylamide gels according to Laemmli.20 The gels were autoradiographed on Kodak X-Omat film (Eastman Kodak Co., Rochester NY) or were evaluated using a Bio-image analyzer (Fujix BAS 2000, Fujicolor, Tokyo, Japan). Identification of a 58-kd Protein. One rat liver (10 g) was homogenized in 40 mL of the TENS buffer, and the homogenates were solubilized in the TENS buffer containing Triton X-100 (final concentration, 0.1%) for 1 hour. The solubilized samples were concentrated using Centricon-100 (molecular-weight cutoff, 100,000) (Grace Japan, Amicon, Tokyo, Japan). The filtrate was then subjected to Centricon 30 (molecular-weight cutoff, 30,000). The residue was fractionated by two rounds of preparative high-pressure liquid chromatographies using a Waters ALC/GPC208 system with a type 440 absorbance detector (Millipore Waters Chromatograph, Tokyo, Japan).
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The first high-pressure liquid chromatography separation was accomplished on a Protein-pak DEAE (10 1 100 mm) (Millipore Waters Chromatograph). The residue from the Centricon 30 centrifugal concentrator was placed on this column, which was equilibrated with buffer containing 20 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L NaN3 , and 0.1% (vol/vol) Triton X-100 (Triton-Tris-EDTA-NaN3 buffer), and eluted with a stepwise gradient of 0.2 to 1.0 mol/L NaCl. The flow rate was 1.0 mL/min, and fractions collected every minute were monitored at 280 nm. Fractions containing high levels of endogenous ADP-ribosylation of a 58-kd protein, which appeared in the flow-through, were pooled and injected onto a protein-Pak carboxymethyl (CM) (10 1 100 mm) (Millipore Waters Chromatograph) equilibrated with the Triton-TrisEDTA-NaN3 buffer, pH 6.0, and then eluted with a stepwise 0.2 to 1.0 mmol/L NaCl gradient. Column fractions collected every 0.5 minute were monitored at 280 nm with a flow rate of 1 mL/min, and those containing high levels of endogenous ADP-ribosylation of a 58kd protein eluted at 0.5 mol/L NaCl were pooled, concentrated, and resolved by SDS–polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and stained with Coomassie brilliant blue R250. A band equivalent to the 32P-incorporated 58-kd protein was excised, and then the amino-terminal of the 58-kd protein was determined using a protein sequencer (model PPSQ-10, Shimadzu Inc., Kyoto, Japan). Sequence homology was determined by searching PIR-international protein sequence database. The final preparation (CM eluate at 0.5 mmol/L NaCl) was subjected to ADP-ribosylation as described above, and a portion of the reaction mixture (30 mg protein) was analyzed by two-dimensional electrophoresis according to O’Farrell.21 The first-dimension isoelectric focusing was performed in the presence of 2% Nonidet P-40 in capillary glass tubing (1 1 70 mm). The second dimension was a 12% SDS-PAGE according to Laemmli.20 Following electrophoresis, the gel was fixed, silver-stained, and subjected to autoradiography as described above. Other Methods. Protein concentrations were determined by means of the Bio-Rad protein assay dye reagent kit (Bio-Rad, Richmond, CA) using bovine serum albumin as the standard. [Adenylate-32P]NAD di(triethylammonium) salt was obtained from Du Pont NEN Research Products (Boston, MA). PGM from rabbit skeletal muscle was obtained from Boehringer Mannheim (Mannheim, Germany). Other reagents were obtained from standard chemical sources. RESULTS
Incubation of rat liver homogenates in the reaction mixture as described above resulted in endogenous 32P incorporation in five protein bands (Fig. 1). It was notable that ADP-ribosylation of the 58-kd form was greater in alcohol-fed rats than in pair-fed controls. Thus, our finding in the plasma membrane fractions were reproducible in homogenates. Therefore, to isolate the 58-kd protein, solubilized rat liver homogenates were partially purified by two sequential rounds of preparative high-pressure liquid chromatographies. Figure 2 shows the protein profiles (Fig. 2A) and 32P-autoradiographs (Fig. 2B) at each step of the purification procedure. The final CM eluate was transferred to a polyvinylidene difluoride membrane following SDS-PAGE. The N-terminal amino acids of the 58-kd protein were sequenced as described in Materials and Methods. The analysis yielded the sequence of up to 15 amino acids (VKIVTVKTQAYPDQK), which was identical to that of residues in rat liver PGM.22 Two-dimensional electrophoresis of the final preparation (CM eluate at 0.5 mol/L NaCl) resulted in one major spot accompanied by two minor ones with pIs close to that of the major spot at 58 kd (Fig. 3A), and selective 32P-labeling was seen in the major spot following ADP-ribosylation (Fig. 3B). Incubating PGM obtained from rabbit skeletal muscle (95% homologous to the rat liver form in terms of amino acid sequences) in the reaction mixture described in Experimental Procedures led to little 32P incorporation of the 58-kd band (Fig. 4), indicating that PGM undergoes negligible auto-ADPribosylation. By contrast, the addition of rat liver plasma membranes obtained from naive rats to the ADP-ribosylation medium together with exogenous PGM strikingly enhanced
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FIG. 1. Endogenous ADP-ribosylation of liver homogenates from control rats and those given alcohol chronically. Rat liver homogenates (120 mg of protein) were ADP-ribosylated with 1 mmol/L [32P]NAD (10m Ci) in the reaction mixture detailed in the text. After an incubation for 30 minutes at 377C, trichloroacetic acid–precipitated proteins were separated on 10% SDS-PAGE and were visualized by autoradiography. Similar results were obtained in three other experiments. C, control; A, alcohol-fed.
the radiolabeling of the 58-kd protein (Fig. 4), suggesting the presence of substantial ADP-ribosyltransferase activities in the membrane fractions. The addition of the cytosol fraction failed to stimulate ADP-ribosylation of the protein (Fig. 5). Furthermore, the enhanced radiolabeling by membrane fractions obtained from alcohol-fed rats was remarkably greater than that by membranes obtained from pair-fed controls (Fig. 5). Incubation of the plasma membranes alone without exogenously added PGM resulted in minimal ADP-ribosylation of the 58-kd protein (Fig. 5). These findings suggested an increase of enzyme activities toward ADP-ribosylation of the 58-kd protein after long-term alcohol intake. DISCUSSION
ADP-ribosylation is a posttranslational protein modification by which living organisms modify the functions of many important regulatory proteins, including guanosine triphosphate-binding protein.2-6 In view of the range of effects of ethanol on cellular metabolism, those on this posttranslational modification deserve attention. We demonstrated that long-term alcohol intake enhanced the endogenous ADP-ribosylation of a 58-kd protein in rat liver plasma membranes.16 To assess the biological significance of this phenomenon, it appeared essential to identify the acceptor protein. In this study, a 58-kd protein was purified by successive column chromatography and microsequenced. N-terminal amino
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FIG. 2. Protein profiles and autoradiographs of a 58-kd protein in liver homogenates and during purification. (A) SDS-PAGE gels stained with Coomassie brilliant blue. Lane 1, liver homogenates (80 mg); lane 2, solubilized liver homogenates (80 mg); lane 3, DEAE flow-through (40 mg); lane 4, CM eluate at 0.5 mol/L NaCl (40 mg). (B) Autoradiographs of preparations during purification, which were ADP-ribosylated as detailed in the text. Lane 1, liver homogenates (80 mg); lane 2, solubilized liver homogenates (80 mg); lane 3, DEAE flow-through (40 mg); lane 4, CM eluate at 0.5 mol/L NaCl (40 mg).
acids sequence analysis revealed that it was the multifunctional enzyme, PGM (EC 5.4.2.2). It was confirmed by twodimensional electrophoresis that a major protein at 58-kd was selectively 32P-labeled following ADP-ribosylation. Furthermore, ADP-ribosylation of a 58-kd protein was strikingly enhanced when PGM was added as an exogenous acceptor to the incubation mixture. Taken together, these observations indicate that the ADP-ribosylated 58-kd protein is PGM. The results of experiments using PGM obtained from rabbit skeletal muscle (95% identical to the rat liver form in terms of amino acid sequences) showed that this enzyme undergoes negligible auto-ADP-ribosylation, but that the protein labeling was remarkably increased by the addition of rat liver plasma membranes. Furthermore, the extent of the increase caused by membrane fractions obtained from alcohol-fed rats was greater than that by those obtained from pair-fed controls, suggesting enhanced ADP-ribosyltransferase activity toward PGM after long-term alcohol intake. Alternatively, it is also possible that PGM was modified nonenzymatically by free ADP-ribose that was generated by NAD glycohydrolase. This is unlikely, because 32P–ADP-ribose failed to bind to the 58-kd protein (data not shown). Also, the ADP-ribosylation of the 58-kd protein was stimulated by GTP,16 which inhibits NAD glycohydrolase.23 It is still possible, however, that a novel linkage of NAD to a protein, as recently clarified,24 can partially account for the protein modification. Several key enzymes are ADP-ribosylated, which, in turn,
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FIG. 3. Two-dimensional electrophoresis of a 58-kd protein in the partially purified preparation. The final preparation (CM eluate at 0.5 mol/L NaCl) was subjected to ADP-ribosylation, and a portion of the reaction mixture (30 mg protein) was analyzed by two-dimensional electrophoresis as described in Materials and Methods. (A) Silver-stained gel. (B) An autoradiogram of the gel shown in (A). A major 58-kd spot in (A) and selective radiolabeling of the 58kd spot in (B) are indicated by arrows. The positions of the molecular mass standards in kilodaltons are shown.
modifies their activities, as reviewed recently.25 Substrates modified by arginine-specific ADP-ribosyltransferase include Ha-ras p21,26 tubulin,27 phosphorylase kinase,28 and Ca2/dependent adenosine triphosphatase.29 Enzymatic and nonenzymatic ADP-ribosylation of cysteine residue of proteins is also known.30 The results of this study showed that PGM is a novel substrate for ADP-ribosylation in the liver and that the enzyme activity of ADP-ribosyltransferase toward PGM is increased after chronic alcohol consumption. Among hepatic membrane-bound enzymes, Na-K adenosine triphosphatase and g-glutamyl transferase were tested for their capacity to be exogenous acceptor proteins. They were not found
FIG. 4. Auto-ADP-ribosylation and enhanced ADP-ribosylation of the 58kd protein by rat liver plasma membranes. PGM obtained from rabbit skeletal muscle (95% homologous to rat liver PGM in terms of amino acid sequences) was ADP-ribosylated as detailed in the text at 377C for 30 minutes in the absence (lanes 1-3) or presence (lanes 4-6) of rat liver plasma membranes (40 mg of protein) obtained from control rats. Lanes 1 and 4, 20 mg PGM; lanes 2 and 5, 40 mg PGM; lanes 3 and 6, 80 mg PGM.
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FIG. 5. Enhanced ADP-ribosylation of PGM by liver plasma membranes obtained from alcohol-fed rats and from pair-fed controls. PGM (40 mg) obtained from rabbit skeletal muscle (95% homologous to rat liver PGM in terms of amino acids sequences) was ADP-ribosylated as detailed in the text. Lane 1, PGM alone; lane 2, PGM / cytosol fraction from control rats (40 mg); lane 3, PGM / membrane fraction from control rats (40 mg); lane 4, PGM / membrane fraction from alcohol-fed rats (40 mg); lane 5, plasma membranes alone from control rats; lane 6, plasma membranes alone from alcohol-fed rats. Similar results were obtained in three other experiments.
to be substrates for ADP-ribosylation (data not shown). The reasons for this apparent specificity for PGM are not currently clear. Identification of the binding site for ADP-ribose, which is currently underway, will provide some explanations for the specificity. PGM is a widely distributed multifunctional enzyme. The classical function of PGM is to link glycolysis and glycogen metabolism by interconverting glucose-1phosphate and glucose-6-phosphate. The activity of PGM is under hormonal control and may play a physiological role in glucose metabolism.31 Alterations of this enzyme may have to be considered in alcohol effects on carbohydrate metabolism. In this context, it is notable that PGM activity was reportedly decreased after an acute ethanol load in the rat.32 Long-term alcohol intake, however, resulted in an increase of PGM activities (Nomura et al., Unpublished observation, June 1996). Mechanisms for the regulation of PGM activities appear to be complex, because this protein is also a substrate for Ca2//calmodulin–dependent protein kinase. The contribution of ADP-ribosylation to the regulation of PGM activity remains to be detailed. In addition to its classical role in glucose metabolism, PGM has another important role. Lee et al.33 have indicated that PGM plays a crucial role in Ca2/ regulation. It is tempting to speculate that hepatic Ca2/ homeostasis can be altered by the modification of PGM by ADP-ribosylation. Thus, our finding that long-term alcohol intake enhanced endogenous ADP-ribosylation of a 58-kd protein in the rat liver16 has been substantiated. We are further clarifying the pathophysiological roles of the posttranslational modification of this multifunctional enzyme in terms of the effects of alcohol on hepatocytes. Acknowledgment: We wish to thank Drs. Motoo Yamasaki and Keiichi Yano, Tokyo Research Laboratory of Kyowa Hakko Kogyo Co., Ltd., for their amino acids sequence analysis. We also thank Yukiko Miyokawa and Sachiko Takegami for their excellent technical assistance. REFERENCES 1. Honjo T, Nishizuka Y, Hayaishi O, Kato I. Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. J Biol Chem 1968;243:3553-3555. 2. Ueda K, Hayaishi O. ADP-ribosylation. Annu Rev Biochem 1985;54:73100. 3. Moss J, Vaughan M. Mechanism of choleragen: evidence for ADP-ribosyltransferase activity with arginine as an acceptor. J Biol Chem 1977;252: 2455-2457. 4. Ui M. Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol Sci 1984;5:277-279.
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5. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 1987;56:615-649. 6. Moss J, Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol 1988;61:303-379. 7. Moss J, Stanley SJ, Watkins PA. Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes. J Biol Chem 1980;255:5838-5840. 8. Tanuma S, Kawashima K, Endo H. Eukaryotic mono(ADP-ribosyl)transferase that ADP-ribosylates GTP-binding regulatory Gi protein. J Biol Chem 1988;263:5485-5489. 9. Maehama T, Takahashi K, Ohoka Y, Ohtsuka T, Ui M, Katada T. Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADPribosylation of GTP-binding proteins. J Biol Chem 1991;266:10062-10065. 10. Seki K, Hirai A, Noda M, Tamura Y, Kato I, Yoshida S. Epoxyeicosatrienoic acid stimulates ADP-ribosylation of a 52kDa protein in rat liver cytosol. Biochem J 1992;281:185-190. 11. McDonald LJ, Moss J. Nitric oxide-independent, thiol-associated ADPribosylation inactivates aldehyde dehydrogenase. J Biol Chem 1993;268: 17878-17882. 12. Molina y Vedia L, Nolan RD, Lapetina EG. The effect of iloprost on the ADP-ribosylation of Gsa (the a-subunit of Gs). Biochem J 1989;261:841845. 13. Tanuma S, Endo H. Mono(ADP-ribosyl) ation of Gi by eukaryotic cysteinespecific mono(ADP-ribosyl)transferase attenuates inhibition of adenylate cyclase by epinephrine. Biochim Biophys Acta 1989;1010:246-249. 14. Richter C, Winterhalter KH, Baumhuter S, Lotscher HR, Moser B. ADPribosylation in inner membrane of rat liver mitochondria. Proc Natl Acad Sci U S A 1983;80:3188-3192. 15. Tanigawa Y, Tsuchiya M, Imai Y, Shimoyama M. ADP-ribosylation regulates the phosphorylation of histones by the catalytic subunit of cyclic AMP-dependent protein kinase. FEBS Lett 1983;160:217-220. 16. Nomura F, Noda M. Stimulation of mono-ADP-ribosylation in rat liver plasma membranes after long-term alcohol intake. HEPATOLOGY 1993;18: 870-873. 17. Lieber CS, DeCarli LM. The feeding of ethanol in liquid diets: 1986 update. Alcohol Clin Exp Res 1986;10:550-553. 18. Prpic V, Green KC, Blakemore PF, Exton JH. Vasopressin-, angiotensin II- and a1-adrenergic-induced inhibition of Ca2/ transport by rat liver plasma membrane vesicles. J Biol Chem 1984;259:1382-1385. 19. Kato I, Noda M. ADP-ribosylation of cell membrane proteins by staphylococcal a-toxin and leukocidin in rabbit erythrocytes and polymorphonuclear leukocytes. FEBS Lett 1989;281:185-190. 20. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4 . Nature 1970;227:680-685. 21. O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007-4021. 22. Revera AA, Elton TS, Dey NB, Bounelis P, Marchase RB. Isolation and expression of a rat liver cDNA encoding phosphoglucomutase. Gene 1993; 133:261-266. 23. Watkins P, Moss J. Effects of nucleotides on activity of a purified ADPribosyltransferase from turkey erythrocytes. Arch Biochem Biophys 1982; 216:74-80. 24. McDonald LJ, Moss J. Stimulation by nitric oxide of a novel linkage of NAD to glyceraldehyde 3-phosphate dehydrogenase. Proc Natl Acad Sci U S A 1993;90:6238-6241. 25. Tsuchiya M, Shimoyama M. Target protein for eukaryotic arginine-specific ADP-ribosyltransferase. Mol Cell Biochem 1994;138:113-118. 26. Tsai SC, Adamic R, Moss J, Vaughan M, Manne V, Kung HF. Effects of phospholipid and ADP-ribosylation on GTP hydrolysis by Escherichia colisynthesized Ha-ras–encoded p21. Proc Natl Acad Sci U S A 1985;82:83108314. 27. Scaife RM, Wilson L, Purich DL. Microtuble protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization. Biochemistry 1992;31:310-316. 28. Tsuchiya M, Tanigawa Y, Ushiroyama T, Matsuura R, Shimoyama M. ADP-ribosylation of phosphorylase kinase and block of phosphate incorporation into the enzyme. Eur J Biochem 1985;147:33-40. 29. Hara N, Tsuchiya M, Mishima K, Tanigawa Y, Shimoyama M. ADP-ribosylation of Ca2/-dependent ATPase in vitro suppress the enzyme activity. Biochem Biophys Res Commun 1987;148:989-994. 30. McDonald LJ, Moss J. Enzymatic and nonenzymatic ADP-ribosylation of cysteine. Mol Cell Biochem 1994;138:221-226. 31. Hashimoto T, Sasaki H, Yoshikawa H. Hormonal control of phosphoglucomutase activity. Biochem Biophys Res Commun 1967;27:368-371. 32. Kanazawa K, Ashida H. Relationship between oxidative stress and hepatic phosphoglucomutase activity in rats. Int J Tissue React 1991;13:225-231. 33. Lee YS, Marks AR, Gureckas N, Lacro R, Nadal-Ginard B, Kim DH. Purification, characterization, and molecular cloning of a 60kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase. J Biol Chem 1992;267:21080-21088.
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