Intrahepatic transport and utilization of biliary glutathione and

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 83, pp , March 1986 Biochemistry ntrahepatic transport and utilization of biliary glutathione and its metabolites (developmental changesy-glutamylglutathione-glutamylcyst(e)inecyst(e)inylglyciney-glutamyl transpeptidase) WLLAM A. ABBOTT AND ALTON MESTER Department of Biochemistry, Cornell University Medical College, 13 York Avenue, New York, NY 121 Contributed by Alton Meister, October 21, 1985 ABSTRACT Glutathione transported by hepatocytes into the bile canaliculi is metabolized by the actions of -glutamyl transpeptidase and dipeptidase located on the biliary ductular epithelium. This pathway is revealed by the finding of high levels of cyst(e)inylglycine, y-glutamylglutathione, y-glutamylcyst(e)ine, glutamate, glycine, and cyst(e)ine in bile, by studies in which intrahepatic metabolism of glutathione was inhibited by administration of a potent inhibitor of y-glutamyl transpeptidase and by experiments in which glutathione synthesis was inhibited. Canalicular transport of glutathione, as estimated from totals of metabolites found, is much greater than the glutathione found in bile. Glutathione and glutathione metabolites found in bile increase with age, in association with an increase in hepatic glutathione. n younger rats there is apparent uptake of cysteine and glycine moieties that may reflect uptake of cysteinylglycine at the ductular level. This intrahepatic pathway of glutathione transport and metabolism, which resembles that which occurs in the kidney, seems to function as a cellular protective mechanism in the processing of glutathione conjugates and as a recovery system for cysteine moieties. Glutathione (GSH) is transported from the sinusoidal surface of hepatocytes to the hepatic venous blood plasma and from the canalicular hepatocyte surface to the bile (1-3). Studies on the interorgan transport ofgsh showed that plasma GSH, which arises chiefly from the liver, is effectively used by the kidney (1, 4-7). Such utilization is mediated by the activities of- t-glutamyl transpeptidase located extracellularly in the renal tubular epithelium (8) and in the basolateral circulation (9). Since liver has a much lower level of y-glutamyl transpeptidase than the kidney in the adult animal, it would appear that relatively little GSH metabolism occurs in the liver. This conclusion is apparently supported by the fact that bile obtained from the common bile duct in the rat may contain millimolar levels of GSH (13). t has been estimated that the flow of GSH from liver to blood plasma is about four times greater than that into bile (see, for example, ref. 1). The present studies, which suggest the need for some revision of these conclusions, show that GSH transported from hepatocytes into the bile ductules undergoes active metabolism. Analytical techniques used here separate, identify, and quantitate GSH metabolites, several of which had not previously been reported in bile. Reliable estimates of canalicular GSH secretion were made possible by combining and comparing the biliary outputs of the amino acid constituents of GSH and of the metabolites. Differences were observed in the biliary appearance of these metabolites during growth and maturation. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. EXPERMENTAL PROCEDURES Materials. The reagents used for enzyme assays and analyses were obtained from Sigma; L-buthionine sulfoximine was obtained as described (14, 15). We thank L. J. Hanka (Upjohn) for L-(aS,5S)-a-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (). Operative Procedures. Male Sprague-Dawley (Taconic Farms, Germantown, NY) rats (2-4 g) were anesthetized by intraperitoneal injection of pentobarbital (6 mgkg), tracheotomized (PE 25), and the left jugular vein was cannulated (PE 1) for saline infusions (1.2 mlhr) to maintain hydration and for other injections. The common bile duct was cannulated (PE 1) at its duodenal end. A second cannula was placed above a ligature about 1 cm from the liver hilus for collection of bile uncontaminated with pancreatic juice ("pure bile"). Pancreatic juice was collected as described (16). Access to the hepatic artery was made by retrograde infusion through the gastroduodenal aboration. This artery was exposed by transecting the pylorus between ligatures placed.5 cm apart around the gastroduodenal junction and across the intervening mesentary; it was cannulated with PE 1 tubing pulled to a tapered end and attached to a syringe valve. Portal vein deliveries were made by a needle inserted under the splenic capsule. Retrograde biliary infusions were made through the bile cannula. The rats were maintained at 38 C to optimize bile flow rates (17). n the experiments described in Figs. 2 and 3, the rats were given either saline (2 mlkg) or (2 ml of a 5 mm solution in saline per kg) via the jugular vein 15 min before common bile duct cannulation. Bile was collected after 3 min for 3 min. After the experiment, portions of the liver were homogenized and assayed for GSH, y-glutamyl transpeptidase, and dipeptidase. Glutathione Determinations. Total GSH was assayed by the glutathione disulfide (GSSG) reductase5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) recycling method (18, 19) using 15 mm Tris HCl (ph 7.5). A sample (2 1,u) of bile was drawn directly from the cannula with a Microcap pipet (Drummond, Bolab, Lake Havasu, AZ) and immediately placed into a cuvette (at 3 C) containing 1 ml of assay buffer,.1 ml of 3.5 mm DTNB, and.1 ml of 4 mm NADPH. The reaction was initiated by adding GSSG reductase. GSSG was assayed after rapid derivatization of the GSH present with.1 M N- ethylmaleimide in.1 ml of 1 mm Tris-HCl, ph 7.5. After 1 min, unreacted reagent was removed as described (2) by applying the mixture to small QAE-Sephadex columns (prepared by swelling 4 ± 2 mg of dry gel in calibrated 1-ml syringe barrels fitted at the bottom with a filter paper disc). The columns were equilibrated with 5 ml of 1 mm Tris HCl, ph 7.5, and after the sample had been applied, were rinsed with 7 ml of this buffer. The fluid level was then brought to 1246 Abbreviations: GSH, glutathione; GSSG, glutathione disulfide;, L-(aS,5S)-a-amino-3-chloro4,5-dihydro-5-isoxazoleacetic acid.

2 Biochemistry: Abbott and Meister the.3-ml mark by opening an attached three-way stopcock (Pharmaseal K-75). Column-bound GSSG was then quantitatively eluted into a cuvette by adding 1 ml of 2 mm Tris HCl, ph 7.5, to the column and again bringing the level to the.3-ml mark; the buffer concentration was about 15 mm as determined by conductivity. DTNB and NADPH were then added, and the reaction was initiated at 3 C. GSSG standards were used to confirm that the recovery was quantitative. Sulfhydryl compounds were determined prior to assay of total GSH from the absorbance increase after adding the sample. Enzyme Assays. y-glutamyl transpeptidase was assayed as described (21) using 2.5 mm L-y-glutamyl-p-nitroanmlide and 2 mm glycylglycine. Dipeptidase was measured using L- leucyl-p-nitroanilide (22). Absorbance was determined at 45 nm (E, 99 Mcm). Protein was determined as described (23). Amino Acid Analyses. Bile samples (8 p1) were treated with 1 pl each of 1 M dithiothreitol and 2-vinylpyridine. After 3 min, 5,ul of 5o (volvol) sulfosalicylic acid was 'added, and the samples were centrifuged (5 min; Beckman Microfuge). The supernatant (75,u) was removed, adjusted to ph 2.2 by adding 1 ul of 1 M LiOH and applied to a Durrum (Palo Alto, CA) model 5 amino acid analyzer using a lithium citrate buffer system. The vinylpyridine derivatives of GSH, y-glutamylcysteine, cysteinylglycine, and cysteine as well as the other amino acids present were effectively separated and quantitated (24, 25). t is important to note that in the standard amino acid analysis system usually used underivatized cysteinylglycine moves together with methionine and that underivatized GSH and y-glu-gsh move with aspartate. y-glu-gsh was prepared enzymatically in reaction mixtures (final volume, 5 ml) containing GSH (5,umol), purified renal y-glutamyl transpeptidase (26) (2 units), and sodium phosphate (25 nmol, ph 7.4). After incubation at 37 C for 1 min,.5 ml of 5% (wtvol) sulfosalicylic acid was added to precipitate the enzyme. The protein-free supernatant solution was adjusted to ph 8 and derivatized with 2-vinylpyridine as described above. The enzymatically prepared compound eluted from the amino acid analyzer at 12 min. Components exhibiting this elution time obtained from bile and the enzymatically produced material gave, after exhaustive hydrolysis with y-glutamyl transpeptidase, 1 mol of vinylpyridine derivative of cysteinylglycine and 2 mol of glutamate. RESULTS "Pure Bile" as Compared to "Mixed Bile." Bile collected from a cannula placed above the sites of entrance of the pancreatic ducts (pure bile) contains low levels of y-glutamyl transpeptidase and dipeptidase (Table 1). On the other hand, bile samples from the same animals withdrawn from a cannula placed near the duodenum (mixed bile) have much higher levels of transpeptidase and dipeptidase. Pancreatic juice, which contains no GSH, has high levels of these enzymes. The presence of y-glutamyl transpeptidase in dog pancreatic juice has been reported (27), and its presence in rat pancreatic juice has been suspected (28). Table 2. Proc. Natl. Acad. Sci. USA 83 (1986) 1247 GSH and metabolites in bile Compound Mixed bile, mm Pure bile, mm GSH 2.45 ± ±.22* y-glu-gsh.312 ± ±.39t t-glu-cys.15 ±.11.9 ±.23 Cys-Gly 1.4 ± ±.11t Glutamate 1.25 ± ±.12t Cyst(e)ine.29 ± ±.91 Glycine.61 ± ±.15 Total [Glu] 4.44 ± ±.1 Total [Cys] 4.19 ± ±.4 Total [Gly] 4.41 ± ±.3 Bile flow (glmin) was 32.4 ± 1.5 and 31.4 ±.5 for mixed and pure bile, respectively. The body and liver weights were, respectively, 381 ± 2 and 15. ± 1.3 g (n = 3). The other free amino acids found in pure bile (,um) were Ala (132 ± 44), Asp (29 ± 1), Gln (127 ± 54), His (91 ± 19), le (153 ± 12), Leu (32 ± 28), Lys (7 ± 21), Met (52 ± 12), Orn (22 ± 19), Phe (1 ± 13), Pro ( ), P-Ser (29 ± 5), Ser (11 ± 12), taurine (15 ± 53), Thr (12 ± 12), Tyr (66 ± 1), urea (9624 ± 149), and Val (177 ± 22). *These values (for total GSH + GSSG) obtained by amino acid analysis agreed closely with those obtained by immediate assay by the enzymatic method (i.e., 2.57 ±.31 mm; 8 rats, weight 394 ± 23 g); 18 ± 3% of the total was in the GSSG form. tsignificantly different from values obtained on mixed bile (P <.5).All values are means ± SD. Both pure bile and mixed bile contain high levels of GSH and its metabolites (Table 2). Mixed bile samples that are not analyzed immediately tend to have much lower levels of GSH (cf. ref. 28). That the GSH values (analyzed immediately) for mixed and pure bile (Table 2) are similar indicates that the GSH metabolites are present in the secreted bile and are not formed in the bile by the actions of pancreas-derived enzymes. Nevertheless, the slight decrease in y-glutamylcyst(e)ine and cyst(e)inylglycine (Table 2) are probably related to the effects of pancreatic enzyme activities. t is of interest that bile contains relatively high levels of cyst(e)inylglycine, y-glutamylcyst(e)ine, and y-glu-gsh. These compounds were not detected in liver by the same methods. ntrahepatic Metabolism of GSH. A series of studies was carried out in which the potent y-glutamyl transpeptidase inhibitor was injected by various routes (portal vein, hepatic artery, common bile duct); the results were similar., which is eluted on the amino acid analyzer just before aspartic acid, was found in bile after portal or hepatic arterial injection; this is consistent with biliary secretion of at the canalicular level. After administration of (Fig. 1, Left), biliary GSH increased rapidly with concomitant decreases in the levels of the GSH metabolites, except for y-glu-gsh, whose level increased somewhat in association with the increased GSH level. Despite a second dose of, complete disappearance of GSH metabolites did not occur indicating, as discussed (9, 29), that this inhibitor does not completely inhibit the enzyme in vivo although it may do so in vitro. Complete amino acid analyses show that the levels of the other amino acids in bile were not significantly or consistently altered by administration of. n this experiment, the level of GSH increased Table 1. Proteins in bile and pancreatic juice y-glutamyl Protein, transpeptidase, Dipeptidase, Flow rate, mgml milliunitsml milliunitsml mlmin Mixed bile 13.4 ± ± ± ± 8.7 Pure bile 4.2 ± ± ± ± 6.3 Pancreatic juice 55.9 ± ± 5 38 ± ±.7

3 1248 Biochemistry: Abbott and Meister Proc. Natl. Acad Sci. USA 83 (1986)._._.5 E C 14 F 12-1 F 8 F 6, f.5 E c BSO y-glu-cys Cysteine Glutamate t-glu-gsh- ~~~~~~~[Gly] [CYS]~A Glycine Cys-Gly y-gu-cys- Gly [GSH1 L--'-J 4 2 m L Control u.) trol Hours Hours BSO FG. 1. Biliary content of GSH and its metabolites before and after inhibition of 'y-glutamyl transpeptidase and GSH biosynthesis. Pure bile was analyzed, for the compounds listed in Table 2, 3 min prior to treatment with (Left) and buthionine sulfoximine (BSO) (Right). The bars indicate, in the appropriate column, the presence of metabolites containing Glu, Cys, and Gly residues (equivalents) (see key, Right). (Left) A fed rat (male, 367 g) was injected (hepatic artery) with (.1 mmol per kg of body weight) at zero time and at 1.5 hr. Mean bile flow rates for each period were as follows: 32., 3.9, 29.9, 26.2, 25.6, 22.9, 22.,ulmin, respectively. Liver weight was 15 g (representative of 6 experiments). (Right) A fasted rat (male, 333 g) was injected intraperitoneally with BSO (4 mmol per kg) at zero time and at 4.5 hr with (.1 mmol per kg). Mean bile flow rates were as follows: 25.3, 2.4, 18.7, 19.9, 19.9, 17.5, 17.7, and 16.9,ulmin, respectively. Liver weight was g (representative of three experiments). No significant changes in the levels of the other amino acids were detected in these experiments. about 2-fold after injection of. n studies on younger rats, the increase in the level of biliary GSH found after giving was much greater. These findings indicate that substantial metabolism of GSH takes place after GSH is transported from hepatocytes and that the amount of GSH exported from hepatocytes is much greater than that found in the common bile duct. Biliary levels of GSH and its metabolites were determined prior to and after intraperitoneal injection of buthionine sulfoximine (Fig. 1, Right). Under these conditions, intrahepatic GSH levels are decreased to about 2% of the control level within 4 hr (5), and virtually all of the cytoplasmic GSH disappears (3). n this experiment, the level of biliary GSH decreased steadily as did the levels of the GSH metabolites. Late in this study, was injected intravenously and this was followed by a significant increase in the level of biliary GSH and a further decrease in the levels ofgsh metabolites, except for y-glu-gsh, which increased. This result indicates that most of the free cyst(e)ine, glutamate, and glycine of bile arises from GSH rather than by canalicular secretion. The intracellular levels of these amino acids are not markedly affected by injection of buthionine sulfoximine; thus, the decrease in levels of these amino acids in the bile under these conditions appears to be a direct result of decreased transport of GSH from the hepatocytes. Developmental Changes. n the course of these studies it was noted that the level of biliary GSH varied considerably and apparently depended upon the age of the animal (see also ref. 31). A series of studies on rats weighing from 4-37 g was carried out on the biliary levels of GSH and its metabolites in control rats and in rats treated with. n these studies linear relationships for controls and -treated rats were found between body weight and liver weight and between bile flow and body weight. However, bile flow was constant per gram of liver. n the controls and the treated rats, the total equivalents of glutamate, glycine, and cysteine increased with increasing body weight and also increased per 21 gram of liver. As indicated in Fig. 2, the biliary output of cysteine equivalents (see key, Fig. 1, Right) in controls increases 2- to 3-fold with age. The biliary output of GSH is quite low (.3 nmol per g of liver per min) for rats weighing 14 - C*m a 6; Bo *..- O a.-.h as Body weight, g a. - 'J : FG. 2. Biliary output of total GSH, sulfhydryl compounds, and cysteine equivalents as a function of body weight. Bile was collected from rats weighing 4-37 g. Output of GSH (e) plus GSSG in GSH equivalents and of sulfhydryl compounds (o) was determined immediately on 2-p4 samples drawn directly from the biliary cannula. Total output of cysteine equivalents (o) was determined by amino acid analysis of bile following reduction and derivatization, i.e., GSH, y-glutamyl-gsh, y-glutamylcysteine, cysteinylglycine, and cysteine. nterrupted lines are based on linear regression of the data for the larger animals (1 g for total [Cys] and 18 g for GSH). * 3

4 less than about 18 g. Rats weighing more than this have substantially higher biliary outputs of GSH. n all of the animals, the total cysteine equivalent content of the bile was much higher than the level of GSH. n the smaller rats, the amount appearing as GSH is a smaller fraction of the total cysteine equivalents than it is in larger animals. With increasing body weight there are concomitant increases in the biliary levels of GSH, total cysteine equivalents, and total SH compounds. Clearly the total SH content of bile is not an accurate index of its GSH content, and a substantial amount of the cysteine moieties is present in disulfide linkage. The increase in biliary GSH with increasing weight seems to be related to increased intracellular GSH. Liver GSH levels were determined; linear regression analysis yielded the following equation: liver GSH = [9.52 (kg body weight) + 3.8]Amol per g of liver; the slope was significantly different from zero (P <.1). y-glutamyl transpeptidase and dipeptidase were determined in the homogenates. The transpeptidase activities were very low (.46 ±.8 milliunitsg) and showed no trends. [Earlier work (8) revealed a marked decline during earlier stages of development for hepatic transpeptidase, which is abundant in fetal liver.] Hepatic dipeptidase was found to increase about 3- to 4-fold with increasing weight. n the -treated rats, the output ofgsh was increased by about 6-fold as compared to the controls in animals weighing 48 g; in the rats weighing more than 18 g, the increase was 2- to 3-fold. n the controls, there were significant differences between the total equivalents found for glutamate, glycine, and cysteine, and these differences decreased by treatment with. n Fig. 3A, the biliary excretion oftotal cysteine equivalents is compared to that of glutamate. The values for cysteine equivalents were consistently lower than those of glutamate, and this difference was maximal for rats weighing between 8 and 2 g. (n this weight range, the -2 P= L. a.> U - -2 CdO _ E Biochemistry: Abbott and Meister A Totail tal[;s [Cys] -total 'ttl[.[lilu]. u.. t - CP.. * * o (b o O o. B_Toal [Cs tl[ly O o _~ ~ ~~ ~~~~~~~~~~~~~ - -C otl Gl] tta [lu Body weight, g FG. 3. Differences in the biliary output of total equivalents of glutamate, cysteine, and glycine as a function of body weight. Bile was collected from rats 3 min after intravenous injection of either saline (controls, o) or (.1 mmolkg, *). Bile samples were reduced and derivatized prior to amino acid analysis and quantitated as described in Fig. 1. The cross differences between these totals for each animal are plotted. (A) Total cysteine total glutamate equivalents. (B) - Total cysteine - total glycine equivalents. (C) Total glycine - total glutamate equivalents. The differences in A and B were significantly decreased by treatment (P <.1). The decreases in these differences observed with treatment for A and C were significant (P <.1) as determined by unpaired T tests for all animals in each group. Proc. Natl. Acad. Sci. USA 83 (1986) 1249 mean difference was 3. nmol per g per min; the total values averaged 4.5 and 7.5 nmol per g per min for cysteine and glutamate equivalents, respectively.) The differences were significantly reduced after treatment with (P <.1). The output of cysteine equivalents was also consistently lower than glycine equivalents (Fig. 3B). This difference is about 1 nmol per g per min throughout the weight range and did not change significantly after treatment. Comparison of glycine equivalents to glutamate equivalents (Fig. 3 showed a pattern similar to that found with cysteine (Fig. 3A). The deficit of glycine relative to that of glutamate is only notable over the weight range 8-2 g. DSCUSSON These studies reveal that the metabolism of GSH in the biliary system is highly complex and leads to formation of substantial amounts of GSH metabolites not previously observed in bile. Although it has long been known that -glutamyl transpeptidase can catalyze "autotranspeptidation" in vitro, the presence of yglu-gsh in bile seems to be the first evidence that this compound is synthesized under physiological conditions. y-glutamylcystine was previously found in the urine of animals given transpeptidase inhibitors and in the urine of patients with -glutamyl transpeptidase deficiency (25). Cystine is one of the most active transpeptidase acceptor substrates (32-34), and there is evidence that y-glutamylcystine formed by transpeptidation is readily transported into kidney cells and efficiently utilized for GSH synthesis (35). The presence ofhigh levels of cysteinylglycine in bile may be associated with the relatively low levels of dipeptidase present. Transpeptidation is a reversible reaction, and since cysteinylglycine is an excellent acceptor substrate, it is probable that some cysteinylglycine may be incorporated into GSH. The findings suggest that the following reactions occur within the biliary system: 2 GSH =- Glu-GSH + CysH-Gly [1] CysH-Gly -- CysH + Gly GSH + (Cys)2 =- Glu(Cys)2 + CysH-Gly GSH -* Glu + CysH-Gly Although reactions 2 and 4 function to convert GSH to its constituent amino acids (cf. Fig. 1), substantial amounts of cysteine moieties are retained in peptide linkage. Thus, the system does not function efficiently in the conversion of GSH to free amino acids. Possibly the peptides are more efficiently taken up than are the free amino acids; therefore, the system may function in recovery mechanisms for cysteine moieties. Older rats (>2 g; cf. Fig. 1) exhibit about equal amounts of total cysteine equivalents before and after inhibition of transpeptidase. On the other hand, in younger animals, especially those weighing in the range 48 g, there is a disappearance of total glycine and cysteine equivalents relative to glutamate equivalents, and this disappearance is significantly reduced by inhibition of transpeptidase. As noted above, the output of cysteine equivalents is also consistently lower than that of glycine equivalents (Fig. 3). The observed differences in the outputs of the amino acid equivalents might be ascribed to independent transport of the respective free amino acids at the canalicular membrane. For example, glutamate export might account for the results shown in Fig. 3 A and C. However, such independent glutamate transport would not be expected to decrease upon inhibition of transpeptidase. The data are consistent with the possibility that there is appreciable ductular uptake of cysteinylglycine. Transport of y-glutamylcystine and of the [2] [3] [4]

5 125 Biochemistry: Abbott and Meister free amino acids may also occur. Although further studies are required to elucidate the nature and extent of such putative transport processes, the data point clearly to an age-dependent system that seems to function in the recovery of cysteine moieties. We had previously considered the existence of a feedback system based on allosteric modulation of ductular transpeptidase by bile acids (16). n such a system, unconjugated bile acids would accelerate the metabolism of biliary GSH by activating ductular transpeptidase. The ductular reabsorption of one or more of the GSH metabolites would furnish the liver with the amino acid substrates required for bile acid conjugation, i.e., glycine for formation of glycocholic acid and taurine (an oxidative metabolite of cysteine) for taurocholic acid. The present studies provide evidence that biliary reabsorption of the appropriate metabolites may indeed occur, especially during development when the bile acid pool is actively increasing. The authors thank Mr. Thomas J. Keady for skillful technical assistance and Dr. Francesco nfante for performing the amino acid analyses. This research was supported in part by the Public Health Service (National nstitutes of Health). 1. Meister, A. & Anderson, M. E. (1983) Annu. Rev. Biochem. 52, Kaplowitz, N., Eberle, D. E., Petrini, J., Tooloukian, J., Corvasce, M. C. & Kuhlenkamp, J. (1983) J. Pharmacol. Exp. Ther. 224, Lauterberg, B. H., Smith, C. V., Hughes, H. & Mitchell, J. R. (1984) J. Clin. nvest. 73, noue, M., Kinne, R., Tran, T. & Arias,. M. (1983) Eur. J. Biol. 134, Griffith,. W. & MNeister, A. (1979) Prop. Eatl. Acad. Sci. USA 76, Anderson, M. E., Bridges, R. J. & Meister, A. (198) Biochenz. Biophys. Res. Commun. 96, Griffith,. W. & Meister, A. (1979) Proc. Natl. Acad. Sci. USA 76, Meister, A., Tate, S. S. & Ross, L. L. (1976) in The Enzymes of Biological Membranes, ed. Martinosi, A. (Plenum, New York), Vol. 3, pp Abbott, W. A., Bridges, R. J. & Meister, A. (1984) J. Biol. Chem. 259, Refsvik, T. & Norseth, T. (1975) Acta Pharmacol. Toxicol. 36, Proc. Natl. Acad. Sci. USA 83 (1986) 11. Sies, H., Koch,. R., Martino, E. & Boveris, A. (1979) FEBS Lett. 13, Eberle, D., Clarke, R. & Kaplowitz, N. (1981) J. Biol. Chem. 256, Abbott, W. A. & Meister, A. (1982) Fed. Proc. Fed. Am. Soc. Exp. Biol. 41, 143 (abstr.). 14. Griffith,. W. & Meister, A. (1979) J. Biol. Chem. 254, Griffith,. W. (1982) J. Biol. Chem. 257, Abbott, W. A. & Meister, A. (1983) J. Biol. Chem. 258, Roberts, R. J., Klaassen, C. D. & Plaa, G. L. (1967) Proc. Soc. Exp. Biol. Med. 125, Owens, C. W.. & Belcher, R. V. (1965) Biochem. J. 94, Tietze, F. (1969) Anal. Biochem. 27, Akerboom, T. P. M., Bilzer, M. & Sies, H. (1982) J. Biol. Chem. 257, Orlowski, M. & Meister, A. (1963) Biochim. Biophys. Acta 73, Rankin, B. B., Mcntyre, T. M. & Curthoys, N. P. (198) Biochem. Biophys. Res. Commun. 96, Lowry,. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.:(1951) J. Biol. Chem. 193, Griffith,. (198) Anal. Biochem. 16, Griffith,. W. & Meister, A. (198) Proc. Natl. Acad. Sci. USA 77, Meister, A., Tate, S. S. & Griffith,. W. (1981) Methods Enzymol. 77, Nakajima, S., Toda, Y., Hayakawa, T., Suzuki, T. & Noda, A. (1973) Pflugers Arch. 345, Hirata, E. & Takashi, H. (1981) Toxicol. Appl. Pharmacol. 58, Capraro, M. A. & Hughey, R. P. (1985) J. Biol. Chem. 26, Griffith,. W. & Meister, A. (1985) Proc. Natl. Acad. Sci. USA 82, Ballatori, N., & Clarkson, T. W. (1982) Science 216, Thompson, G. A. & Meister, A. (1975) Proc. Natl. Acad. Sci. USA 72, Thompson, G. A. & Meister, A. (1976) Biochem. Biophys. Res. Commun. 71, Thompson, G. A. & Meister, A. (1977) J. Biol. Chem. 252, Anderson, M. E. & Meister, A. (1983) Proc. Nat. Acad. Sci. USA 8,