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CLIN. CHEM. 25/1, 75-79 (1979) Hydrolysisof Glutathioneby Human Liver ‘y-Glutamyltransferase Leslie M. Shaw and David A. Newman We studied the catalytic hydrolysis of glutathione by human tathione. Reasoning from these data, we propose a catabolic pathway for glutathione and glutathione conjugates in human liver. This may have important metabolic implications for the production from glutathione was maximal at pH 7.4 (37 #{176}C). diseased liver, because the activity of this enzyme in liver is significantly increased in various hepatic diseases (8). Kinetically, the liver enzyme is similar to human kidney liver -y-glutamyltransferase acid -y-glutamyltransferase, [(y-glutamyl)-peptide:amino EC 2.3.2.21. Glutamate ‘y-glutamyltransferase: their respective Km values with glutathione as substrate are similar (0.096 X iO mol/L and 0.097 X iO mol/L, respectively). S-Methylglutathione was hydrolyzed at a slightly higher rate than glutathione by liver y-glutamyltransferase. From these findings and other established properties of liver and kidney yglutamyltransferase we propose that human liver is an important site of glutathione catabolism and that ‘y-glutamyltransferase in liver catalyzes the first step of the catabolism of glutathione and glutathione conjugates in this organ. AdditionalKeyphrases: liver disease - catabolic pathways Recent studies in several laboratories have demonstrated that the physiological role of y-glutamyltransferase (EC 2.3.2.2) is to catalyze the first step of the breakdown of glutathione or its S-substituted conjugates to produce L-glutamate and L-cysteinylglycine or S-substituted L-cysteinylglycine (1-4). The original proposal (5) that the enzyme functions in vivo as a transpeptidase, and as such plays a key role in amino acid transport across cell membranes, has never been proven. In fact, recent evidence strongly argues against this as its physiological role in man (6) as well as in experimental animals (1, 2). Most biochemical and clinical studies of it make use of the synthetic substrates -y-glutamyl-4-nitroanilide or ‘y-glutamyl-3-carboxy-4-nitroanilide as y-glutamyl donor substrates and glycylglycine as the acceptor substrate. Assays with these substrates are convenient and straightforward, especially when compared to the much more tedious procedures in which the physiological substrate, glutathione, is used. In studies of y-glutamyltransferase in which glutathione or glutathione conjugates were the substrate, the enzyme source was kidney. The kidney is now considered in the rat-and, by implication, in man-to be the major site of catabolism of glutathione and glutathione conjugates (4, 7). In this work we demonstrate that human liver y-glutamyltransferase has kinetic properties very similar to that in kidney with glutathione as substrate and that the human liver enzyme catalyzes efficiently the hydrolysis of S-methylgluToxicology and Special Enzymology Laboratory, Division of Laboratory Medicine, Wm. Pepper Laboratory, Department of Pathology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104. Received Oct. 3, 1978; accepted Oct. 24, 1978. Materials Glutathione, S-methylglutathione, the sodium salt of adenosine-5’-diphosphate (Grade I), tris(hydroxymethyl) aminomethane hydrochloride, 3-nicotinamide adenine dinucleotide (Grade III), imidazole (Grade III), and maleic acid were obtained from Sigma Chemical Co., St. Louis, MO 63178; glycine and dithiothreitol from Bio-Rad Laboratories, Richmond, CA 94804; hydrazine hydrate and perchioric acid from Fisher Scientific Co., Philadelphia, PA 19406; and glutamate dehydrogenase (EC 1.4.1.3, from bovine liver, in glycerol! water, 1:1) from P-L Biochemicals Inc., Milwaukee, WI 53205. All other chemicals used were of the highest analytical quality available. -y-Glutamyltransferase was prepared from human liver through the batch diethylaminoethylcellulose step and from human kidney through the DE-52 chromatography step, both as described by Shaw et al. (9). Methods For the study of glutamate production from glutathione or its S-methyl conjugate, -y-glutamyltransferase prepared from human liver as described above was incubated in 2 mL of a reaction mixture, at 37 #{176}C, containing, per liter, 5 mmol of glutathione (or S-methylglutathione), 10 mmol of dithiothreitol, and 100 mmol of tris(hydroxymethyl)aminomethane (pH 7.4 unless otherwise noted). The reaction was terminated at 30 mm (unless otherwise noted) by adding of 2 mL of perchloric acid (1.0 mol!L). After centrifugation, a 2-mL aliquot of the supernatant fluid was adjusted to pH 9.0 with 0.5 mL of a K;1P04 solution (1.93 mol/L). Duplicate reaction blanks were included in each experiment. The same incubation, reaction termination, and final pH adjustment procedures as above were followed except that distilled water was used in place of the enzyme. The i-glutamate produced by the enzymic hydrolysis of glutathione was quantitatively determined by the enzymic method of Bernt and Bergmeyer (10). In this procedure a 0.4-mL aliquotof terminated reaction mixture is added to an incubation medium (0.92mL) in each of two testtubes containinga pH 9.0glycine-hydrazinebuffer(perliter, 300 mmol of glycine and 250 mmol of hydrazine), adenosine 5’-phosphate (1.0 mmol/L) and nicotinamide adenine dinucleotide (1.6 mmol!L). The A340 nm (A1) of thismixture in one of the two test tubes is determined. Glutamate dehydrogenase (0.02 mL; from bovine liver; in glycerol/water, 1:1; catalytic activity 4.5 kU/L) is added to the second test tube. The reaction CLINICAL CHEMISTRY, Vol. 25. No. 1, 1979 75 140 120 //‘N a, (I) 0 E 80 a, a E 0 60 /[Glutathione] (m mol/li’ 40 20 D 68 7.6 84 92 > pH Fig. 1. Glutamate production from glutathione as a function of pH Glutamate was measured by the enzymic procedure described under Methods, at pH values (37 #{176}C) of 6.0, 7.0, 7.4, 8.0,8.6, and9.0. Incubationwasfor 30 mm as described under Methods, with a glutathione concentration of 5 mmol/L and buffer limidazole at pH 6.0; trls(hydroxymethyl)aminomethane at the other pH values] concentrationsof 100 mrnol/L Eachpointis the average glutamatevalue for duplicate incubationflasks. I / [Glutathione] Fig. 2. Double-reciprocal plots: The reciprocal of initial velocity (1 / v) is plotted vs. the reciprocal of glutathione concentration (L/mmol) for liver and kidney -y-glutamyltransferase In these experiments mixture is after which A1 and A2 calculated. then incubated for 45 mm at room temperature, A:340 (A2) is determined. The difference between (A2 - A1) for each sample and reaction blank was Then Asampie Ablank = LAgiutamate was used for the calculation of glutamate concentration mula [glutamate, .Agiut.amate mmol!L] = X TV from the forX 2.5 6.22 X SV where TV (1.34 mL) is the total volume of the glutamate assay, SV (0.4 mL) is the sample volume, 2.5 is the dilution factor, and 6.22 is the millimolar absorptivity of NADH at 340 nm. All absorbance measurements were made with a Stasar III spectrophotometer (Gilford Instrument Labs., Oberlin, OH 44074). In two experiments glutamate formation from glutathione was measured with a Model D-500 amino acid analyzer (Durrum Chemical Corp., Palo Alto, CA 94303). The same incubation conditions as above were used to study the production of glutamate by the action of ‘y-glutamyltransferase on glutathione. We terminated the reactions by placing the mixtures in a boiling water bath for 3 mm, then rapidly cooling them in ice. At this point 0.1 mL of norleucine internal standard (6.5 mmol/L) was added to each reaction mixture, followed by 0.05 mL of a 0.01 mol!L solution of dithiothreitol. lodoacetamide, 5 mg, was then added to each reaction mixture, with thorough mixing. After 15 mm at room temperature, enzyme protein was removed from the reaction mixtures by filtration through collodion membranes (ii). These proteinfree samples were analyzed with the amino acid analyzer, according to the regular protocol for amino acid analysis with this system (12). The average elution times for standard so- lutions of the 5-acetamido derivatives of glutathione and L-cysteinylglycine, and of L-glutamate were 12 mm, 40 mm, and 17 mm, 10 s, respectively. Results Precision of the enzymic solution of L-glutamate centration 76 of 100 smol/L. (m mol/l1’ was assay. An aqueous having a nominal conof this solution were frozen. glutamate prepared Aliquots CLINICAL CHEMISTRY, Vol. 25, No. 1, 1979 glutamate was determined with an amino acid analyzer as described under Methods The glutamate content of one of these aliquots was determined each day an experiment was conducted. The mean, standard deviation, and coefficient of variation for seven determinations were 105.6 tmol!L, 6.3 mol/L, and 5.96%, respectively, indicating acceptable precision for this method. Variation of glutathione hydrolysis with time, enzyme amount and pH. We incubated 0.49 U of liver in the glutathione reaction mixture for various intervals up to 45 mm. The increase in glutamate production with time was linear over this interval. To study the variation of glutamate production with enzyme amount, we incubated zero to 0.74 U of liver ‘yglutamyltransferase with glutathione reaction mixture for 30 mm, and found the quantity of glutamate produced to be proportional to the amount of enzyme over the entire range. Glutathione hydrolysis by liver -y-glutamyltransferase was measured as a function of pH over the range of pH 6.0 to pH 9.0. As shown in Figure 1, L-glutamate production is maximal at pH 7.4. Kinetic studies. Figure 2 illustrates double-reciprocal plots of the rate of glutathione hydrolysis vs. glutathione concentration. Glutamate production by the catalytic action of liver and kidney -y-glutamyltransferase on glutathione was measured over the range of glutathione concentrations of 0.025 to 5.0 mmol/L. We determined Km values for the liver and kidney enzymes, using the mathematical model and computer methods previously described (9, 13). Evidently the kinetic behavior of liver ‘y-glutamyltransferase is very similar to that of the kidney enzyme, because their respective Km values were almost identical (0.096 X 10 mol!L for the enzyme from liver, 0.097 X iO mol!L for that from kidney). We incubated the glutathione conjugate S-methylglutathione in place of glutathione in the standard incubation system, to determine whether or not this compound is a substrate for liver y-glutamyltransferase. As summarized in Table 1, S-methylglutathione does serve as a substrate for the enzyme from liver. In fact, the rate of glutamate production was slightly higher (14.2%) with this substrate than for glutathione. To test the effect of including maleate in the reaction Glutathione and Its S-Methyl Conjugate by Liver ‘y-Glutamyltransf erase Activity Glutathione Glutathione + maleate S-Methylglutathione S-Methylglutathione + maleate Boiled enzyme control 0.097 X 10 molfL. This Km value is far lower than the reported 5 mmol/L concentration of glutathione in mammalian liver. In a previous study (9) we demonstrated that glutathione substantially and rather equally inhibits the formation of 4-nitroaniine by the enzyme from human liver or kidney. A further indication of the similarity of the kinetic behavior of the two with glutathione as substrate is the fact that y-glutamyltransferase in rat kidney tubules catalyzes the transpeptidation reaction under the standard conditions of Szasz (14), with use of the synthetic ‘y-glutamyltransferase, Table 1. Effect of Maleate on Hydrolysis of a 118.9 114.8 135.8 127.0 4.2 Nanomolesof glutamateproducedper milliliter of reaction mixture per 30 mm at 37 #{176}C. Each result is the average of duplicate determinations. The total volume of each reaction mixture was 2.0 mL and contained 0.74 U of human liver y-giutamyltransferase,and, per liter, 5 mmol of glutathione or S-methylglutathione, 10 mmol of dithiothreitol, and 100 mmol of tris(hydroxymethyl)aminomethanehydrochloride buffer, pH 7.4. Maleate,when present, hada final reaction mixture concentration of 20 mmol/L. In the boiled enzyme control an aliquot ofliver ‘-gIutamyltransferase was first placed in a boiling water bath for 5 mm before introduction into the glutathione-containingreaction mixture. After 30 mm of incubation the reaction was terminated andthe glutamateconcentrations determined as described under Methods. The amount of enzyme we included in each reaction mixture, 0.74 U. is based on activity determined by the Szasz (14) method at 30 #{176}C. mixture on the rate of glutathione hydrolysis we included this compound in the standard reaction mixture at a final concentration of 20 mmol!L. As shown in Table 1, maleate had little effect on the hydrolysis of either glutathione or its Smethyl conjugate. Hydrolysis of glutathione was slightly slower in the presence of maleate (3.4%), but the difference was within the experimental error of the glutamate assay. In the case of S-methylglutathione the hydrolysis was also slightly slower in the presence of maleate (6.5%) but this difference barely exceeded the experimental error of the glutamate procedure. These results contrast with the data of Tate and Meister (15), who obtained an almost eightfold increase by maleate of the rate of hydrolysis of S-methylglutathione by rat-kidney ‘y-glutamyltransferase. Perhaps this reflects a species difference. Discussion The synthetic -y-glutamyl donor substrates y-glutamyl4-nitroanilide or y-glutamyl-3-carboxy-4-nitroanilide are the ones most commonly used in clinical and biochemical studies of y-glutamyltransferase because of the much simpler assay techniques involved. Unfortunately, use of such unphysiological substrates has not been very helpful in the study of the physiological role of ‘y-glutamyltransferase. The pH optima with these substrates is 8.25 (9, 14) and initial velocity rates are maximal only with high concentrations of the dipeptide glycylglycine (14). In a recent detailed kinetic study, we have shown that ‘y-glutamyltransferase does not catalyze the hydrolysis of these substrates, whereas this is now considered to be the physiological action of this enzyme toward its natural substrate,glutathione (13). Thus, to gain insight into the physiologicalroleof y-glutamyltransferaseinhuman liverwe used the physiological substrate glutathione in this study. This work has further potential significance because the activity of this enzyme is increased significantly in diseased human liver (8); such increases would be expected to increase proportionally the rate of glutathione catabolism in the liver. We have shown that the pH optimum for glutathione hydrolysis by human liver y-glutamyltransferase is 7.4. This contrasts with our earlier finding with human liver -y-glutamyltransferase of the unphysiological pH optimum of 8.25 with the synthetic substrates (9). The Km value for liver ‘yglutamyltransferase with glutathione as substrate is 0.096 X iO mol!L and isclose to the value we obtained for kidney substrate ‘y-glutamyl-4-nitroanilide and the acceptor substrateglycyiglycine,at 94-fold the rate of glutathione hydrolysis by kidney tubules (4). We obtained a value of 93 for the ratio with human liver y-glutamyltransferase, based on the glutathione hydrolysis data in Table 1. Recent studies make it appear that the catabolism of glutathione and its S-substituted derivatives occurs primarily in the lumen of the proximal tubule of kidney (2-4). The first step of this pathway is the cleavage of the y-glutamyl moiety by ‘y-glutamyltransferase (16) to produce L-glutamate and L-cysteinylglycine (when glutathione is substrate) or S-substituted L-cysteinylglycine (when S-substituted glutathione is substrate) (17). The L-cysteinyl dipeptides are then hydrolyzed by a dipeptidase to produce glycine and L-cysteine or S-substituted L-cysteine (18). According to this model glutathione is hydrolyzed outside of the cells in which synthesized; one prerequisite for this is the synthesis of tathione by organs such as the liver, followed by its efflux plasma. That such glutathione synthesis and efflux from it is glu- into liver actually occurs is strongly supported by the evidence for the inter-organ transport of glutathione in dogs (19) and by the demonstration of glutathione efflux from the perfused rat liver (20) and from isolated viable rat hepatocytes (21). Another important feature of this model of glutathione catabolism is the assumption that the orientation of -y-glutamyltransferase is toward the lumen of the proximal tubule. This assumption is favored by experiments that show the rapid hydrolysis of glutathione by isolated rat kidney tubules (4). The finding of marked glutathionuria and glutathionemia in a patient who lacks ‘y-glutamyltransferase in his tissues provides support for the inter-organ transport of glutathione and emphasizes the importance of y-glutamyltransferase in the hydrolysis and conservation of glutathione in man (7). In establishing this scheme for the catabolism of glutathione, the kidney has been postulated as the primary site for the degradation of glutathione or its related S-substituted conjugates, for good experimental reasons. The first of these is that rat kidney contains the highest y-glutamyltransferase activity, as compared to any other tissue (22). The second is that when glutathione was introduced into the perfusate of the perfused rat kidney it was rapidly broken down into its constituent amino acids (23, 24). On the other hand, when glutathione was introduced into the perfusate of perfused rat liver or incubated with freshly prepared erythrocytes it was not metabolized (4, 25). We believe that serious consideration should be given to the role of ‘y-glutamyltransferase in glutathione catabolism in human liver. We propose that even though kidney isprobably the major site of glutathionehydrolysisin the rat,otherorgans such as the liver play a significant role in glutathione catabolism in man. This proposal is based on the following data: (a) ‘y-Glutamyltransferase activityin human liverisabout 10-fold that in rat liver, and its activity in human kidney is about 10-fold lower than in rat kidney (16). (b) The kinetic properties of human liver ‘y-glutamyltransferase with glutathione or 5-methylglutathione as substrates are very similar to those of the kidney enzyme: (i) Human liver y-glutamyltransferase catalyzes the hydrolysis of glutathione and has a Km value similar to that of the kidney CLINICAL CHEMISTRY, Vol. 25, No. 1, 1979 77 )‘-GLu-CYS-GLY] SH I Conjugation y-GW-CYS-GLV CH1I Glutothione-S-tronsferase + References S-CH3 Glutoth,one Hydrolysis L-GLL) GGT GGT Hydrolysis #{149} L -CYS-GLY L-GLU. L-CYS-GLY SH S-C H3 Dupeptidase Dipeptudase 0 L-CYS.GLY SH C erase N-ocetyllronsf L-CYS ‘ + GLY S-CH, L-CYS S-CH, Mercopturuc (.CH, - -s-coa) Acid Fig. 3. Proposed glutathione and conjugated glutathione catabolic pathway in human liver CH3I is used here as an exampleof an electrophilic compound that is conjugated to glutathione with the ultimate production of the corresponding mercapturic acid. I3LU, L-gfutamate;Ct’S, L-cysteine; GL V. glycine. GOT, -y-glutamyitrans- ferase SH enzyme. (ii) In this work we have shown that human liver y-glutamyltransferase catalyzes hydrolysis of S -methylglutathione equally as efficiently as hydrolysis of glutathione. Similarly, others have shown that -y-glutamyltransferase in isolated rat-kidney tubules efficiently catalyzes the hydrolysis of S-methylglutathione (3). (iii) As indicated above, the ratio of the rate of p-nitroaniline production in the standard Szasz (14) assay to the rate of glutamate production from glutathione obtained with liver -y-glutamyltransferase was comparable to that reported by others for rat kidney tubule y glutamyltransferase. (c) Glutathione added to the perfusate of perfused rat liver is not metabolized. This should not he considered as evidence that the liver is not involved in glutathione hydrolysis, because the perfusate only comes into intimate contact with the sinusoidal side of hepatocytes, and y-glutamyltransferase is not present in the sinusoidal membrane of rat or human hepatocytes. Histochemically, it has been shown to be located in the canalicular plasma membranes and in the plasma membrane of biliary duct epithelial cells of normal rat and human liver (26, 27). A permeability barrier exists between the canaliculi and the space of Disse (28). Thus glutathione added to a perfusate of the perfused rat liver is unlikely to come into contact with y -glutamyltransferase. (d) The N-acetylcysteine, glutathione, L-cysteinylglycine, and L-cysteine conjugates of a group of hydrocarbon epoxides have been detected in the bileof ratstreated with the corresponding polycyclic hydrocarbons (29-31). This is but one illustration of the fact that glutathione conjugates, once formed in liver cells, are excreted in high concentrations in bile (17). The presence in bile of the catabolites-namely, the N-acetylcysteine, L-cysteinyl glycine, and L-cysteine conjugates-is evidence in favor of the role of liver y-glutamyltransferase in glutathione catabolism, because the first step in the breakdown of conjugated glutathione is the production of conjugated L-cysteinylglycine by the catalytic action of y-glutamyltransferase. Numerous drugs and toxic agents are metabolized by this pathway. A summary of the proposed steps in the catabolism of glutathione in human liver is displayed in Figure 3. We are indebted to Lorette Petersen for the preparation of human liver and kidney -y-glutamyltransferase used in this work; to Jack London for the determination of the kinetic constants; to Donald Fetterolf and Ada Bello for the amino acid analyses; and to Jeanne Esposito for assistance with the manuscript. 78 CLINICAL CHEMISTRY. Vol. 25. No. 1, 1979 I. RIce,J. S., and Broxmeyer, B., y-Glutamyltransferase of rat kidney. Simultaneous assay of the hydrolysis and transfer reactions with glu1amafeJ4Cjglutathione. Biochem. J. 153, 223 (1976). 2. Wendel, A., Hahn, R., and Guder, W. G., On the role of -y-glutamyltransferase in renal tubular amino acid reabsorption. In Current Problems in Clinical Biochemistry 6. Renal Metabolism in Relation to Renal Punctwn, U. Schmidt and U. C. Dubach, Eds., Hans Huber, Bern, Vienna, 1976, pp 426-436. 3. Wendel, A., Heinle, H., and Silbernagl, S., The degradation of glutathione derivatives in the rat kidney. Hoppe-Seylers Z. Physiol. (‘hem. 358, 1413 (1977). 4. Hahn, R., Wendel, A., and Floh#{233}, L., The fate of extracellular glutathione in the rat. Biochim. !3iophvs. Acta 539, 324 (1978). 5. Meister, A., On the enzymology of amino acid transport. Science 180,33 (1973). 6. Pellefigue, F., Butler, J. D., Spielberg, S. P., et al., Normal amino acid uptake by cultured human fibroblasts does not require ‘y-glutamyl transpeptidase. Riochem. Biophys. Rex. Commun. 73, 997 (1976). 7. Schulman, .1.D., Goodman, S. I., Mace, .J.W., et al., Glutathionuria: Inborn error of metabolism due to tissue deficiency of -glutamyl transpeptidase. Biochem. Biophvs. Rex. Commun. 65,68 (1975). 8. Schmidt, F. W., Rationale for the use of enzyme determinations in the diagnosis (if liver disease. Chap. 4 in Evaluation of Liver unct ion: A Multifaceted Approach to (‘linical Diagnosis, L. Demers and L. M. Shaw, Eds., Urban and Schwarzenherg, Baltimore, MD, 1978. 9. Shaw, L. M., London, ‘J. W.. and Petersen, L. E., Isolation of rglutamvltransferase from human liver, and omparison with the enzyme from human kidney. Clin. Chem. 24, 905(1978). 10. Bernt, E., and Bergmeyer, H. U., 1,-Glutamate UV-assay with glutamate dehydrogenase and NAD. In Methods of Enzymatic Analysis 4. H. U. Bergmeyer, Ed., Academic Press, New York, NY, 1974, pp 1704-1708. ii. Farese, G., and Mager, M., Protein-free filtrates obtained by membrane ultraflltration. Clin. Chem. 16, 280 (1970). 12. Benson, .J. R., High-speed, high-sensitivity single-column analysis of amino acids. Offprint of paper presented at 1972 Am. Chem. Soc. meetings, Boston, MA. 13. Shaw, L. S., London, .J. W., Fetterolf, D., and Garfinkel, D., ‘Glutamyltransferase: Kinetic properties and assay conditions when ‘y-glutamvl-4.nitroanilide and its 3-carboxy derivative are used as donor substrates. Clin. Chem. 23,79 (1977). 14. Szasz, F.. A kinetic photometric method for serum -y-glutamyltranspeptidase. (‘lin. Chem. l5, 124 (1969). 15. Tate, S. S., and Meister, A., Stimulation of the hydrolytic activity and decrease of the transpeptidase activity of -y-glutamyl transpeptidase by maleate; identity of a rat kidney maleate-stimulated glutaminase and -y-glutamyltranspeptidase. (‘tin. (‘him. Acta 71, 3329 (1974). 16. Shaw, L. M., Molecular properties of -glutamyltransferase. Chap. 6 in Evaluation of Liver Function: A Multifaceted Approach to (‘tin ical 1)iagnosis, L. Demers and L. M. Shaw, Eds., Urban and Schwarzenherg, Baltimore, MD, 1978. 17. Chasseaud, L. F., Conjugation with glutathione and mercapturic acid excretion. In Glut at hione: Metabolism and Function, I. M. Arias and W. B. ,Jakoby, Eds., Raven Press, New York, NY, 1976, pp 77114. 18. Hughey, R. P., Rankin, B. B., Elce, J. S., and Curthoys, N. P., Specificity of a particulate rate renal peptidase and its localization along with other enzymes of mercapturic acid synthesis. Arch. Riochem. Biophys. 186, 211 (1978). 19. Elwyn, D. H., Parikh, H. C., and Shoemaker, W. C., Amino acid movements between gut, liver and periphery in unanesthetized dogs. Am. J. Phv.siol. 215, 1260 (1968). 20. Bartoli, G. M., and Sies, H. Reduced and oxidized efflux from liver. FEES Lett. 86,89(1978). glutathione 21. Reed, I). .J., and Orrenius, S., The role of methionine in glutathione biosynthesis by isolated hepatocytes. Biochem. Riophys. Rex. Commun. 77, 1257 (1977). 22. Meister, A., Tate, S. S., and Ross, L. L., Membrane-bound -yglutamyl transpeptidase. In Enzymes of Biological Membranes, 3, A. N. Martonosi, Ed., Plenum Press, New York, NY, 1976, pp 315347. 27. Tanaka, M., A histochemical study on the activity ofy-glutamyltranspeptidase in liver disease. Acta. Pathol. Jpn. 24, 651, 23. Maack, T., Johnson, V., Tate, S. S., and Meister, A., Effects of amino acids on the functions of the isolated perfused rat kidney. Fed. (1974). 28. ,Jones, A. L., and Spring-Mills, E., The liver and gall bladder. In Histology, L. Weiss and R. 0. Greep, Eds., McGraw-Hill, New York, Proc. Fed. Am. Soc. Exp. Biol. 33, 305 (1974). 24. Meister, A., and Tate, S. S., Gluthione and related y-gluthmyl compounds: Biosynthesis and utilization. Annu. Rev. Biochem. 45, 559 (1976). 25. Wendel, A., and Floh#{233}, L., Permeability of the erythrocyte membrane to glutathione. J. Clin. Chem. Clin. Biochem. 8, 441 (1970). 26. Rutenberg, A. M., Kim, H., Fischbein, and ultrastructural tivity. J. Histochem. demonstration Cytochem. J. W., et al., Histochemical of y-glutamyltranspeptidase 17, 517 (1969). ac- NY, 1977, pp 730-732. 29. Boyland, E.,Mercapturic and Sims, P., Theand metabolism of phenanthrene rabbits and rats: acids related compounds. Riochem.in ,.i. 84, 564 (1962). 30. Boyland, E., and Sims, P., The metabolism of pyrene in rats and rabbits. Riochem. J. 90, 391 (1964). 31. Boyland, E., and Sims, P., The metabolism Biochem. J. 91,493 (1964). of benz(a)anthracene. CLINICAL CHEMISTRY, Vol. 25. No. 1, 1979 79