Human kidney gamma-glutamyl transpeptidase. Catalytic properties, subunit structure, and localization of the gamma-glutamyl binding site on the light subunit.

Human kidney gamma-glutamyl transpeptidase has been purified by a procedure involving Lubrol extraction, acetone precipitation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-150. The final preparation is a glycoprotein (molecular weight of approximately 84,000) composed of two nonidentical glycopeptides (molecular weights of 62,000 and 22,000). The isozymic forms, separable by isoelectric focusing, have different contents of sialic acid. The utilization of L-glutamine (which is both a gamma-glutamyl donor and acceptor) is stimulated about 3-fold by maleate in contrast to 10-fold stimulation of glutamine utilization by the rat kidney enzyme. The gamma-glutamyl analogs, 6-diazo-5-oxo-L-norleucine (DON) and L-azaserine inactivate the human kidney enzyme with respect to its transpeptidase and hydrolase activities. Inactivation is prevented by gamma-glutamyl substrates (but not by acceptor substrates) and is accelerated by maleate. [14C]DON reacts covalently and stoichiometrically at the gamma-glutamyl site, which was localized to the light subunit of the enzyme. The light subunit of human transpeptidase closely resembles that of rat kidney enzyme in having the gamma-glutamyl binding site, and similar molecular weight and amino acid composition. The heavy subunits of the two enzymes are markedly different in both molecular weight and amino acid content; this may account for differences observed in acceptor amino acid specificity and in the magnitude of the maleate effect.     

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.     

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.     

Studies of human kidney gamma-glutamyl transpeptidase. Purification and structural, kinetic and immunological properties.

gamma-Glutamyl transpeptidase, present in various mammalian tissues, transfers the gamma-glutamyl moiety of glutathione to a variety of acceptor amino acids and peptides. This enzyme has been purified from human kidney cortex about 740-fold to a specific activity of 200 units/mg of protein. The purification steps involved incubation of the homogenate at 37 degrees followed by centrifugation and extraction of the sediment with 0.1 M Tris-HCl buffer, pH 8.0, containing 1% sodium deoxycholate; batchwise absorption on DEAE-cellulose; DEAE-cellulose (DE52) column chromatography; Sephadex G-200 gel filtration; and affinity chromatography using concanavalin A insolubilized on beaded Agarose. Detergents were used throughout the purification of the enzyme. The purified enzyme separated into three protein bands, all of which had enzyme activity, on polyacrylamide disc electrophoresis in the presence of Triton X-100. The enzyme has an apparent molecular weight of about 90,000 as shown by Sephadex G-200 gel filtration, and appears to be a tetramer with subunits of molecular weights of about 21,000. The Km for gamma-glutamyl transpeptidase using the artificial substrate, gamma-glutamyl-p-nitroanilide, with glycylglycine as the acceptor amino acid was found to be about 0.8 mM. The optimum pH for the enzyme activity is 8.2 and the isoelectric point is 4.5. Both GSH and GSSG competitively inhibited the activity of gamma-glutamyl transpeptidase when gamma-glutamyl-p-nitroanilide was used as the substrate. Treatment of the purified enzyme with papain has no effect on the enzyme activity or mobility on polyacrylamide disc electrophoresis. The purified gamma-glutamyl transpeptidase had no phosphate-independent glutaminase activity. The ratio of gamma-glutamyl transpeptidase to phosphate-independent glutaminase changed significantly through the initial steps of gamma-glutamyl transpeptidase purification. These studies indicate that the transpeptidase and phosphate-independent glutaminase activities are not exhibited by the same protein in human kidney.     

Renal glutamine utilization: glycylglycine stimulation of gamma-glutamyltransferase

Glycylglycine stimulation of renal glutamine utilization was studied on the homogenate, subcellular and purified enzyme level. The results clearly establish the existence of two glutamine utilizing pathways, the mitochondrial dependent L-glutamine amidohydrolase (PDG) and a second, extramitochondrial pathway. In contrast to the mitochondrial pathway which produces stoichiometric amounts of ammonia and glutamate, this second pathway hydrolyzes glutamine to produce ammonia and transfers the gamma-glutamyl moiety, producing gamma-glutamyl peptides. In the crude systems, containing cyclotransferase, the gamma-glutamyl moiety appears mainly as 5-oxoproline; however, in the enzyme preparation, purified 112-fold, gamma-glutamyl peptides (transpeptidation) and a small amount of glutamate (hydrolysis) appear. D-Glutamine was also hydrolyzed, in contrast to the stereospecific PDG, but at less than one-half the rate of the L-isomer. The molecular weight of this extramitochondrial D- and L-glutamine utilizing enzyme was estimated by gel filtration on a Sephadex G-200 column and found to be approximately 70 000. Based on product formation, molecular weight estimation and copurification with the activity responsible for p-nitroanilide release from gamma-glutamyl-p-nitroanilide, we conclude that this reaction is catalyzed by gamma-glutamyltranspeptidase. Glycylglycine stimulated this enzyme to produce more ammonia while decreasing the appearance of glutamate; in contrast, the mitochondrial glutaminase was unaffected by glycylglycine. This extramitochondrial glutamine utilizing pathway can make a significant contribution to in vivo renal ammoniagenesis.     

Evidence that transpeptidation is a significant function of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (purified from rat kidney) was incubated with glutathione and a mixture of amino acids that closely approximates the amino acid composition of blood plasma, and the relative extents of transpeptidation and hydrolysis were determined by quantitative measurement of the products formed (glutamate, cysteinylglycine, gamma-glutamyl amino acids). At pH 7.4, in the presence of 50 microM glutathione and the amino acid mixture, about 50% of the glutathione that was utilized participated in transpeptidation. Studies in which the formation of individual gamma-glutamyl amino acids was determined in the presence of glutathione and the amino acid mixture showed that L-cystine and L-glutamine are the most active amino acid acceptors, and that other neutral amino acids also participate in transpeptidation to a significant extent. These in vitro experiments are consistent with a number of other findings which indicate that transpeptidation is a significant physiological function of gamma-glutamyl transpeptidase.     

Theanine,gamma-glutamylethylamide,is metabolized by renal phosphate-independent glutaminase

The distribution of theanine-degrading activity in Wistar rats was examined and this activity was detected only in the kidney. Judging from polyacrylamide gel electrophoresis, theanine-degrading enzyme from rat kidney was purified almost to homogeneity. Theanine-degrading activity was co-purified with glutaminase activity, and the relative activity for theanine was about 85% of that for L-glutamine throughout purification. Substrate specificity of purified enzyme preparation coincided well with the data of phosphate-independent glutaminase [EC 3.5.1.2], which had been previously reported. It was very curious that gamma-glutamyl methyl and ethyl esters were more effectively hydrolyzed than theanine and L-glutamine, in view of relative activity and K(m) value. It was suggested that gamma-glutamyl moiety in theanine molecule was transferred to form gamma-glutamylglycylglycine with relative ease in the presence of glycylglycine. On the other hand, purified phosphate-dependent glutaminase did not show theanine-degrading activity at all. Thus, it was concluded that theanine was hydrolyzed by phosphate-independent glutaminase in kidney and suggested that, as for the metabolic fate of theanine, its glutamyl moiety might be transferred by means of gamma-glutamyl transpeptidase reaction to other peptides in vivo.     

Uptake and metabolism of glutamine in cultured kidney cells

The metabolism of glutamine was investigated in cultured rat kidney cells. Glutamine utilization and product formation were followed as a function of time at either 10 microM or 1 mM initial glutamine concentration. At 1 mM glutamine, glutamate and gamma-glutamylglutamate were the major products formed at the end of a 5-min incubation period; glutamate accounted for 46% while gamma-glutamylglutamate accounted for 33% of the glutamine utilized. With time, glutamate continued to accumulate while gamma-glutamyl peptide formation leveled off. The role of gamma-glutamyl transpeptidase was assessed by using hippurate, a physiological activator of gamma-glutamyl transpeptidase and acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, an inhibitor of gamma-glutamyl transpeptidase. Hippurate, 4 mM, increased the utilization of glutamine and the formation of glutamate, gamma-glutamyl peptides and ammonia. Exposure of cells to acivicin resulted in 98% inhibition of gamma-glutamyl transpeptidase without effecting phosphate-dependent glutaminase activity. Acivicin inhibition resulted in a decreased utilization of glutamine and product formation as compared to control; 5-oxoproline appearance fell 70%. The fractional distribution of glutamine carbon and nitrogen into its metabolic products in control, hippurate and acivicin-treated cells showed no change at the end of 60 min. The data provide evidence that gamma-glutamyl transpeptidase utilizes glutamine and forms gamma-glutamyl peptides in cultured kidney cells.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

Gamma-glutamyl transpeptidase of rat seminal vesicles; effect of orchidectomy and hormone administration on the transpeptidase in relation to its possible role in secretory activity.

γ-Glutamyl transpeptidase was prepared from rat seminal vesicles by two methods and was found to be similar to rat kidney γ-glutamyl transpeptidase with respect to substrate specificity, stimulation of “glutaminase” activity by maleate, and apparent molecular weight. Histochemical studies demonstrated that γ-glutamyl transpeptidase is concentrated in the secretory epithelium of the seminal vesicle. Like the epithelium itself, the enzyme responds to the presence or absence of testosterone. The content and specific activities of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase in rat seminal vesicles are low in orchidectomized animals, an effect which is reversed by administration of testosterone but accentuated by estradiol administration. These enzymes may be involved in the secretory functions of the seminal vesicles.     

Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.

A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.     

Affinity labeling of rat-kidney gamma-glutamyl transpeptidase.

The reaction of gamma-glutamyl transpeptidase from rat kidney with a glutamine analog, 6-diazo-5-oxo-L-norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half-maximum rate of inactivation was 6 mMat pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active-site-directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6-diazo-5-oxo-L-norleucine. Inactivation of the enzyme with 6-diazo-5-oxo-L-[6=14C]norleucine as well as with 6-diazo-5-oxo-L[1,2,3,4,5-14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label/248000 g enzyme protein. A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C-labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

Purification and cloning of a gamma-glutamyl transpeptidase from onion (Allium cepa)

Gamma-glutamyl transpeptidase (E.C. 2.3.2.2; GGT) catalyses hydrolysis of gamma-glutamyl linkages in gamma-glutamyl peptides and transfer of the gamma-glutamyl group to amino acids and peptides. Although plant gamma-glutamyl peptide metabolism is important in biosynthesis and metabolism of secondary products and xenobiotics, plant GGTs are poorly characterised. We purified a membrane-associated GGT from sprouting onion bulbs that catalyses transpeptidation of methionine by the synthetic substrate gamma-glutamyl-p-nitroanilide (GGPNA) and obtained N-terminal peptide sequence. We also cloned the full-length coding region of an onion GGT by homology with the Arabidopsis enzyme and confirmed that this shared the same N-terminal sequence. Enzyme kinetic studies show that the enzyme has high affinity for glutathione and glutathione conjugates, and that affinity for S-substituted glutathione analogs decreases as the substituted chain length increases. The major onion gamma-glutamyl peptide, gamma-glutamyl trans-S-1-propenyl cysteine sulfoxide (GGPrCSO) exhibited uncompetitive inhibition of transpeptidation by GGPNA. This suggests that GGPrCSO is a poor glutamyl donor and therefore unlikely to be an in vivo substrate for peptidase activity by this enzyme.     

Modification of the acceptor binding site of gamma-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem.254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem.255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(αS, 5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [¹⁴C]p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M_r 68,000, shown to result from crosslinking of the two subunits of transpeptidase (M_r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.     

Ammonia production by the small intestine of the rat.

1. Slices of duodenum and jejunum produce ammonia from glutamine in vitro. 2. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. 3. Gut contains glutaminase 1 as well as gamma-glutamyl transpeptidase. 4. These enzymes do not show any increase during starvation.     

Single-chain precursor of renal gamma-glutamyl transpeptidase

The two subunits of gamma-glutamyl transpeptidase (EC 2.3.2.2) are derived from a single-chain glycosylated precursor. A small fraction of the propeptide survives proteolytic processing in the rat kidney and has been purified by an immunoaffinity technique. The propeptide contains determinants for both the subunits and its amino acid composition resembles that of the dimeric enzyme. However, the propeptide exhibits less than 2% of the transpeptidase activity shown by the dimeric enzyme.     

Gamma-glutamyl-glutathione. Natural occurrence and enzymology

The natural occurrence of gamma-glutamyl-glutathione (gamma-glutamyl-gamma-glutamylcysteinylglycine) in bile was established by analytical and chromatographic studies on the isolated and chemically synthesized materials. Evidence that it is formed in kidney was obtained. The origin of gamma-glutamyl-glutathione was explored through studies on the interaction of glutathione with gamma-glutamyl transpeptidase. When purified gamma-glutamyl transpeptidase was incubated with various concentrations (4 microM-50 mM) of glutathione, the initial rates of formation of gamma-glutamyl-glutathione were substantial at all concentrations of glutathione studied and were greater than the rates of formation of glutamate at physiological levels of glutathione (1-10 mM). The findings indicate that gamma-glutamyl transpeptidase catalyzes transpeptidation in vivo. That gamma-glutamyl-glutathione is formed in vivo and that it is a significant product of the reaction between glutathione and gamma-glutamyl transpeptidase under physiological conditions suggest that this polyanionic tetrapeptide may have a physiological role. gamma-Glutamyl-glutathione is not a substrate of glutathione reductase or of glutathione S-transferase, but it is a substrate of gamma-glutamyl-cyclotransferase. That gamma-glutamyl-glutathione has an additional negative charge as compared to glutathione suggests that it may be more effective than glutathione in forming complexes with certain metal ions and other cations.     

Conversion of glutathione to glutathione disulfide, a catalytic function of gamma-glutamyl transpeptidase.

A purification procedure, based on that previously used for rat kidney gamma-glutamyl transpeptidase, was used for the purification of glutathione oxidase (which converts glutathione to gluthathione disulfide). The two activities co-purified, the ratio of the activities remaining constant through all steps of the isolation procedure. The purified enzyme was separable into 12 isozymic species by isoelectric focusing. All 12 isozymes exhibited a constant ratio of transpeptidase to glutathione oxidase activities, strongly supporting the conclusion that conversion of glutathione to glutathione disulfide is a catalytic function of gamma-glutamyl transpeptidase. Modulation of oxidase activity by inhibitors and acceptor substrates of transpeptidase is discussed in relation to the possible glutathione binding sites involved in gamma-glutamyl transfer and oxidase activities of the enzyme.