A spectrophotometric method for the determination of gamma-glutamyl cyclotransferase with alanine dehydrogenase in the presence of anthglutin

gamma-Glutamyl cyclotransferase activity is assayed in tissues by a colorimetric method using gamma-glutamyl alanine as a substrate coupled with alanine dehydrogenase from B. sphericus, to measure the formation of NADH. In order to avoid interference by the reaction catalyzed by gamma-glutamyl transpeptidase, anthglutin, a specific inhibitor of the transpeptidase was included in the reaction mixture. The Km value of rat kidney gamma-glutamyl cyclotransferase with respect to gamma-glutamyl alanine appeared to be the same when determined by either the colorimetric or the radiometric method. This assay presents a reliable alternative to the use of radiolabeled substrate and is used for the assay of gamma-glutamyl cyclotransferase in a variety of physiological and experimental samples.     

Metabolism of gamma-glutamyl amino acids and peptides in mouse liver and kidney in vivo.

The metabolism in vivo of gamma-glutamyl amino acids and peptides was studied in the mouse after administration of loading doses of L-gamma-glutamyl-2-aminobutyrate and several other gamma-glutamyl compounds, including glutathione. A great and rapid accumulation of glutamate, glutamine, aspartate and pyrrolidone carboxylate was observed in the kidney. Similarly, after administration of a tracer dose of L-gamma-[14C]glutamyl-L-2-aminobutyrate a rapid incorporation of label into kidney glutamate, glutamine and aspartate was found. These results suggest that both the hydrolytic and gamma-glutamyl transfer reactions catalyzed by gamma-glutamyl transpeptidase are active in the renal handling of gamma-glutamyl compounds. Indirect evidence was obtained that L-gamma-glutamyl-2-aminobutyrate is partially taken up by the kidney cell in an intact form. In contrast to the kidney, administration of several gamma-glutamyl derivatives did not cause an increase in liver glutamate, glutamine and pyrrolidone carboxylate. After administration of L-gamma-glutamyl-2-aminobutyrate only a slight increase in liver aspartate and pyrrolidone carboxylate was observed. Experiments with L-gamma-[14C]glutamyl-L-2-aminobutyrate suggest that this derivative is largely first degraded to its component amino acids (probably in the kidney) before entering into the metabolism of the liver cell. gamma-Glutamyl transpeptidase may function in the metabolism and transport of glutathione and other gamma-glutamyl compounds in a manner analogous to the function of dipeptidases and disaccharidases in the metabolism and transport of dipeptides and disaccharides respectively.     

Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids.

GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.     

Synthesis of ophthalmic acid in liver and kidney in vivo.

The synthesis of ophthalmic acid, an analogue of glutathione, was studied in vivo in mouse liver and kidney after administration of either L-alpha-aminobutyrate or L-gamma-glutamyl-L-alpha-aminobutyrate as precursor. L-alpha-aminobutyrate accumulated to a much greater extent, and induced a much greater synthesis of ophthalmic acid in the liver than in the kidney. In contrast, L-gamma-glutamyl-L-alpha-aminobutyrate initiated a large and more rapid synthesis of ophthalmic acid in the kidney than in the liver. Experiments with L-gamma-[G(-14)C]glutamyl-L-alpha-aminobutyrate showed that, although part of the dipeptide is degraded to its constituent amino acids, a significant proportion is directly incorporated into kidney ophthalmic acid. In contrast L-gamma-glutamyl-L-alpha-aminobutyrate serves poorly as a direct precursor of liver ophthalmic acid. The present results show that kidney gamma-glutamyl tripeptide synthesis can proceed directly from an exogenous gamma-glutamyl dipeptide precursor.     

gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney

Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35S after incubation of the slices in media containing gamma-glutamyl methionine [35S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices.     

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.     

Specificity of gamma-glutamyl cyclotransferase.

Using the phthaloyl method, 18 gamma-L-glutamyl peptides labelled with 14-C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified gamma-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that gamma-glutamyl cyclotransferase acts only on the L-form of gamma-glutamyl substrates; the enzyme could, therefore, be used for preparation of gamma-D-glutamyl peptides from their racemic mixtures. The specificity of gamma-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as gamma-L-glutamyl-NH--CHR--COOH. The amino acid linked to the gamma-glutamyl group must be in the L configuration.     

The identification and structural characterization of C7orf24 as gamma-glutamyl cyclotransferase. An essential enzyme in the gamma-glutamyl cycle

The hypothetical protein C7orf24 has been implicated as a cancer marker with a potential role in cell proliferation. We have identified C7orf24 as gamma-glutamyl cyclotransferase (GGCT) that catalyzes the formation of 5-oxoproline (pyroglutamic acid) from gamma-glutamyl dipeptides and potentially plays a significant role in glutathione homeostasis. In the present study we have identified the first cDNA clones encoding a gamma-glutamyl cyclotransferase. The GGCT gene is located on chromosome 7p14-15 and consists of four exons that span 8 kb. The primary sequence is 188 amino acids in length and is unlike any protein of known function. We crystallized functional recombinant gamma-glutamyl cyclotransferase and determined its structure at 1.7 A resolution. The enzyme is a dimer of 20,994-Da subunits. The topology of GGCT is unrelated to other enzymes associated with cyclotransferase-like activity. The fold was originally classified as "BtrG-like," a small family that only includes structures of hypothetical proteins from Mus musculus, Escherichia coli, Pyrococcus horikoshii, and Arabidopsis thaliana. Since this is the first member of this family with a defined function, we propose to refer to this structure as the gamma-glutamyl cyclotransferase fold. We have identified a potential active site pocket that contains a highly conserved glutamic acid (Glu(98)) and propose that it acts as a general acid/base in the reaction mechanism. Mutation of Glu(98) to Ala or Gln completely inactivates the enzyme without altering the overall fold.     

alpha-Aminomethylglutarate, a beta-amino analog of glutamate that interacts with glutamine synthetase and the enzymes that catalyze glutathione synthesis.

The glutamate analog, alpha-aminomethylglutaric acid, was synthetized by Michael addition of ammonia to 2-methylene glutaronitrile followed by hydrolysis of the intermediate alpha-aminomethylglutaryl nitrile; the analog cyclizes readily on heating to 2-piperidone-5-carboxylic acid. Sheep brain glutamine synthetase utilizes one isomer of DL-alpha-aminomethylglutarate at about 10% of the rate with L-glutamate. gamma-Glutamylcysteine synthetase uses both isomers of DL-alpha-aminomethylglutarate, preferentially acting on the same isomer used by glutamine synthetase. gamma-(alpha-Aminomethyl)glutaryl-alpha-aminobutyrate, prepared enzymatically with gamma-glutamylcysteine synthetase, was found to be a substrate and an inhibitor of glutathione synthetase. alpha-Aminomethylglutarate does not inhibit gamma-glutamyl cyclotransferase and gamma-glutamyl transpeptidase appreciably. When alpha-aminomethylglutarate was administered to mice, there were substantial decreases in the levels of glutamine, glutathione, glutamate, and glycine in the kidney, and of glutamine and glutamate in the liver, indicating that this glutamate analog is effective as an inhibitor of glutamine and glutathione synthesis in vivo, and suggesting that it may also inhibit other enzymes.     

ENZYMES OF THE γ-GLUTAMYL CYCLE IN THE CHOROID PLEXUS AND BRAIN

—The presence of enzymes of the γ-glutamyl cycle in the bovine and rabbit brain and choroid plexus is described. The activities of γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase and γ-glutamyl-cysteine synthetase in the choroid plexus were found to be higher than in the brain. The activity of γ-glutamyl transpeptidase in the choroid plexus was many times higher than the activity of the other enzymes. Brain and choroid plexus γ-glutamyl transpeptidase were activated by Na⁺ and K⁺. Both brain and choroid plexus showed only a very limited capacity to metabolize [¹⁴C]5-oxoproline to ¹⁴CO₂.     

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.     

Extracellular metabolism of glutathione accounts for its disappearance from the basolateral circulation of the kidney

Glutathione labeled in each of its amino acid residues, the corresponding free amino acids, and gamma-glutamyl-amino acids were used to evaluate their renal basolateral transport and metabolism at physiological levels of glutathione. Recovery of label in the venous outflow was compared to that of co-administered inulin after a single-pass in vivo infusion of rat kidney. Metabolites of glutathione and of its constituent amino acids were determined. No net basolateral transport of glutathione was detected; instead there was extensive breakdown of glutathione by the actions of basolateral gamma-glutamyl transpeptidase and dipeptidase. Glutamate and 5-oxoproline showed net basolateral uptake. Recoveries of 35S greater than those of inulin were found after perfusion of [35S]cysteine and [35S]glutathione suggesting rapid net tubular reabsorption of cyst(e)ine. Recovery of label from perfused [U-14C]glycine was equivalent to that of inulin consistent with little or no net flux. Co-administration of large amounts of unlabeled metabolites together with the labeled glutathiones led to label recoveries closer to those of inulin, consistent with competitive inhibition of labeled metabolite transport. Treatment of rats with an inhibitor of gamma-glutamyl transpeptidase decreased basolateral glutathione metabolism and thus indirectly decreased transport of labeled metabolites. No net basolateral transport of gamma-glutamyl-amino acids was detected. Significant amounts of label perfused as [Glu-U-14C]glutathione appeared in the gamma-glutamyl-amino acid fraction of the renal venous outflows, providing direct evidence that glutathione is used in vivo for the formation of gamma-glutamyl-amino acids.     

Purification and properties of gamma-glutamylcyclotransferase from human erythrocytes.

1. GAMMA-Glutamylcyclotransferase was purified 10000-fold from human erythrocytes. 2. The purification steps involved fractionation with (NH4)(2)SO(4) and chromatography on Sephadex G-75, DEAE-cellulose and hydroxyapatite. The purified enzyme was found to be homogeneous on density-gradient polyacrylamide-gel electrophoresis. 3. The maximum reaction rate was observed at pH9.0 and the apparent Km value for gamma-glutamyl-L-alanine was 2.2mM. 4. The molecular weight (25250) of the purified enzyme agreed well with the value (25500) in fresh haemolysates, indicating no apparent structural modification of the enzyme during purification. However, rapid processing of the blood through the initial (NH4)(2)SO(4) and Sephadex-chromatography steps was required to prevent formation of a high-molecular-weight aggregate with substantially lower specific activity. 5. gamma-Glutamylcyclotransferase catalyses the formation of 5-oxoproline from gamma-glutamyl dipeptides. The role of this enzyme in erythrocytes is of particular interest, because gamma-glutamyl-L-cysteine serves as a substrate for both gamma-glutamylcyclotransferase and glutathione synthetase. Thus the cyclotransferase could modulate glutathione synthesis.     

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.     

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.     

Differential effects of sodium butyrate and dimethylsulfoxide on gamma-glutamyl transpeptidase and alkaline phosphatase activities in MCF-7 breast cancer cells

Sodium butyrate and dimethylsulfoxide (DMSO), two known chemical inducers of cell differentiation, were examined on MCF-7 breast cancer cells. Both agents reduce the proliferative capacity of MCF-7 cells, as reflected by inhibition of colony formation in semisolid agar. Sodium butyrate is shown to enhance markedly the activity of two plasma membrane-bound enzymes, alkaline phosphatase and gamma-glutamyl transpeptidase. DMSO does not enhance the activity of these enzymes, but rather induces a small decrease in gamma-glutamyl transpeptidase activity. The present results show that although both agents inhibit cell proliferation, they have a distinct effect on phenotypic expression.     

Perturbation of hepatic glutathione level and glutathione-related enzyme activities by repeated administration of aminopyrine in rats

The influence of repeated administration of aminopyrine on the tissue glutathione level and related enzyme activities was investigated in rats. Reduced glutathione level in the liver was not changed after 5 days of treatment but a significant increase was seen after 15 days of aminopyrine treatment. Oxidized glutathione level was unaltered throughout the experiment. Repeated administration of aminopyrine for 5 days caused a marked increase in gamma-glutamyl transpeptidase activities in liver whole homogenates as well as in the nuclear fraction, but not in liver microsomes. These results suggest that gamma-glutamyl transpeptidase located in plasma membrane may be induced by repeated administration of aminopyrine for 5 days. The activities of cytosolic glutathione peroxidase, which modulates glutathione level, were also significantly increased by aminopyrine treatment. Under the same conditions, glutathione peroxidase activity with H2O2 as a substrate was unaltered, while a time-dependent increase in the activity was found when cumene hydroperoxide was used as a substrate, even after a single administration of aminopyrine. The intracellular cysteine level was increased accompanying the increased gamma-glutamyl transpeptidase activities. Therefore, induced gamma-glutamyl transpeptidase may play a role in the reclamation of extracellular oxidized glutathione.     

Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.     

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.     

Transport of amino acids in gamma-glutamyl transpeptidase-implanted human erythrocytes

gamma-Glutamyl transpeptidase purified from hog kidney cortex was implanted in the human erythrocyte membrane by incubation of erythrocytes at 37 degrees c with gamma-glutamyl transpeptidase-incorporated dipalmitoyl phosphatidylcholine vesicles. Membranes prepared from these implanted cells exhibited 4- to 5-fold increase in gamma-glutamyl transpeptidase activity. The association/insertion of gamma-glutamyl transpeptidase into erythrocyte membrane was further demonstrated by antibody to gamma-glutamyl transpeptidase. Implantation of gamma-glutamyl transpeptidase into erythrocyte membrane led to stimulation of uptake of glutamate and alanine, which are normally transported at a slow rate in human erythrocytes. The uptake of these amino acids in the implanted system was inhibited by inhibitors (serine-borate and azaserine) of transpeptidase activity as well as by antibody to gamma-glutamyl transpeptidase. These results in the implanted human erythrocytes demonstrate that gamma-glutamyl transpeptidase enzyme can mediate the translocation of amino acids and provide further evidence in support of its postulated role in the transport of amino acids in natural membranes.