Variations in gamma-glutamyl transpeptidase glycosylation and kinetic parameters in cultured liver cells.

In rat hepatocytes; the tumorigenic rat liver cell line ARL-16; and the human hepatoma line, Hep G2, 50% of the total gamma-glutamyl transpeptidase (GGT) activity was bound by a Concanavalin-A Sepharose 4B column, calling for alpha-methylmannoside elution (Peak I). Non-binding GGT was distributed between a rapidly eluting Peak II and a slightly retained Peak III. The Km for gamma-glutamyl-p-nitroanalide for either hydrolysis or transpeptidation, or glutathione (GSH) transpeptidation did not vary with peak number or cell type. The GSH hydrolysis Km was essentially constant in Peak I and II GGT. Peak III GGT exhibited a lower Km for GSH hydrolysis with Hep G2 Peak III GGT being the lowest. Peak III GGT increased to 50% of the GGT activity in Hep G2 cells cultured with GSH as the sole cysteine source.     

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.     

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.     

gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties.

gamma-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity. The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B). The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000. The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000. Fraction A catalyzes the hydrolysis and transpeptidation of all gamma-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors. The apparent Km values for glutathione and gamma-glutamyl-p-nitroanilide as gamma-glutamyl donors in the transpeptidation reaction were both 35 microM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively. The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT. The temperature stability of the purified GGT supports our hypothesis that E. coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.     

Activity determination, kinetic analyses and isoenzyme identification of gamma glutamyltransferase in human neutrophils

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) which hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH concentration. GGT is a membrane enzyme present in leukocytes and platelets. Its activity has also been observed in human neutrophils. In this study, GGT was purified from Triton X-100 solubilized neutrophils and its kinetic parameters were determined. For kinetic analyses of transpeptidation reaction, gamma-glutamyl p-nitroanilide was used as the substrate and glycylglycine as the acceptor. Apparent K(m) values were determined as 1.8 mM for gamma-glutamyl p-nitroanilide and 16.9 mM for glycylglycine. The optimum pH of GGT activity was 8.2 and the optimum temperature was 37 degrees C. It had thermal stability with 58 % relative activity at 56 degrees C for 30 min incubation. L-serine, in the presence of borate, was detected as the competitive inhibitor. Bromcresol green inhibited neutrophil GGT activity as a noncompetitive inhibitor. The neutrophils seem to contain only the isoenzyme that is present in platelets. We characterized the kinetic properties and compared the type of the isoenzyme of neutrophil GGT with platelet GGT via polyacrylamide gel electrophoresis (PAGE) under a standard set of conditions.     

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

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.     

Design, synthesis, and evaluation of gamma-phosphono diester analogues of glutamate as highly potent inhibitors and active site probes of gamma-glutamyl transpeptidase

Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the transfer of the gamma-glutamyl group of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and plays a central role in glutathione metabolism. GGT is involved in a number of biological events, such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics and reactive oxygen species through glutathione metabolism, and is also implicated in physiological disorders, such as Parkinson's disease, neurodegerative disease, diabetes, and cardiovascular diseases. In this study, we designed, synthesized, and evaluated a series of gamma-phosphono diester analogues of glutamate as transition-state mimic inhibitors of GGT. The electrophilic phosphonate diesters served as highly potent mechanism-based inhibitors that caused the time-dependent and irreversible inhibition of both the E. coli and human enzymes, probably by phosphonylating the catalytic Thr residue of the enzyme. In particular, one of the inhibitors exhibited more than 6000 times higher activity toward human GGT than acivicin, a classical but nonselective inhibitor of GGT. The dependence of the inactivation rate on the leaving group ability of the phosphonates (Br?nsted plot) revealed that the phosphonylation of the catalytic Thr residue proceeded via a dissociative transition-state with substantial bond cleavage between the phosphorus and the leaving group for both E. coli and human GGTs. The binding site of GGT for the Cys-Gly moiety of glutathione or for the acceptor molecules was probed by the phosphonate diesters to reveal a significant difference in the mechanism of substrate recognition between E. coli and human GGT. Thus, in the human enzyme, a specific residue in the Cys-Gly binding site played a critical role in recognizing the Cys-Gly moiety or the acceptor molecules by interacting with the C-terminal carboxy group, whereas the Cys side chain and the Cys-Gly amide bond were not recognized significantly. In contrast, the E. coli enzyme was a nonselective enzyme that accommodated substrates without specifically recognizing the C-terminal carboxy group of the Cys-Gly moiety of gamma-glutamyl compounds or the acceptor molecules. The phosphonate diester-based GGT inhibitors shown here should serve as a blue print for the future design of highly selective GGT inhibitors for use as drug leads and biological probes that gain insight into the hitherto undefined physiological roles of GGT and the relationships between GGT and a variety of diseases.     

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.     

Expression and activity of gamma-glutamyl transpeptidase in the rat epididymis.

Following Northern analysis, GGT mRNA was found predominantly within the caput epididymides and kidney. The size of mRNAs for kidney, caput, corpus, and ductus deferens were 2.2, 2.3, 2.2, and 2.3 kb, respectively, whereas cauda showed a doublet of 2.2 and 2.3 kb. GGT transpeptidation and hydrolytic activity within epididymal luminal fluids collected by micropuncture showed caput = corpus greater than cauda and corpus greater than caput greater than cauda, respectively. Caput luminal GGT transpeptidation activity was significantly inhibited by serine-borate and was optimal at pH 8.0. The calculated Km and Vmax values for hydrolysis of GSH by caput luminal GGT were 0.06 microM and 2.19 nmoles/min/microliters luminal fluid at pH 8.5 compared to 0.49 microM and 0.49 nmoles/min/microliters luminal fluid, respectively, at the physiological pH 6.5 of caput fluid. These studies would suggest that the epididymis can control the activity of luminal GGT by pH. Lower Km (0.12 microM) and higher Vmax (1.13 nmoles/min/microliters luminal fluid) values were also calculated when GSSG was used compared to GSH. Results from Triton X-114 partitioning experiments suggest that luminal GGT probably exists in both membrane bound and nonmembrane bound forms. Western blot analysis of proteins within epididymal luminal fluids revealed both subunits of GGT in all epididymal regions studied. However, two lower molecular bands, approximately 22 kDa and 21 kDa, were also observed in cauda fluid. It is suggested that as GGT is transported along the epididymal duct it undergoes degradation, which accounts for its loss of activity in the distal epididymal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of gamma-glutamyltranspeptidase from Proteus mirabilis.

gamma-Glutamyltranspeptidase was purified ca. 15,200-fold from cell-free extracts of Proteus mirabilis to electrophoretic homogeneity and then crystallized. The enzyme has an estimated molecular weight of 80,000 and consists of two different subunits with molecular weights of ca. 47,000 and 28,000. The purified enzyme catalyzed hydrolysis and transpeptidation of various gamma-glutamyl compounds, including the oxidized and reduced forms of glutathione, gamma-glutamyl compounds of L-phenylalanine, L-tyrosine, L-histidine, L-alpha-aminobutyrate, L-leucine, and p-nitroaniline. Glycylglycine, L-phenylalanine, L-methionine, L-histidine, L-tryptophan, and L-isoleucine were good acceptors of the gamma-glutamyl moiety in the transpeptidation reaction. Km values for gamma-glutamyl compounds were on the order of 10(-4) to 10(-5) M, and those for acceptor peptides and amino acids were on the order of 10(-2) to 10(-3) M. The enzyme was inhibited by L-serine plus borate and 6-diazo-5-oxo-L-norleucine, which are inhibitors of gamma-glutamyltranspeptidases isolated from mammals. Various amino acids alone were found to inhibit the transpeptidation competitively with a gamma-glutamyl donor. Kinetic analysis suggested that the reaction sequence of substrate binding and product release proceeds according to a ping pong bi bi mechanism.     

gamma-Glutamyl transpeptidase GGT4 initiates vacuolar degradation of glutathione S-conjugates in Arabidopsis

The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.     

Phosphonate-based irreversible inhibitors of human γ-glutamyl transpeptidase (GGT). GGsTop is a non-toxic and highly selective inhibitor with critical electrostatic interaction with an active-site residue Lys562 for enhanced inhibitory activity

γ-Glutamyl transpeptidase (GGT, EC 2.3.2.2) that catalyzes the hydrolysis and transpeptidation of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione metabolism and is an attractive pharmaceutical target. We report here the evaluation of a phosphonate-based irreversible inhibitor, 2-amino-4-{[3-(carboxymethyl)phenoxy](methoyl)phosphoryl}butanoic acid (GGsTop) and its analogues as a mechanism-based inhibitor of human GGT. GGsTop is a stable compound, but inactivated the human enzyme significantly faster than the other phosphonates, and importantly did not inhibit a glutamine amidotransferase. The structure–activity relationships, X-ray crystallography with Escherichia coli GGT, sequence alignment and site-directed mutagenesis of human GGT revealed a critical electrostatic interaction between the terminal carboxylate of GGsTop and the active-site residue Lys562 of human GGT for potent inhibition. GGsTop showed no cytotoxicity toward human fibroblasts and hepatic stellate cells up to 1 mM. GGsTop serves as a non-toxic, selective and highly potent irreversible GGT inhibitor that could be used for various in vivo as well as in vitro biochemical studies.     

Localization of gamma-glutamyl transferase activity and protein in Zea mays organs and tissues

Gamma-glutamyl transferase/transpeptidase (GGT, (5-l-glutamyl)-peptide:amino-acid 5-glutamyltransferase; EC 2.3.2.2.) is an ectoenzyme promoting the cleavage of the gamma-glutamyl moiety of glutathione (GSH) and gamma-glutamyl related compounds. In this work, we describe the localization of GGT by enzymehistochemical and immunohistochemical analysis in maize plants. Our results show that the tissue spatial distribution of GGT activity closely correlates with the localization of the GGT protein. We also demonstrate that GGT activity and protein are unevenly distributed in tissues, being higher in the epidermis and stomata, parenchyma of conductive elements and root meristem. These results can contribute to our understanding of GGT function and regulation as well as its role in glutathione metabolism. To date, these are largely unknown in plants.     

Localization of members of the gamma-glutamyl transpeptidase family identifies sites of glutathione and glutathione S-conjugate hydrolysis

gamma-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (gamma-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the gamma-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:beta-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with gamma-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Kinetic characterization and identification of the acylation and glycosylation sites of recombinant human gamma-glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.     

Induction of gamma-glutamyl transferase by dexamethasone in cultured fetal rat hepatocytes

Hepatocytes isolated as a relatively pure population from normal fetal rats were maintained in primary monolayer culture for 4-10 days. Hepatocytes exhibited a small increase in basal gamma-glutamyl transferase (GGT) activity over time. Exposure to dexamethasone (10(-6) mol/l) elicited a rise in GGT activity after a lag of 24 h. The presence of the steroid was necessary to maintain induction, and its removal resulted in reversal of induction. The maximal response was 2- to 3-fold, 72 h after exposure to the steroid. After this maximal response, a gradual decay in enzyme activity occurred, despite the continuous presence of the hormone. Actinomycin D or cycloheximide given prior to/or simultaneously with the steroid prevented the induction, thus suggesting that both RNA and protein biosynthesis are necessary for induction to occur.     

Kinetic studies of sheep kidney gamma-glutamyl transpeptidase.

The kinetics of sheep kidney gamma-glutamyl transpeptidase was studied using a novel substrate L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate. When the substrate was incubated with the enzyme in the presence of an amino acid or peptide acceptor, the corresponding L-alpha-methyl-gamma-glutamyl derivatives of the acceptors were formed. In the absence of acceptor only hydrolysis occurred, and no transpeptidation products were detected. The presence of the methyl group on the alpha-carbon apparently prevents enzymatic transfer of the L-alpha-methyl-gamma-glutamyl residue to the amino group of the substrate itself (autotranspeptidation). When the enzyme was incubated with conventional substrates, such as glutathione or gamma-glutamyl-p-nitroanilide and an amino acid acceptor, hydrolysis, autotranspeptidation, and transpeptidation to the acceptor occurred concurrently. Initial velocity measurements in which the concentration of L-alpha-methyl-gamma-glutamyl-L-alpha-aminobutyrate was varied at several fixed acceptor concentrations, and either the release of alpha-aminobutyrate or the formation of the transpeptidation products was determined, yielded results which are consistent with a ping-pong mechanism modified by a hydrolytic shunt. A scheme of such a mechanism is presented. This mechanism predicts the formation of an alpha-methyl-gamma-glutamyl-enzyme intermediate, which can react with an amino acid to form the transpeptidation product; or in the absence of, or in the presence of low concentrations of amino acids, can react with water to form the hydrolytic products. Kinetic derivations for the reaction of the enzyme with the conventional substrate gamma-glutamyl-p-nitroanilide predict either linear or nonlinear double-reciprocal plots, depending on the prevalence of the hydrolytic, autotranspeptidation, or transpeptidation reactions. The results of kinetic experiments confirmed these predictions.     

Studies on serum gamma-glutamyl transpeptidase in primary idiopathic hypothyroidism patients.

Variations in the levels of serum gamma-glutamyl transpeptidase (GGT) were measured in control subjects and in 39 adult primary idiopathic hypothyroidism (PIH) patients. The serum GGT activity was low in PIH patients compared to that of control subjects. A more significant correlation was found between serum GGT and T3 (r = 0.766) but not with T4 (r = 0.476). The comparison of serum GGT with TSH has revealed that those two parameters are not parallel with each other (r = -0.454). No significant correlation between serum GGT activity, age, and sex in PIH patients and control subjects was observed. The present available data indicate that measurement of serum GGT might be useful as a marker index in PIH patients.