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.     

Latent proteinase activity of gamma-glutamyl transpeptidase light subunit.

gamma-Glutamyl transpeptidase, which is composed of two unequal subunits, exhibits proteinase activity when treated with agents such as urea and sodium dodecyl sulfate. The heavy subunit is preferentially and rapidly degraded. The enzyme also degraded bovine serum albumin in the presence of urea; however, several other proteins and model proteinase substrates were not cleaved. Treatment of the enzyme with 6-diazo-5-oxo-L-norleucine, a gamma-glutamyl analog, results in parallel loss of transpeptidase and proteinase activities indicating that the site at which gamma-glutamylation of the enzyme occurs (presumably a hydroxyl group on the light subunit) is also involved in proteinase activity. The purified light subunit, but not the heavy subunit, exhibits proteinase activity even in the absence of urea. Results suggest that dissociation of the enzyme unmasks the proteinase activity of the light subunit involving the site at which gamma-glutamylation of the enzyme occurs, and that the heavy subunit may impose transpeptidase reaction specificity by contributing the binding domains for gamma-glutamyl substrates.     

gamma-glutamyl transpeptidase activity in human colostrum

gamma-Glutamyl transpeptidase (GGT) was determined in the colostrum and milk of 38 patients, 14 days postpartum. The results obtained were compared with the enzymatic activity in colostra of some animals. The human colostrum has been found to contain the highest enzymatic activity which decreases during the first 8 days and then remains stationary. The high GGT activity in the colostrum and milk and histochemical localization of the enzyme in the secretory epithelium of the milk gland indicate its participation in resorption processes of amino acids and peptides.     

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)     

Effects of bile acids and their glycine conjugates on gamma-glutamyl transpeptidase

Glycine and taurine conjugates of bile acids modulate gamma-glutamyl transpeptidase by interacting with the cysteinylglycine binding site (acceptor site) of the enzyme. These compounds stimulate hydrolysis of glutamine and S-methylglutathione and the rate of the inactivation of the enzyme by the gamma-glutamyl site-directed reagent, AT-125 (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Transpeptidation between S-methylglutathione and methionine was inhibited by these compounds. These effects resemble those caused by hippurate; the glycine derivatives of bile acids, however, exhibit a much greater affinity for transpeptidase than hippurate. Cholate, as shown previously for benzoate, also seems to bind to a portion of the acceptor site as indicated by its effects on S-methylglutathione utilization and AT-125-dependent inactivation of the enzyme. The Kd values for cholate and benzoate are, however, at least one order of magnitude larger than those for their respective glycine derivatives. The acceptor site-directed modulators increase the affinity of the enzyme for AT-125 and kinetic and binding studies show that binding of gamma-glutamyl site-directed reagents increases the affinity of the enzyme for cholate. These results thus indicate cooperative interactions between the gamma-glutamyl donor and acceptor binding domains of the transpeptidase active center.     

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.     

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.     

Low activity of gamma-glutamyl transpeptidase in serum of acute intrahepatic cholestasis.

Low gamma-glutamyl transpeptidase (gamma-GTP) activity in serum was observed in 11 patients with acute intrahepatic cholestasis (cholestatic hepatitis and fulminant hepatitis), despite a marked increase in bilirubin levels. Inhibitors of gamma-GTP were not detected in sera of these patients. Their gamma-GTP levels in the liver were significantly higher than those in chronic liver diseases. An electrophoretic study of liver gamma-GTP in acute intrahepatic cholestasis showed the same mobility as in chronic liver diseases. These results suggest that the low serum gamma-GTP activity in acute intrahepatic cholestasis is due to factors inhibiting the release of the enzyme from the liver.     

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.     

Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.     

Duplication of the bcr and gamma-glutamyl transpeptidase genes.

The Philadelphia (Ph') translocation involves rearrangement of the bcr gene located on chromosome 22. Hybridization experiments revealed the presence of multiple bcr gene-related loci within the human genome. Two of these were molecularly cloned and characterized. Both loci contain exons and introns corresponding to the 3' region of the bcr gene. Restriction enzyme and DNA sequence analysis indicate a very high degree of conservation between bcr and the two related genomic sequences. Both bcr-related loci are located on chromosome 22, one centromeric, the other telomeric, of the bcr gene. Within the two bcr related genomic sequences, fragments or the complete coding sequences of an unrelated gene were found to be present. This gene was identified; it encodes gamma-glutamyl transferase, an enzyme involved in the glutathione metabolism.     

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.     

A new chromogenic substrate for gamma-glutamyl transpeptidase.

A benzfurazan derivative of glutathione l-γ-glutamyl-(S-4-nitrobenz-2-oxa-1,3-diazole)-l-cysteinylglycine (GS-NBD) with an absorption maximum at 419 nm is readily acted upon by γ-glutamyl transpeptidase to yield the S-benzfurazan derivative of cysteinylglycine. An internal S→N shift occurs immediately to yield the N-benzfurazan derivative, which in turn reacts with the sulfhydryl reagent 4,4′-dithiodipyridine to produce the mixed disulfide with an intense absorption at 461 nm. The maximum difference in molar extinction coefficient is 13,200 and occurs at 470 nm. This general device should be applicable to the assay of many other peptidases.     

Cell surface gamma-glutamyl transpeptidase in live cultures.

A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human foetal organs.

The activity and distribution of y-GT was investigated in a number of organs from human foetuses aged from 14 to 24 weeks post menstruationem. Over this period, enzyme activity increased in the kidney, pancreas and thymus, but decreased in the small intestine. No trend could be established for the liver, although activity was high. In the lung, spleen, brain and adrenals, y-GT was either detectable at very low levels or could not be demonstrated. The possible relationship between y-GT activity in some human tumours and the enzyme level in the corresponding foetal organs is discussed.