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.     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice

Summary Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.This work was supported by a grant from A.S.F.C., Switzerland, and the Jubiläumsspende 1970 der Zürcher KantonalbankN.F. was supported during part of the work by a Royal Society European Science Exchange Programme Fellowship     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice.

Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.     

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.     

Synthesis of human gamma-glutamyl transpeptidase (GGT) during the fetal development of liver

We have determined expression of human GGT gene encoding gamma-glutamyl transpeptidase (GGT) during fetal development of liver using the Northern-blot analysis with a cloned human GGT cDNA and immunohistochemistry with a monoclonal antibody. GGT mRNA could be detected as early as the 12th week of gestation. It then increased gradually to a peak of approx. threefold the amount at week 12, at week 40, just before birth. The size of the mRNA in the fetal liver was 2.7 kb and mRNA of the same size was detected both in the human fetal kidney and human hepatocellular carcinoma as well as normal adult liver. Immunohistochemical analyses show that GGT increased as the fetal liver developed in parallel with the increase in mRNA. Histochemically, GGT was shown to be located in the wall of bile canaliculi when synthesis was low in early development, but to be distributed, in addition, all over the cell membrane of the fetal hepatocytes when synthesis was high at the later stage of development.     

Purification and characterization of gamma-glutamyl transpeptidase from human pancreas

Gamma-glutamyl transpeptidase was purified from human pancreas to an electrophoretically homogeneous state. The enzyme was separated into two active fractions on a DEAE-cellulose column. Both enzyme preparations had the same molecular weight (9 x 10(4)) and were composed of two nonidentical subunits (molecular weight, 61,000 and 27,000). While the optimum pH and pH- and thermal-stability range of both enzymes were identical, their isoelectric points were considerably different. Following incubation with neuraminidase, however, the isoelectric point of F-11 became similar to that of F-I, suggesting that this difference in electrophoretic mobility is due to a difference in the content of sialic acid moiety.     

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.     

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)     

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.     

In vitro translation and processing of human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase

Human hepatoma cell (Hep G2) gamma-glutamyl transpeptidase (gamma-GT), a 120 ka single-chain glycoprotein, is much larger than the expected precursor of the dimeric enzyme in other human tissues. However, the Hep G2 gamma-GT mRNA encodes a 63 kDa peptide, similar to that of rat gamma-GT mRNA product and to the predicted, unglycosylated precursor of the enzyme in human tissues. Translation in presence of dog pancreas microsomes results in processing of the 63 kDa to an 80 kDa core-glycosylated species which is subsequently cleaved to 58 and 22 kDa subunits resembling those in other human tissues. The unusually large Mr of gamma-GT in Hep G2 would thus seem to be due to further glycosylation and processing in the Golgi. A deficiency of the processing protease is the most likely reason for the persistence of the single-chain form of gamma-GT in Hep G2 cells.     

Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney

In human, the two subunits of gamma-glutamyl transpeptidase (GGT) arise from a common precursor encoded by a multigene family. Until now, a single specific coding sequence for this precursor (type I) has been identified in human placenta and liver. In the present study, we have isolated from a human kidney cDNA library, a GGT specific clone (0.8 Kb). The sequence of which (type II) i) covers the carboxy terminal part of the GGT precursor, ii) exhibits 22 point mutations and a 30 bp deletion as compared to the type I GGT sequence. The sequencing of a human genomic clone reveals that this type II GGT mRNA is encoded by a different gene than the type I GGT mRNA. Both type I and type II GGT mRNAs are expressed in human liver, while almost exclusively type II GGT mRNA is detected in human kidney.     

Uptake and utilization of L-glutamine by human lymphoid cells; relationship to gamma-glutamyl transpeptidase activity.

Initial rates of glutamine uptake were studied in human lymphoid cell lines whose γ-glutamyl transpeptidase activities vary from 93 to 11,300 units/mg. In general, glutamine was transported at lower rates than other amino acids (met, phe, leu) in all cell lines studied. A cell line with very high transpeptidase activity exhibited an increased rate of glutamine uptake as compared to other amino acids, and a markedly decreased intracellular concentration of glutamine. In all cell lines transported glutamine was extensively (80%) converted to glutamate. Treatment of cells with 6-diazo-5-oxo-L-norleucine (DON) decreased transpeptidase and conversion of transported glutamine to glutamate by about 80%. Inhibition of glutamine transport was less pronounced (0–20%). The findings indicate that transported glutamine does not equilibrate with glutamine in the intracellular pool, but may enter a separate pool in which it is rapidly converted to glutamate.     

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.     

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.