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)     

gamma-glutamyl transpeptidase and related enzyme activities inthe reproductive system of the male rat.

The γ-glutamyl transpeptidase activity of the epididymis is much higher than that of the several other organs of the reproductive system of the male rat. The epididymal caput has much more activity than the epididymal cauda. Relatively low activity was found in spermatozoa. The enzyme is present in the epididymal fluid in a particulate form suggesting that it originates from membranes of epididymal epithelial cells. The epididymal caput exhibits high γ-glutamylcysteine synthetase activity indicating an active γ-glutamyl ycle in this tissue, which plays an important role in transport phenomena.     

Glutathione S-transferase and gamma-glutamyl transpeptidase activities in cultured rat hepatocytes treated with tocotrienol and tocopherol

1. The effect of tocotrienol and tocopherol on glutathione S-transferase (GST) and gammaglutamyl transpeptidase (GGT) activities in cultured rat hepatocytes were investigated.2. Tocotrienol and tocopherol significantly decreased GGT activities at 5 days in culture but tocotrienol also significantly decreased GGT activities at 1–2 days.3. Tocotrienol and tocopherol treatment significantly decreased GST activities at 3 days compared to the control but tocotrienol also decreased GST activities at 1–3 days.4. Tocotrienol showed a more pronounced effect at a dosage of greater than 50 μM tocotrienol at 1–3 days in culture compared to the control.     

Identity of maleate-stimulated glutaminase with gamma-glutamyl transpeptidase in rat kidney.

Gamma-Glutamyl transpeptidase was purified from rat kidney by a procedure involving Lubrol extraction, acetone precipitation, ammonium sulfate fractionation, treatment with bromelain, and column chromatography on DEAE-cellulose and Sephadex G-100. The final preparation (enzyme III), which exhibits a specific activity about 8-fold higher than that of the purified rat kidney transpeptidase previously obtained in this laboratory (enzyme I), was apparently homogeneous on polyacrylamide gel electrophoresis. Enzyme III is a glycoprotein containing 10% hexose, 7% aminohexose, and 1.5% sialic acid; a tentative molecular weight value of about 70,000 was obtained by gel filtration. Enzyme III has a much lower molecular weight and a different amino acid and carbohydrate content than the less active rat kidney transpeptidase preparation previously obtained, but obtained, but the catalytic properties of these preparations are virtually identical. It is suggested that bromelain treatment may liberate the transpeptidase from a brush border complex that contains other proteins. An improved method is described for the isolation of the higher molecular weight form of the enzyme (enzyme I) in which affinity chromatography on concanavalin A-Sephrose is employed. The purified transpeptidase (enzyme III) is similar to the phosphate-independent maleate-stimulated glutaminase preparation obtained from rat kidney by Katunuma and colleagues with respect to amino acid and carbohydrate content, apparent molecular weight, and relative transpeptidase and maleate-stimulated "glutaminase" activities. Both of these enzyme preparations are much more active in transpeptidation reactions with glutathione and related gamma-glutamyl compounds than with glutamine. In the absence of maleate, the enzyme catalyzes the utilization of glutamine (by conversion to gamma-glutamylglutamine, glutamate, and ammonia) at about 2% of the rate observed for catalysis of transpeptidation between glutathione and glycylglycine; the utilization of glutamine occurs about 8 times more rapidly in the presence of 0.1 M maleate. The transpeptidation and maleate-stimulated glutaminase reactions catalyzed by both enzyme preprations are inhibited by 5 mM L-serine in the presence of 5 mM sodium borate. Studies on gamma-glutamyl transpeptidase and maleate-stimulated glutaminase in the kidneys of fetal rats, newborn rats, and rats after weaning showed parallel development of these activities. The evidence reported here and earlier work in this laboratory strongly support the conclusion that maleate-stimulated glutaminase activity is a catalytic function of gamma-glutamyl transpeptidase. The studies on the ontogeny of gamma-glutamyl transpeptidase and other data are considered in relation to the proposal that this enzyme is involved in amino acid and peptide transport. Its possible role in renal formation of ammonia is also discussed.     

Glutathione and gamma-glutamyl transpeptidase in the adult female rat brain after intraventricular injection of LHRH and somatostatin

Glutathione content and glutamyl transpeptidase activity in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH and somatostatin. Hypothalamic glutathione levels were significantly elevated at 10 and 30 min after a single injection of a 0.1 micrograms dose of LHRH. On the contrary, glutathione levels significantly decreased in the hypothalamus, cerebral cortex and cerebellum at 10 and 30 min after 0.5 or 1 microgram dose. However, significant decrease in brain stem glutathione was evident at 30 min after 0.5 microgram and 10 min after the 1 microgram dose. Somatostatin at doses of 0.5 microgram and 1 microgram significantly decreased glutathione levels in all four brain regions both at 10 and 30 min following injection into the 3rd ventricle. Gamma-glutamyl transpeptidase activity in the hypothalamus and cerebral cortex was significantly elevated after intraventricular injection of LHRH. However, a significant increase in gamma-glutamyl transpeptidase activity in cerebellum and brain stem was seen only with 0.5 and 1 micrograms doses of LHRH. Somatostatin also significantly increased gamma-glutamyl transpeptidase activity in hypothalamus, cerebral cortex, brain stem and cerebellum. The decrease in glutathione levels with corresponding increase in gamma-glutamyl transpeptidase activity after intraventricular administration of LHRH and somatostatin suggests a possible interaction between glutathione and hypothalamic peptides.     

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.     

Characterization of proteins structurally related to human N-acetyl-beta-D-glucosaminidase.

Those proteins of human liver that cross-reacted with antibodies raised to apparently homogenous hexosamindases A and B were detected by immunodiffusion. Cross-reacting proteins with high molecular weights (greater than 2000000) and intermediate molecular weights (70000--200000) were present both in the unadsorbed fraction and in the 0.05--0.2M-NaCl eluate obtained by DEAE-cellulose chromatography at pH7.0. The unadsorbed fraction also contained a cross-reacting protein of low molecular weight (10000--70000). The possible structural and functional relationships between hexosaminidase and the cross-reacting proteins are discussed. An apparently cross-reacting protein present in the 0.05M-NaCl eluate from the DEAE-cellulose column was serologically unrelated to hexosaminidase, but it gave a reaction of immunological identify with one of the apparently cross-reacting proteins having the charge and size characteristics of hexosaminidase A. It is suggested that immunochemical methods may provide criteria for the homogeneity of enzyme preparations superior to those of conventional methods.     

In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase

Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.     

Taurocholate uptake by adult rat hepatocytes in primary culture

Adult rat hepatocytes were cultured on Petri dishes for 25--30 h prior to measuring their ability to transport taurocholate. A rapid uptake of the bile acid (25 muM) was observed: about 20% was accumulated in the cells within 15 min. The taurocholate transport was saturable with an apparent Km of 28 +/- 10 muM and a maximal velocity V of 0.07 +/- 0.02 nmol/(micrograms DNA x min). Uptake was shown to be energy dependent as it was inhibited about 65% by antimycin A (20 micrograms/ml). The monohydroxylated bile acid taurolithocholate and the dihydroxylated taurochenodeoxycholate inhibited taurocholate transport to about 30 and 40% resp. of the control. The transport process was strongly dependent on sodium ions. It is concluded that the characteristics of taurocholate uptake into adult rat hepatocytes are very similar either in freshly prepared cells or in hepatocytes which are cultured on Petri dishes for 25--30 h.     

Tissue difference in gamma-glutamyl transpeptidase attributed to sialic acid content.

Gamma-glutamyl transpeptidase (GGT) was extracted from the microsomal fraction of various bovine tissues and partially purified. Purified enzymes demonstrated different mobilities toward the anode in polyacrylamide gel electrophoresis in 0.5% Emulphogene BC720, pH 7.5. The ciliary-body GGT migrated fastest, while the brain enzyme was electrophoresed most slowly. The apparent Km values (Km′) of GGT for L-gamma-glutamyl-p-nitroanilide were 1.4–2.0 mM when assayed with glycylglycine as the gamma-glutamyl acceptor. After neuraminidase treatment, electrophoretic mobility was decreased considerably for all enzyme preparations, compatibly with the removal of negatively charged sialic-acid residues. The Km′ values of the enzyme were not affected by the hydrolytic treatment. Electrophoresis of digested enzymes showed essentially identical mobilities. From these results we conclude that tissue differences in GGT are attributable to the varying extent of sialylation of enzyme.     

Monoclonal antibodies against rat kidney gamma-glutamyl transpeptidase show species and tissue specificity.

The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.     

Immunohistochemical study of localization of gamma-glutamyl transpeptidase in the rat brain

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.     

Density-dependent growth control of adult rat hepatocytes in primary culture

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.     

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.