Interaction of glutathione analogues with Hydra attenuata gamma-glutamyltransferase.

gamma-Glutamyltransferase activity was studied in extracts of the cnidarian Hydra attenuata. The binding of gamma-glutamyl peptide analogues to the enzyme was studied by observing their effects on heat denaturation and their inhibition of p-nitroaniline release from gamma-glutamyl p-nitroanilide. Neither position-1 analogues, in which the gamma-glutamyl moiety was changed to a beta-aspartyl (beta-Asp-Abu-Gly) or an alpha-glutamyl (Glu-Abu-Gly) linkage, nor glutamate protected the enzyme against inactivation at 58 degrees C. GSH (reduced glutathione), gamma-Glu-Abu-Gly and gamma-Glu-Met on the other hand did prevent heat denaturation. GSH and analogues of GSH were competitive inhibitors of p-nitroaniline release, but those analogues in which glycine was replaced by 2-aminoisobutyrate, phenylalanine, leucine or tyrosine had Ki values that were approximately five times those of analogues with the cysteine residue replaced.     

Partial purification and some properties of gamma-glutamyl transpeptidase from human bile.

1. Gamma-Glutamyl transpepetidase ((5-glutamyl)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2) from human bile has been partially purified using protamine sulphate treatment, DEAE-cellulose chromatography and Sephadex G-200 filtration. The procedure resulted in 150-fold increase in specific acitivity with a 37% yield. 2. The partially purified enzyme showed a single zone of enzyme activity by polyacrylamide gel electrophoresis and eluted in the inner volume of Sephadex G-200. 3. The enzyme had a pH optimum of 8.1 and Km of 1.52 mM using gamma-glutamyl p-nitroanilide as substrate. 4. The effects of cations and different gamma-glutamyl acceptors on the activity of the enzyme are reported. 5. As bile gamma-glutamyl transpeptidase appears to be soluble in the absence of detergents, it is suggested that bile may prove to be a useful source for further studies of the kinetic properties and physiological role of human gamma-glutamyl transpeptidase.     

Presence of gamma glutamyl transferase in Mycobacterium smegmatis

The presence of gamma glutamyl transferase (GGT) has been established in Mycobacterium smegmatis. The 10,000 x g supernatant demonstrated only hydrolase activity and did not exhibit any transpeptidase activity. Most of the transferase activity was recovered in 100,000 x g supernatant demonstrating that GGT is a cytosolic enzyme. Maximum activity of GGT was observed at two days of growth and the activity decreased significantly till the seventh day of growth when mycobacteria was grown as stationary culture. The km for gamma glutamyl-p-nitroanilide was found to be 0.074 mM and Vmax for the reaction approached 11.9 nmol per min per mg protein. L-serine + borate was found to be a competitive inhibitor (Ki 12.05 mM) for GGT activity. The pH optimum for GGT activity was observed between 7.5 to 8.5 and temperature above 35 degrees C rapidly inactivated the enzyme activity. To the best of our knowledge, this is the first report which unequivocally establishes the presence of GGT activity in 10,000 x g supernatant of M. smegmatis.     

Adaptation of a gamma-glutamyl transpeptidase assay to microtiter plates

A kinetic assay for measuring gamma-glutamyl transpeptidase (GGT) activity has been adapted to microtiter plates and an automated microtiter plate reader. This method permits the simultaneous analysis of enzyme activity in a large number of samples incubated with the chromogenic GGT substrate gamma-glutamyl-p-nitroanilide. A major advantage of this assay over previously reported methods is the substantial reduction in the time needed for measuring sample enzyme activity. In addition, reduction of the total assay volume to 0.28 ml conserves both sample and reagents. This method has been calibrated at 23 degrees C using purified GGT, and used to analyze GGT activity in human sera. The assay is sensitive over a range of 3-200 U/liter.     

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.     

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.     

The exocytosis induced in HL-60 cells by 4-hydroxynonenal, a lipid peroxidation product, is not prevented by reduced glutathione

The effect of reduced glutathione (GSH) was studied on exocytosis triggered by 4-hydroxynonenal in HL-60 cells induced to differentiate towards the granulocytic cell line by dimethylsulfoxide; we measured beta-glucuronidase secretion from cells incubated at 37 degrees C in the presence of 5 mM GSH. GSH addition to the cell suspensions failed to induce any significant change of the exocytosis stimulated by HNE concentrations between 10(-8) and 10(-6) M. In contrast however, 5 mM GSH was able to fully prevent the release of lactate dehydrogenase observed in the presence of 50 microM HNE, a concentration much higher than that able to stimulate the exocytotic secretion. As the activation of phosphoinositide-specific phospholipase C (PLC) has been shown to play a major role in HNE-induced exocytosis, we studied the GSH effect on the breakdown of phosphatidylinositol-4,5-bisphosphate added to plasma membranes isolated from rat neutrophils and incubated in the presence of increasing concentrations of the aldehyde. In neutrophil membranes HNE induced a significant increase of PLC activity when used in the same concentrations as those able to stimulate beta-glucuronidase secretion in DMSO-differentiated HL-60 cells; the presence of 5 mM GSH failed to prevent its action. Our results suggest that these low aldehyde concentrations, which have actually been found in exudates, may increase tissue damage in inflammation through the release of lytic enzymes by neutrophils; it seems unlikely that their effects could be influenced by the levels of -SH groups present in the exudate and by its protein concentration.     

High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.     

A mutant Bacillus subtilis gamma-glutamyltranspeptidase specialized in hydrolysis activity

gamma-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of gamma-glutamyl compounds and the transfer of their gamma-glutamyl moieties to amino acids and peptides. The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity. In B. subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity. The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT. The K(m) value for L-glutamine was 15.3 mM. D445A GGT was salt tolerant like the wild-type GGT. These results indicate that D445A GGT will be highly useful as a 'glutaminase' in food industry.     

Calcium chelation induced glutathione efflux from tumor cells and prevention by ruthenium red or neomycin.

Cultured human lung carcinoma cells (A549) were incubated in a calcium-free medium containing calcium chelators (EGTA, 1-10 mM or BAPTA, 5 mM) for 1 hour at 37 degrees C. With limited toxicity, the presence of calcium chelators resulted in a decrease of cellular GSH and detachment of the cells from the tissue culture flask. The permeable EGTA tetraacetoxymethyl ester (0.5mM-5 mM) caused a decrease in the cellular GSH content without cell detachment. GSH was not oxidized to GSSG nor formed mixed disulfides with protein thiols. AT-125, a gamma-glutamyl transpeptidase inhibitor, prevented detachment, but not the efflux of cellular GSH. Pretreatment with two impermeable compounds (ruthenium red, 100 microM and neomycin, 0.5-10 mM) protected the cells from detachment and prevented the decrease in intracellular GSH. The presence of calcium in the medium during the EGTA and BAPTA treatments also protected the cells. Calcium associated with the cytoplasmic membrane phospholipids or proteins appears important to limit membrane permeability for GSH efflux and to maintain cell attachment.     

Ontogenic changes in metabolism and transport of glutathione in the rat

Changes in the level of glutathione (GSH), the turnover rate, and gamma-glutamyltransferase (GGT) activity were examined in newborn, weanling, and adult male Wistar rats, the objective being to elucidate the mechanisms which control the hepatic GSH level during maturation as well as under conditions of different degrees of protein ingestion. The hepatic GGT activity in the newborn rats was high at birth, decreased within a few days to 1 to 2% of the initial level, and remained unchanged thereafter, when these rats were fed a normal diet after 3 weeks of age. In contrast, the hepatic GSH level increased 3-4-fold while total GGT activity in the kidney increased 6-8-fold. When weanling rats were fed a low protein diet (containing 10% soy protein) for 3 weeks, the hepatic GSH level decreased markedly while the GGT activity increased 5-6-fold. The turnover rate of hepatic GSH also increased, as determined by the use of buthionine sulfoximine, a specific inhibitor of GSH synthesis; a value of 2.1 h was obtained in comparison with 3.5 h for that of rats fed the normal laboratory chow (CRF-1). On the other hand, feeding adult rats on the low protein diet resulted in a marked decrease in hepatic GSH level with no effect on either hepatic or renal GGT activity. These results together with other observations may suggest that GSH translocated out of liver cells in the newborn rats is degraded mainly by these cells, while the tripeptide secreted by hepatocytes of adult rats is metabolized predominantly in extrahepatic tissues, such as the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of some kinetic and characteristic properties of glutathione S-transferase from bovine erythrocytes

Glutathione S-transferase was purified from bovine erythrocytes and some kinetic and characteristic properties of the enzyme were investigated. The purification procedure was composed of preparation of homogenate and Glutathione-Agarose affinity chromatography. Thanks to the procedure, the enzyme was purified 6,800 fold with 97% yield and a specific activity of 136 EU/mg proteins. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), one band with a mass of 27 kDa was found. The native molecular weight of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimum pH, stable pH, optimum temperature, and optimum ionic strength were determined as 7.0, 6.5 in K-phosphate buffer, 20 degrees C, 0.1 M K-phosphate, respectively. The best activity was obtained with 1-chloro-2,4-dinitrobenzene (CDNB) in a study performed with different substrates. Vmax, Km, and kcat values were calculated as 402.63 +/- 4.99 EU/mg proteins, 0.7447 +/- 0.0007 mM, and 11436 min(-1) for CDNB, and 88.00 +/- 2.30 EU/mg proteins, 0.3257 +/- 0.0012 mM, and 477 min(-1) for GSH, respectively, by using Lineweaver-Burk graphs obtained from 1/V versus 1/[CDNB] and 1/[GSH].     

Purification and characterization of the raffinose oligosaccharide chain elongation enzyme,galactan : galactan galactosyltransferase (GGT), from Ajuga reptans leaves

Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an alpha-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (alpha-galactosyl-myo-inositol)-independent alpha-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20 degrees C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10 degrees C/3 degrees C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant alpha-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from alpha-galactosidases; it neither hydrolysed nor transferred alpha-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10-50 mM.     

Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes

Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites, and neuroactive compounds. Two cell surface enzymes that metabolize gamma-glutamyl compounds have been identified: gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetic analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative, and conducted at physiological pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The K_m value for reduced glutathione was 11 μM for both GGT1 and GGT5. However, the K_m values for oxidized glutathione were 9 μM for GGT1 and 43 μM for GGT5. Our data show that the K_m values for leukotriene C₄ are equivalent for GGT1 and GGT5 at 10.8 and 10.2 μM, respectively. This assay was also used to evaluate serine–borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than in inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism, and other pathways that involve gamma-glutamyl compounds.     

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.     

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.     

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.     

Expression of gamma-glutamyl transpeptidase protects ramos B cells from oxidation-induced cell death

The ectoenzyme, gamma-glutamyl transpeptidase (GGT, EC ) cleaves glutathione (GSH) to facilitate the recapture of cysteine for synthesis of intracellular GSH. The impact of GGT expression on cell survival during oxidative stress was investigated using the human B cell lymphoblastoid cell line, Ramos. Ramos cells did not express surface GGT and exhibited no GGT enzyme activity. In contrast, Ramos cells stably transfected with the human GGT cDNA expressed high levels of surface GGT and enzymatic activity. GGT-transfected Ramos cells were protected from apoptosis when cultured in cyst(e)ine-deficient medium. The GGT-expressing cells also had lower levels of intracellular reactive oxygen species (ROS). Homocysteic acid and alanine, inhibitors of cystine and cysteine uptake, respectively, caused increased ROS content and diminished viability of GGT expressing cells. Exogenous GSH increased the viability of the GGT-transfected cells more effectively than that of control cells, whereas the products of GSH metabolism prevented death of both the control and GGT-transfected cells comparably. These data indicate that GGT cleavage of GSH and the subsequent recapture of cysteine and cystine allow cells to maintain low levels of cellular ROS and thereby avoid apoptosis induced by oxidative stress.     

Protective effect of GABA-enriched fermented sea tangle against ethanol-induced cytotoxicity in HepG2 Cells

This study was conducted to evaluate the protective effects of GABA (gamma-amino butyric acid)-enriched sea tangle juice (STJ) by Lactobacillus brevis BJ-20 fermentation against alcohol hepatotoxicity. The protective effects were determined by assessing glutathione (GSH) content levels and gamma-glutamyl transpeptidase (GGT) activity against ethanol-induced cytotoxicity in HepG2 cells. In ethanol-treated cells, GSH content decreased to 44.35% of control (ethanol-untreated cell) values; however, treatment with fermented sea tangle juice (FSTJ) at a concentration of 25 μg/mL increased GSH levels to 67.08%.These results suggest that FSTJ may prevent intracellular GSH depletion caused by ethanol consumption. Treatment with FSTJ against alcohol-injured HepG2 cells resulted in a dose-dependent decrease in GGT activity. The expression of cytochrome P450 2E1 (CYP2E1) enzyme, a major contributor to ethanol-induced oxidative stress, was also completely inhibited in FSTJ-treated cells at a concentration of 25 μg/mL. Thus, this study demonstrated that ethanolinduced cytotoxicity could be attenuated by inhibition of GSH depletion, GGT activity, and CYP2E1 expression.     

Mutagenicity of tetrachloroethene in the Ames test--metabolic activation by conjugation with glutathione

The mutagenicity of tetrachloroethene (tetra) and its S conjugate, S-(1,2,2-trichlorovinyl)glutathione (TCVG) was investigated using a modified Ames preincubation assay. TCVG was a potent mutagen in presence of rat kidney particulate fractions containing high concentrations of gamma-glutamyl transpeptidase (GGT) and dipeptidases. Purified tetra was not mutagenic without exogenous metabolic activation or under conditions favoring oxidative metabolism. Preincubation of tetra with purified rat liver glutathione (GSH) S-transferases in presence of GSH and rat kidney fractions resulted in a time-dependent formation of TCVG as determined by (HPLC) analysis and in an unequivocal mutagenic response in the Ames test. Experiments with tetra in the isolated perfused rat liver demonstrated TCVG formation and its excretion with the bile; bile collected after the addition of tetra to the isolated perfused liver was unequivocally mutagenic in bacteria in the presence of kidney particulate fractions. The mutagenicity was reduced in all cases by the GGT inhibitor serine borate or the beta-lyase inhibitor aminooxyacetic acid. These results support the suggestion that cleavage of the GSH S conjugate formed from tetra by the enzymes of the mercapturic acid pathway and by beta-lyase may be involved in the nephrocarcinogenic effects of this haloalkene in rats.