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PubMed1996

Effect of glutathione, glutathione sulphonate and S-hexylglutathione on the conformational stability of class pi glutathione S-transferase.

The glutathione S-transferases (GST) are a supergene family of phase II detoxification enzymes which catalyse the S-conjugation between glutathione and an electrophilic substrate. The active site can be divided into two adjacent functional regions, a highly specific G-site for binding the physiological substrate glutathione and a nonspecific H-site for binding nonpolar electrophilic substrates. Equilibrium and kinetic unfolding experiments employing tryptophan fluorescence and enzyme activity measurements were preformed to study the effect of ligand binding to the G-site on the unfolding and stability of the porcine class pi glutathione S-transferase against urea. The presence of glutathione caused a shift in the equilibrium-unfolding curves towards lower urea concentrations and enhanced the first-order rate constant for unfolding suggesting a destabilisation of the pGSTP1-1 structure against urea. The presence of either glutathione sulphonate or S-hexylglutathione, however, produced the opposite effect in that their binding to the G-site appeared to exet a stablising effect against urea. The binding of these glutathione analogues also reduced significantly the degree of cooperativity of unfolding indicating a possible change in the protein's unfolding pathway.

Animals↗
PubMed2003

Study of the activity of glutathione-peroxidase, glutathione-transferase, and glutathione-reductase in renal transplants.

The aim of this work was to study the temporare variation of oxidative stress in the plasma and erythrocytes (CR) of renal transplant patients We determined total glutathione (GST), as well as oxidized (GSSG) and reduced (GSH) fractions and the activity of glutathione peroxidase (G-px), glutathione reductase (G-red) and glutathione transferase (GSt). Determinations were performed 48 hours before transplant as well as 1 and 2 weeks after the renal transplant. The results showed a high "oxidative stress" rate, resulting from the equilibrium between the production of free radicals and the activity of antioxidants, the former being higher proportionally. Immediately after the transplant, there was an increase in oxidative stress, which resulted in an increased G-red, a marked decrease in plasma and in erythrocyte G-px (CR9 and an abrupt drop both in GST levels in plasma and in GSG (as well as in the [GSH]/[GSSG] relationship). Thereafter there was a significant improvement in the activity of antioxidant enzymes, but without normalization; the total glutathione levels and the activity of various enzymes approached the average values of the control group.

Erythrocytes↗
PubMed2003

Formation of S-[2-carboxy-1-(1H-imidazol-4-yl) ethyl]glutathione, a new metabolite of L-histidine, from cis-urocanic acid and glutathione by the action of glutathione S-transferase.

Exposure of the skin to sunlight results in an increase of the content of epidermal trans-urocanic acid, a key metabolite of L-histidine, and also in occurrence of the isomerization of trans-urocanic acid to the cis isomer. S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (GS(CIE)), an adduct of urocanic acid and glutathione, is a presumed origin of a urinary compound S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteine (Cys(CIE)). The formation of GS(CIE) is stimulated by exposing the skin to sunlight irradiation. In this study we investigated an enzymatic formation of GS(CIE) from glutathione and cis-urocanic acid by incubation with rat liver extract that contained glutathione S-transferase (GST) at high activity. The formation of GS(CIE) was suppressed significantly when a liver extract depleted of GST activity was used. Enzymatic degradation of GS(CIE) with gamma -glutamyl transpeptidase resulted in the formation of N-[S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-L-cysteinyl]glycine, a metabolic intermediate between the glutathione adduct and Cys(CIE). A hydrolyzed product of GS(CIE) by HCl was identical with the urinary Cys(CIE). Compounds were analyzed by high-voltage paper electrophoresis, capillary electrophoresis, and fast atom bombardment mass spectrometry. From these results, we suggest that GS(CIE) formed from cis-urocanic acid and glutathione is an origin of the urinary compound Cys(CIE) and that the formation reaction is catalyzed mostly by the action of GST.

Animals↗
PubMed1986

Erythrocyte glutathione synthetase deficiency leads not only to glutathione but also to glutathione-S-transferase deficiency.

Glutathione synthetase (GSH-S) is one of the two known hereditary causes of glutathione deficiency. We describe a family whose two children have hemolytic anemia. The children's erythrocytes lack GSH and are severely deficient in GSH-S activity. No neurologic findings or 5-oxoprolinuria were present. A concurrent deficiency of glutathione-S-transferase (GST) was also detected in the erythrocytes. Residual glutathione could be detected in the erythrocytes using a sensitive cycling assay. The deficiency was found to be most severe in reticulocyte-depleted preparations. The GSH-S activity of the erythrocytes of the parents was one-half normal, while the glutathione S-transferase activity was normal. We conclude that the primary defect is one of GSH-S. Glutathione stabilizes GST in vitro, and it is assumed that the deficiency of GST in the erythrocytes of the patients is due to the instability of this enzyme in the absence of adequate intracellular GSH levels.

Adolescent↗
PubMed1995

Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and in the rat in vivo.

Inhibitors of rat and human Alpha- and Mu-class glutathione S-transferases that effectively inhibit the glutathione (GSH) conjugation of bromosulphophthalein in the rat liver cytosolic fraction, isolated rat hepatocytes and in the rat liver in vivo have been developed. The GSH analogue (R)-5-carboxy-2-gamma-(S)-glutamylamino-N-hexylpentamide [Adang, Brussee, van der Gen and Mulder (1991) J. Biol. Chem. 266, 830-836] was used as the lead compound. To obtain more potent inhibitors, it was modified by replacement of the N-hexyl moiety by N-2-heptyl and by esterification of the 5-carboxy group with ethyl and dodecyl groups. In isolated hepatocytes, the branched N-2-heptyl derivatives were stronger inhibitors of GSH conjugation of bromosulphophthalein than the N-hexyl derivatives. The ethyl ester compounds were more efficient than the corresponding unesterified derivatives. The dodecyl ester of the N-2-heptyl analogue was the most effective inhibitor in isolated hepatocytes, but was relatively toxic in vivo. However, the corresponding ethyl ester was a potent in vivo inhibitor: GSH conjugation of bromosulphophthalein (as assessed by biliary excretion of the conjugate) was decreased by 70% after administration of a dose of 200 mumol/kg. The isoenzyme specificity of the inhibitors towards purified rat and human glutathione S-transferases was also examined. The unesterified compounds were more potent than the esterified analogues, and inhibited Alpha- and Mu-class isoenzymes of both rat and human glutathione S-transferase (Ki range 1-40 microM). Other GSH-dependent enzymes, i.e. GSH peroxidase, GSH reductase and gamma-glutamyltranspeptide, were not inhibited. Thus (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide, the in vivo inhibitor of GSH conjugation, may be useful in helping to assess the role of the Alpha and Mu classes of glutathione S-transferases in cellular biochemistry, physiology and pathology.

Animals↗
PubMed1981

Enzymatic conjugation of erythrocyte glutathione with 1-chloro-2,4-dinitrobenzene: the fate of glutathione conjugate in erythrocytes and the effect of glutathione depletion on hemoglobin.

Erythrocyte glutathione (GSH) can be rapidly depleted by incubating the cells with 1-chloro-2,4-dinitrobenzene (CDNB), which forms 2,4-dinitrophenyl-S-glutathione with GSH through the reaction catalyzed by glutathione S-transferase. GSH-CDNB conjugate thus formed stays undegraded within the erythrocytes. This indicates that in the erythrocytes, mercapturic acid pathway is inoperative. Depletion of GSH in the intact erythrocytes by CDNB results in rapid oxidation of large amounts of hemoglobin to methemoglobin. When glutathione S-transferase-free hemolysate of erythrocytes is incubated with CDNB, the depletion of GSH as well as methemoglobin formation are minimal. Glutathione peroxidase and glutathione reductase activities of the erythrocytes are not affected by CDNB. These studies provide a specific enzymatic method for rapid removal of erythrocyte GSH and also indicate that GSH is vital in maintaining a reduced environment within the erythrocytes.

Chromatography, High Pressure Liquid↗
PubMed1995

The content of glutathione and glutathione S-transferases and the glutathione peroxidase activity in rat liver nuclei determined by a non-aqueous technique of cell fractionation.

Hepatocellular nuclei require glutathione, glutathione S-transferases (GSTs) and Se-dependent glutathione peroxidase (GPx) for intranuclear protection against damage from electrophiles or products of active oxygen. Data so far available from the literature on nuclei isolated in aqueous systems range from glutathione, GSTs and GPx either being absent altogether to being present in quantities in excess of those in the cytoplasm. This paper describes a small-scale preparation of a nuclear fraction from rat liver by a non-aqueous technique, designed to retain nuclear water-soluble molecules in situ, since low-molecular-mass compounds can diffuse freely into other compartments during aqueous separation. This non-aqueous procedure shows the nucleus to contain glutathione at 8.4 mM and soluble GSTs at 38 micrograms/mg of protein, the enrichment over the homogenate being 1.2-1.4-fold. Se-dependent GPx activity was also present in the nucleus (56 m-units/mg), although with slightly lower activity than in the homogenate (0.7-fold).

Animals↗
PubMed1990

Effect of ischaemia-reperfusion on glutathione peroxidase, glutathione reductase and glutathione transferase activities in human heart protected by hypothermic cardioplegia.

The activities of glutathione peroxidase (GSH-px), glutathione reductase (GSSG-rx) and glutathione transferase (GST) were measured in myocardial specimens obtained from right atria of patients subjected to different period of ischaemic arrest (aortic clamping ranging from 10 min to 90 min) followed by 60 min. of reperfusion, during open heart surgery 41-90 min. period of aortic clamping induced a significant increase of GSH-px activity with both H2O2 (p less than 0.05) and cumene hydroperoxide (p less than 0.025) as substrates when compared with baseline levels. Aortic clamping and reperfusion, however did not significantly change the myocardial activities of glutathione transferase and glutathione reductase. It is suggested that the increase of GSH-px in ischaemic-reperfused human hearts may render the myocardium less susceptible to oxidative attack particularly during the reoxygenation period when the level of active oxygen species is greatly elevated.

Female↗
PubMedDate not supplied

[Glutathione reductase and glutathione S-aryltransferase in the rat liver depending upon the glutathione level].

After a decrease of protein in rations of rats content of total glutathione was decreased in liver tissue, whereas the glutathione reductase activity was increased in liver cell cytosol. The glutathione reductase activation occurred due to an increase in the enzyme affinity to the substrate. In the rations containing 15%, 10% and 5% of protein the apparent Km values of the glutathione reductase reaction in liver cytosol constituted 91 microM, 74 microM and 50 microM, respectively. The glutathione-S-aryltransferase activity was decreased in liver tissue only under conditions of a distinct protein deficiency.

Animals↗
PubMedDate not supplied

Activity of glutathione-dependent enzymes in long term diabetes. I. Activity of glutathione S-transferase and glutathione peroxidase in the liver of alloxan induced diabetic rats.

Cytosolic liver glutathione S-transferase (GST) activity was decreased for CDNB and DCNB as substrates in long term alloxan induced diabetes. Similar to cytosolic, microsomal glutathione S-transferase activity was also decreased for CDNB. In contrast, both microsomal and cytosolic GST activities for ETA as well as cytosolic and microsomal glutathione (GSH) contents were unaffected. The activity of Se-dependent glutathione peroxidase activity, but not nonSe-dependent peroxidase activity was increased in diabetic rats. The results suggest that diabetic state has a different effect on each isoenzyme of hepatic glutathione S-transferase activity. After insulin treatment of diabetic animals the activities of both cytosolic and microsomal GST was not restored and the activity of non Se-GSHPx was significantly lower than the control value.

Alloxan↗
PubMed1982

Glutathione S-transferases of the bovine retina. Evidence that glutathione peroxidase activity is the result of glutathione S-transferase.

We have purified two isoenzymes of glutathione S-transferase from bovine retina to apparent homogeneity through a combination of gel-filtration chromatography, affinity chromatography and isoelectric focusing. The more anionic (pI = 6.34) and less anionic (pI = 6.87) isoenzymes were comparable with respect to kinetic and structural parameters. The Km for both substrates, reduced glutathione and 1-chloro-2,4-dinitrobenzene, bilirubin inhibition of glutathione conjugation to 1-chloro-2,4-dinitrobenzene, 1-chloro-2,4-dinitrobenzene inactivation of enzyme activity and molecular weight were similar. However, pH optimum and energy of activation were found to differ considerably. Retina was found to have no selenium-dependent glutathione peroxidase activity. The total glutathione peroxidase activity fractionated with the transferases in the gel-filtration range of mol.wt. 49000 and expressed activity with only organic hydroperoxides as substrate. Only the more anionic isoenzyme expressed both transferase and peroxidase activity.

Animals↗
PubMed1990

Glutathione peroxidase, glutathione reductase and glutathione transferase activities in the human artery, vein and heart.

The continuous exposure to blood components, including prooxidants, makes the blood vessel wall susceptible to oxidative stress and free radical mediated reactions (Henning and Chow, 1988; Stamm et al., 1989; Halliwell and Gutteridge, 1984). Free radicals can be produced extracellularly via the respiratory bursts of activated neutrophils, or intracellularly, via oxidation of hypoxanthine by xanthine oxidase (Henning and Chow, 1988; Stamm et al., 1989; Rubanyi, 1988). Microsomal enzymes such as lipoxygenase and cyclooxygenase may also be a source of reactive species of oxygen (Henning and Chow, 1988; Stamm et al., 1989; Rubanyi, 1988; Mason et al., 1980). It has been proposed that free radicals are involved in the initiation and progression of various cardiovascular diseases including arteriosclerosis (Henning and Chow, 1988; Stamm et al., 1989; Yagi, 1988; Jürgens et al., 1987). Thus the adequacy of the defence systems against free radicals is critical for the susceptibility of blood vessel wall to oxidative damage. Among the enzymatic systems capable of protecting the cell against oxidative injury, selenium dependent glutathione peroxidase (Se-GSH-px), glutatione reductase (GSSG-rx) and glutathione transferase (GST) play a crucial role (Flohe' et al., 1976; Mannervik and Danielson, 1988). Using glutathione (GSH) as a cofactor, Se-GSH-px reduces H2O2 to water and organic hydroperoxides to the corresponding alcohols (Flohe' et al., 1976). This reaction leads to conversion of GSH into its oxidized form (GSSG). In the presence of NADPH, GSSG-rx is able to reduce the oxidized glutathione.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMedDate not supplied

Glutathione-dependent biosynthesis and bioactivation of S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine, the glutathione and cysteine S-conjugates of dichloroacetylene, in rat tissues and subcellular fractions.

Dichloroacetylene is neurotoxic, nephrotoxic, and nephrocarcinogenic. The present experiments were designed to test further the hypothesis that the glutathione conjugate of dichloroacetylene, S-(1,2-dichlorovinyl)glutathione (DCVG), may be involved in dichloroacetylene-induced neurotoxicity. Hence, the biosynthesis of DCVG from dichloroacetylene and glutathione was studied in cytosolic, microsomal, and mitochondrial fractions of liver, lung, brain, and kidney. Enzymatic formation of DCVG was observed in liver microsomes and mitochondria, but only nonenzymatic formation of DCVG was seen in lung, brain, and kidney. The KM and Vmax for DCVG biosynthesis with glutathione as the variable substrate were 0.40 +/- 0.28 mM and 487 +/- 90.3 nmol/mg protein/min, respectively. No region-specific differences in the rates of DCVG biosynthesis in the brain were observed. In whole brain homogenates, DCVG was hydrolyzed to DCVC, which was biotransformed to pyruvate and S-(1,2-dichlorovinyl)-3- mercaptopyruvate, indicating cysteine conjugate beta-lyase-catalyzed beta-elimination and transamination reactions in cerebellar tissues. These findings indicate that the glutathione-dependent bioactivation of dichloroacetylene may be involved in the dichloroacetylene-induced neurotoxicity.

Acetylene↗
PubMed1997

Glutathione reductase, selenium-dependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, Unio tumidus, as biomarkers of aquatic contamination in field studies.

The aim of this study was to evaluate the usefulness of antioxidant parameters in the freshwater bivalve, Unio tumidus, as biomarkers of exposure to pollutants and to study their potential interest in predicting toxicity. Selenium-dependent glutathione peroxidase (Se-GPx), non-selenium-dependent glutathione peroxidase (non-Se-GPx), glutathione reductase (GRd), catalase, and superoxide dismutase (SOD) activities; reduced (GSH) and oxidized (GSSG) glutathione levels; and lipid peroxidation were measured in the gills and digestive glands of Unio. Control mussels were encaged and transplanted for 15 and 30 days to sites where the contamination of sediments was analyzed, along a river receiving domestic and industrial sources of pollution. After 15 days of exposure, all antioxidant parameters of the bivalves transferred to the most polluted sites had strongly decreased compared with control values. This was particularly true for Se-GPx and GRd activities, which were inhibited by 60 and 80% in the two tissues, and for GSH levels (80% reduction in the gills and 60% in digestive glands). These decreases were associated in the gills with lipid peroxidation (measured by malondialdehyde content) and with a high level of contamination of sediments by polycyclic aromatic hydrocarbons and polychlorinated biphenyls. In the mussels exposed at the least polluted sites, the same parameters decreased in the gills, but to a lesser extent: 50% for Se-GPx and 32% for GRd activities, and 45% for GSH levels. The gills appeared more sensitive than the digestive glands. After 30 days of exposure, while Se-GPx, GRd, and GSH remained reduced, a significant induction of non-Se-GPx and catalase activities was recorded in the gills, which reflected an adaptation of the transplanted species to their unsafe environment. All the results indicated that antioxidant defense components, namely, Se-GPx, GRd, and GSH, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The relationship between the degree of deficiency of antioxidant defenses and lipid peroxidation suggests that these parameters could also be biomarkers for toxicity.

Animals↗
PubMed1985

Glutathione S-sulfonate, a sulfur dioxide metabolite, as a competitive inhibitor of glutathione S-transferase, and its reduction by glutathione reductase.

Glutathione S-sulfonate (GSSO3H) is a reaction product of glutathione disulfide (GSSG) and sulfite, the hydrated form of sulfur dioxide. In the present study, GSSO3H was found to be a potent competitive inhibitor of the glutathione S-transferases (GST) in the rat liver (Ki = 14 microM) and lung (Ki = 9 microM), and in human lung tumor-derived A549 cells (Ki = 4 microM). GSSO3H was also reduced by a cytosolic enzyme in the rat liver (Km = 313 microM) and lung (Km = 200 microM), and human lung A549 cells (Km = 400 microM). These results suggest that SO2 may affect the detoxification of xenobiotic compounds by inhibiting, via formation of GSSO3H, the enzymatic conjugation of glutathione (GSH) and reactive electrophiles. Although GSSO3H can be enzymatically degraded, the high substrate Km value suggests that this compound may not be readily reduced at low concentrations.

Animals↗
PubMed2004

Native specific activity of glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo- and hypomotile human sperm samples.

Glutathione-dependent selenoenzymes in human spermatozoa are responsible for a generalized protection against reactive oxygen species (ROS) as well as some other metabolic and structural regulation during spermiogenesis and sperm cell maturation. Glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (GPx-4 or PHGPx) and glutathione reductase (GR) native specific activities have been studied in human Percoll-purified spermatozoa from healthy fertile subjects and asthenozoospermic patients. The mean values obtained for the three enzymes in normal specimens are 1.52 +/- 0.90 mU/10(6) sperm cells (PHGPx), 4.26 +/- 1.73 mU/10(6) sperm cells (GPx-1) and 1.95 mU/10(6) sperm cells (GR). No statistically significant differences for any of the three enzymes were encountered between these values and those of asthenozoospermic patients. These results are discussed and compared with recent literature data on both rescued and native PHGPx specific activity in human spermatozoa, as well as with data obtained for GPx in human seminal plasma.

Adult↗
PubMed2005

Engineering glutathione transferase to a novel glutathione peroxidase mimic with high catalytic efficiency. Incorporation of selenocysteine into a glutathione-binding scaffold using an auxotrophic expression system.

Glutathione peroxidase (GPx, EC 1.11.1.9) protects cells against oxidative damage by catalyzing the reduction of hydroperoxides with glutathione (GSH). Several attempts have been made to imitate its function for mechanical study and for its pharmacological development as an antioxidant. By replacing the active site serine 9 with a cysteine and then substituting it with selenocysteine in a cysteine auxotrophic system, catalytically essential residue selenocysteine was bioincorporated into GSH-specific binding scaffold, and thus, glutathione S-transferase (GST, EC 2.5.1.18) from Lucilia cuprina was converted into a selenium-containing enzyme, seleno-LuGST1-1, by genetic engineering. Taking advantage of the important structure similarities between seleno-LuGST1-1 and naturally occurring GPx in the specific GSH binding sites and the geometric conformation for the active selenocysteine in their common GSH binding domain-adopted thioredoxin fold, the as-generated selenoenzyme displayed a significantly high efficiency for catalyzing the reduction of hydrogen peroxide by glutathione, being comparable with those of natural GPxs. The catalytic behaviors of this engineered selenoenzyme were found to be similar to those of naturally occurring GPx. It exhibited pH and temperature-dependent catalytic activity and a typical ping-pong kinetic mechanism. Engineering GST into an efficient GPx-like biocatalyst provided new proof for the previous assumption that both GPx and GST were evolved from a common thioredoxin-like ancestor to accommodate different functions throughout evolution.

Catalysis↗
PubMed1989

Influence of cell cycle on glutathione-S-transferase, selenium-dependent glutathione peroxidase, superoxide dismutase and glutathione levels in human myeloid leukaemia cell lines.

An important biological function of glutathione (GSH) resides in the detoxication reactions mediated by enzymes such as glutathione-S-transferase (GSTs) and glutathione peroxidase (GPX). An increasing body of evidence implies that GSH and these enzymes play important roles in determining the sensitivity of tumours against cytotoxic drugs like quinone antibiotics, in particular adriamycin (Adr). In the present study, we have analysed the effects of cell-cycle on GSH and GSH-dependent enzymes in an attempt to explain cell-cycle specificity of these antileukaemic drugs which were shown to be involved in free-radical-type reactions. Determination of GSH, GST, GPX and superoxide dismutase in cell-cycle-enriched fractions of five different human myeloid leukaemia cell lines (KG1, K562, U937, ML-1 and ML-2) yielded results identical to those obtained in random cultures, which implies that neither GSH nor GSH-related enzymes are cell-cycle regulated. These findings argue against the presumption that cell-cycle specificity of cytotoxic drugs like Adr could be due to the glutathione-dependent metabolism in myeloid leukaemia cell lines.

Cell Cycle↗