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PubMed1980

Oxidized forms of ovine erythrocyte glutathione peroxidase. Cyanide inhibition of a 4-glutathione:4-selenoenzyme.

[75Se]Glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) containing 4 mol selenium per mol was isolated in 33% yield using 10% ethanol to stabilize the purified enzyme. When reduced with GSH and rapidly separated from GSH by gel filtration chromatography, GSH peroxidase was eluted in a labile oxidized (iodoacetate-insensitive) form which was stable at 4 degrees C but unstable at 25 degrees C (form A). When GSH-reduced enzyme was allowed to oxidize in the course of dialysis a more stable oxidized form was obtained (form C) which was rapidly inactivated by cyanide. Using [35S]GSH, form C was shown to contain tightly bound glutathione in approx. equimolar ratio with selenium. The cyanide sensitivity of GSH peroxidase is therefore correlated with the presence of a glutathione moiety in the enzyme. The isolation of GSH peroxidase containing bound glutathione suggests that intermediates containing glutathione bound to selenium may be formed during the catalytic cycle.

Animals↗
PubMed1986

Inhibition of rat liver glutathione S-transferases by glutathione conjugates and corresponding L-cysteines and mercapturic acids.

Glutathione S-transferases from rat liver were partially purified by ion exchange chromatography. Active peaks, tentatively identified as containing the 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 isoenzymes were kept for study. The glutathione conjugates, S-hexyl-, S-benzyl- and S-(2,4-dinitrophenyl) L-glutathione were tested as inhibitors of the enzymes. The 1-2, 2-2, 3-3 and 3-4 fractions were inhibited to similar extents by these conjugates. For all enzymes the hexyl conjugate at 0.1 mM concentration was strongly inhibitory, the benzyl conjugate moderately so and the dinitrophenyl compound was only weakly inhibitory. In contrast, the epoxide conjugating activity in the 4-4 and 5-5 peak was barely affected by the substituted glutathiones at 0.1 mM concentrations. Studies on a purified ligandin (isoenzyme 1-2) from rat liver showed that further metabolism of the glutathione conjugates, to the corresponding cysteines or mercapturic acids, resulted in products with inhibitory properties approximately three orders of magnitude less potent than those of the parent S-substituted glutathiones.

Acetylcysteine↗
PubMed1994

Effect of the sulfonylurea glyburide on glutathione and glutathione peroxidase activity in alloxan-induced diabetic rat hepatocytes.

1. The effect of glyburide treatment on glutathione peroxidase activity and glutathione levels of non-insulin diabetic rats has been studied. 2. Hepatic glutathione and glutathione peroxidase concentrations were significantly reduced in diabetic animals. 3. Glyburide treatment of diabetic rats for 4 weeks corrected the changes on the glutathione levels observed in diabetic liver. 4. High blood glucose levels of untreated diabetic rats were decreased following glyburide treatment as well. 5. Administration of glyburide to diabetic rats reversed the diabetes-induced changes suggesting that glyburide may directly increase liver glutathione concentrations.

Alloxan↗
PubMed1984

Detoxication of industrial pollutants by the glutathione glutathione-S-transferase system in the liver of Anabas testudineus (Bloch).

The interrelationship of reduced glutathione (GSH) and glutathione-S-transferase in the liver of a freshwater climbing perch Anabas testudineus (Bloch) exposed to common industrial pollutants has been studied. In both short- and long-term treatments there was a concomitant decrease in reduced glutathione profile and an increase in glutathione-S-transferase activity. It may be surmised that the majority of xenobiotics of industrial origin are detoxicated by the glutathione glutathione-S-transferase pathways enabling the fish to survive exposure to the additive and/or synergistic toxicity of mixtures of poisons.

Ammonia↗
PubMed2004

Glutathione and glutathione peroxidase in sputum samples of adult patients with cystic fibrosis.

BACKGROUND: Reduced glutathione (GSH) is a major antioxidant in the lung. In cystic fibrosis (CF) patients, extracellular GSH levels of lower airways, obtained by bronchoalveolar lavage (BAL), were reported to be lower than non-CF individuals. METHODS: Upper airway secretions of stable adult CF patients (29 spontaneous and 13 induced sputum) and non-CF individuals (14 healthy and 12 asthmatics; all induced sputum) were analyzed for total glutathione (i.e. the sum of reduced, GSH, and oxidized, GSSG, forms), GSH and GSSG levels by enzymatic kinetic assay. RESULTS: In CF, both spontaneous and induced sputum samples were comparable in total glutathione levels which were surprisingly high (median concentration of 9.2 (range 1.4-65.2) and 11.6 (1.1-69.8) microM, respectively). In non-CF individuals, total glutathione levels were significantly lower (healthy 2.8 (1.0-12.3), asthmatics (5.3 (1.3-19.2) microM; p<0.001, both vs. CF). In CF, more than 90% of total glutathione was represented by GSH, whereas in non-CF controls, GSH made up less than 50% of total glutathione (p<0.001). CONCLUSIONS: In contrast to BAL, CF sputum contains high levels of GSH. Sputum induction is a potentially useful procedure to monitor antioxidant levels in upper airways of CF patients.

Adult↗
PubMed2005

Modulation of cadmium-induced oxidative stress in Ceratophyllum demersum by zinc involves ascorbate-glutathione cycle and glutathione metabolism.

To understand the interaction between Zn, an essential micronutrient and Cd, a non-essential element, Cd-10 microM and Zn supplemented (10, 50, 100, and 200 microM) Cd 10 microM treated Ceratophyllum demersum L. (Coontail), a free floating freshwater macrophyte was chosen for the study. Cadmium at 10 microM concentration decreased thiol content, enhanced oxidation of ascorbate (AsA) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively, a clear indication of oxidative stress. Zinc supplementation to Cd (10 microM) treated plants effectively restored thiols, inhibited oxidation of AsA and GSH maintaining the redox molecules in reduced form. Cd-10 microM slightly induced ascorbate peroxidase (APX, E.C. 1.11.1.11) but inhibited monodehydroascorbate reductase (MDHAR, E.C. 1.6.5.4), dehydroascorbate reductase (DHAR, E.C. 1.8.5.1) and glutathione reductase (GR, E.C. 1.6.4.2), enzymes of ascorbate-glutathione cycle (AGC). Zn supplementation restored and enhanced the functional activity of all the AGC enzymes (APX, MDHAR, DHAR and GR). Gamma-glutamylcysteine synthetase (gamma-GCS, E.C. 6.3.2.2) was not affected by Cd as well as Zn, but Zn supplements increased glutathione-S-transferase (GST, E.C. 2.5.1.18) activity to a greater extent than Cd and simultaneously restored glutathione peroxidase (GSH-PX, E.C. 1.11.1.9) activity impaired by Cd toxicity. Zn-alone treatments did not change above investigated parameters. These results clearly indicate the protective role of Zn in modulating the redox status of the plant system through the antioxidant pathway AGC and GSH metabolic enzymes for combating Cd induced oxidative stress.

Ascorbic Acid↗
PubMed1998

Inhibition of glutathione conjugation in the rat in vivo by analogues of glutathione conjugates.

Glutathione (GSH) conjugation plays an important role in (de-)toxification of its substrates in vivo. We have developed inhibitors of GSH conjugation that are active in the rat in vivo which are derived from the structure of GSH conjugates: they contain a backbone of gamma-L-Glu-D-2-aminoadipic acid that is virtually isosteric with the gamma-L-Glu-L-Cys-Gly structure of GSH. In addition, a hydrophobic alkyl group is attached such that it may interact with the H-site of the enzyme. Finally, the carboxyl groups were esterified with alcohols of varying chain length. The results show that all these compounds preferentially inhibit alpha-GST's 1-1 and 2-2, have less effect on mu isoenzymes 3-3 and 4-4, and finally, have little effect on rat theta (G.J. Mulder, S. Ouwerkerk-Mahadevan, Modulation of glutathione conjugation in vivo: How to decrease glutathione conjugation in vivo or in intact cellular systems in vitro, Chem. Biol. Interact. 105 (1997) 17-34) and pi (S. Ouwerkerk-Mahadevan, J.H. van Boom, M.C. Dreef-Tromp, J.H.T.M. Ploemen, D.J. Meyer, G.J. Mulder, Glutathione analogues as novel inhibitors of rat and human glutathione S-transferase isoenzymes, as well as of glutathione conjugation in isolated rat hepatocytes and the rat in vivo, Bioche. J., 308 (1995) 283-290). Several of the compounds inhibit the GSH conjugation of bromsulfophthalein and (S)-2-bromisovalerylurea in hepatocytes, in the situ recirculating rat liver perfusion and in the rat in vivo (after i.v. administration). The most effective compound contains a 2-heptylamine group linked as an amide to the 1-carboxyl group of the aminoadipic acid moiety at the H-site, and an ethyl ester at the 5-carboxylic acid group of aminoadipic acid.

Animals↗
PubMedDate not supplied

Modulation of cytochrome P-450-dependent monooxygenases, glutathione and glutathione S-transferase in rat liver by geniposide from Gardenia jasminoides.

Geniposide is an iridoid glycoside extracted from the fruits of Gardenia jasminoides, which are used as a food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. The effects of geniposide and G. jasminoides fruit crude extract on liver cytochrome P-450 (P-450)-dependent monooxygenases, glutathione and glutathione S-transferase were investigated using rats treated orally with the iridoid glycoside (0.1 g/kg body weight/day) or the fruit crude extract (2 g/kg/day) for 4 days. The treatments decreased serum urea nitrogen level but increased liver to body weight ratio, total hepatic glutathione content and hepatic cytosolic glutathione S-transferase activity. Treatments with geniposide and G. jasminoides decreased P-450 content, benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and erythromycin N-demethylation activities in liver microsomes without affecting aniline hydroxylation activity. The natural products had no effect on glutathione content and monooxygenase activities in kidney microsomes. Immunoblotting analyses of liver microsomal proteins using mouse monoclonal antibody 2-13-1 to rat P4503A1/2 revealed that geniposide and G. jasminoides crude extract decreased the intensity of a P4503A-immunorelated protein. Protein blots probed with mouse monoclonal antibody 1-12-3 to rat P4501A1 and rabbit polyclonal antibody against human P4502E1 showed that both treatments had little or no effect on P4501A and 2E proteins. The present findings demonstrate that geniposide from G. jasminoides has the ability to inhibit a P4503A monooxygenase and increase glutathione content in rat liver.

Animals↗
PubMed2003

Covalent coupling of reduced glutathione with ribose: loss of cosubstrate ability to glutathione peroxidase.

Glycation (nonenzymatic glycosylation of proteins) is known to be increased as a result of hyperglycaemia in diabetes. Moreover, cell glutathione concentration has been found to be lower in diabetics and such depletion may impair the cell defence against toxic radical species. Ribose being a potent reducing sugar expected to be increased in cells of diabetics where the pentose phosphate pathway is enhanced, its putative condensation with glutathione was investigated. Reduced glutathione (GSH) was incubated with ribose and the structure of the resultant product was assessed by mass spectrometry, as well as the measurement of its remaining thiol group. A covalent reaction clearly occurred between the reducing sugar and GSH, to give an adduct named N-ribosyl-1-glutathione. This adduct appears to be the Amadori product resulting from the condensation of the primary amine group of GSH with the aldehyde group of ribose. Interestingly, the adduct could not be used as a proper substrate by glutathione peroxidase although it keeps its thiol group. We conclude that the coupling of GSH with a monosaccharide such as ribose might contribute to the decreased cell GSH and glutathione peroxidase activity observed in diabetics.

Chromatography, High Pressure Liquid↗
PubMed1995

Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate.

BACKGROUND: Glutathione transferases (GSTs) constitute a family of isoenzymes that catalyze the conjugation of the tripeptide glutathione with a wide variety of hydrophobic compounds bearing an electrophilic functional group. Recently, a number of X-ray structures have been reported which have defined both the glutathione- and the substrate-binding sites in these enzymes. The structure of the glutathione-free enzyme from a mammalian source has not, however, been reported previously. RESULTS: We have solved structures of a human alpha-class GST, isoenzyme A1-1, both in the unliganded form and in complexes with the inhibitor ethacrynic acid and its glutathione conjugate. These structures have been refined to resolutions of 2.5 A, 2.7 A and 2.0 A respectively. Both forms of the inhibitor are clearly present in the associated electron density. CONCLUSIONS: The major differences among the three structures reported here involve the C-terminal alpha-helix, which is a characteristic of the alpha-class enzyme. This helix forms a lid over the active site when the hydrophobic substrate binding site (H-site) is occupied but it is otherwise disordered. Ethacrynic acid appears to bind in a non-productive mode in the absence of the coenzyme glutathione.

Apoenzymes↗
PubMed1997

Effect of decreased glutathione levels in hereditary glutathione synthetase deficiency on dibromoethane-induced genotoxicity in human fibroblasts.

The genotoxic effect of dibromoethane is thought to be due to glutathione S-transferase mediated metabolism. The purpose of this study was to determine whether variations in endogenous glutathione in human cells could modify the genotoxicity of dibromoethane. Genotoxicity of dibromoethane, assessed by sister chromatid exchange, was examined in normal human skin fibroblasts and fibroblasts obtained from individuals with hereditary generalized glutathione synthetase deficiency. Cell proliferation was examined as a measure of dibromoethane toxicity. The number of sister chromatid exchanges induced by dibromoethane was significantly lower in the fibroblasts with glutathione synthetase deficiency compared to control cells. Inhibition of cell proliferation was similar in the glutathione-deficient and normal fibroblasts. In conclusion, low endogenous glutathione levels are protective against dibromoethane-induced genotoxicity in human fibroblasts.

Analysis of Variance↗
PubMed1992

Affinity labeling of Cys111 of glutathione S-transferase, isoenzyme 1-1, by S-(4-bromo-2,3-dioxobutyl)glutathione.

Incubation of S-(4-bromo-2,3-dioxobutyl)glutathione (S-BDB-G), a reactive analogue of glutathione, with the 1-1 isoenzyme of rat liver glutathione S-transferase at pH 6.5 and 25 degrees C results in a time-dependent inactivation of the enzyme. k(obs) exhibits a nonlinear dependence on S-BDB-G from 50 to 1200 microM, with a kmax of 0.111 min-1 and KI = 185 microM. The addition of 5 mM S-hexylglutathione, a competitive inhibitor with respect to glutathione, gives almost complete protection against inactivation by S-BDB-G. About 1.2 mol of [3H]S-BDB-G/mol of enzyme subunit is incorporated when the enzyme is 85% inactivated, whereas 0.33 mol of reagent/mol of subunit is incorporated in the presence of S-hexylglutathione when the enzyme has lost only 17% of its original activity. Modified enzyme, prepared by incubating glutathione S-transferase with [3H]S-BDB-G in the absence or in the presence of S-hexylglutathione, was reduced with sodium borohydride, reacted with N-ethylmaleimide, and digested with alpha-chymotrypsin. Analysis of the chymotryptic digests, fractionated by reverse-phase high-performance liquid chromatography, revealed Cys111 as the amino acid whose reaction with S-BDB-G correlates with enzyme inactivation. It is concluded that Cys111 lies within or near the hydrophobic substrate binding site of glutathione S-transferase, isoenzyme 1-1.

Affinity Labels↗
PubMedDate not supplied

Glutathione conjugation of the cytostatic drug ifosfamide and the role of human glutathione S-transferases.

Development of drug resistance against alkylating cytostatic drugs has been associated with higher intracellular concentrations of glutathione (GSH) and increased expression of glutathione S-transferase (GST) enzymes. Therefore, enhanced detoxification by the glutathione/glutathione S-transferase pathway has been proposed as a major factor in the development of drug resistance toward alkylating agents. In this paper we describe 31P NMR and HPLC studies on the spontaneous and glutathione S-transferase catalyzed formation of glutathionyl conjugates of two metabolites of ifosfamide, i.e., 4-hydroxyifosfamide and ifosfamide mustard. At 25 degrees C activated ifosfamide (= 4-hydroxyifosfamide + aldoifosfamide) disappeared faster in the presence of a 10-fold excess of GSH (t1/2 = 107 min) compared to incubations without GSH (t1/2 = 266 min). No evidence for the formation of 4-glutathionyl ifosfamide was found. The ultimate alkylating species of ifosfamide is ifosfamide mustard (IM). In the absence of glutathione, the rate constant for the disappearance of the ifosfamide mustard signal at 25 degrees C (pH 7) was 1.98 x 10(-3) min-1 (t1/2 = 350 min). In the presence of a 10-fold molar excess of glutathione, this rate constant was 1.95 x 10(-3) min-1 (t1/2 = 355 min), indicating that the spontaneous formation of an aziridinium ion is the rate-limiting event in the reaction with glutathione. The aziridinium ion formed from IM can deprotonate upon formation, leading to the formation of a (noncharged) aziridine species. This intermediate (N-(2-chloroethyl)-N'-phosphoric acid diamide) was characterized by 31P, 1H, and 13C NMR spectra. When 2 mM ifosfamide mustard was incubated with 1 mM GSH in the presence of 40 microM GST P1-1, the formation of monoglutathionyl ifosfamide mustard was 2.3-fold increased above the spontaneous level. The other major human isoenzymes tested (A1-1, A2-2, and M1a-1a) did not influence the formation of monoglutathionyl ifosfamide mustard. The results of these studies demonstrate that increased levels of GST P1-1 can contribute to an enhanced detoxification of ifosfamide.

Antineoplastic Agents, Alkylating↗
PubMed1979

Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism.

Glutathione derivatives inhibit glutathione S-transferase A [cf. Biochem. J. (1975) 147, 513--522]. The steady-state kinetics of this inhibition have been investigated in detail by using S-octyglutathione, glutathione disulphide and S-(2-chloro-4-nitrophenyl)glutathione: the last compound is a product of the enzyme-catalused reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were found to be competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of two inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations has been based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and was found to increase with velocity. The established error structure was used as a basis for weighting in the regression and to construct confidence levels for the judgement of goodness-of-fit of rate equations fitted to experimental data. The results obtained support a steady-state random model for the mechanism of action of glutathione S-transferase A and exclude a number of simple kinetic models.

Animals↗
PubMed1990

Inhibition and recognition studies on the glutathione-binding site of equine liver glutathione S-transferase.

Equine liver glutathione S-transferase has been shown to consist of two identical subunits of apparent Mr 25,500 and a pl of 8.9. Kinetic data at pH 6.5 with 1-chloro-2,4-dinitrobenzene as a substrate suggests a random rapid-equilibrium mechanism, which is supported by inhibition studies using glutathione analogues. S-(p-Bromobenzyl)glutathione and the corresponding N alpha-, CGlu- and CGly-substituted derivatives have been found, at pH 6.5, to be linear competitive inhibitors, with respect to GSH, of glutathione transferase. N-Acetylation of S-(p-bromobenzyl)glutathione decreases binding by 100-fold, whereas N-benzoylation and N-benzyloxycarbonylation abolish binding of the derivative to the enzyme. The latter effect has been attributed to a steric constraint in this region of the enzyme. Amidation of the glycine carboxy group of S-(p-bromobenzyl)glutathione decreases binding by 13-fold, whereas methylation decreases binding by 70-fold, indicating a steric constraint and a possible electrostatic interaction in this region of the enzyme. Amidation of both carboxy groups decreases binding significantly by 802-fold, which agrees with electrostatic interaction of the glutamic acid carboxy group with a group located on the enzyme.

Animals↗
PubMed1998

The three-dimensional structure of a class-Pi glutathione S-transferase complexed with glutathione: the active-site hydration provides insights into the reaction mechanism.

The structure of mouse liver glutathione S-transferase P1-1 complexed with its substrate glutathione (GSH) has been determined by X-ray diffraction analysis. No conformational changes in the glutathione moiety or in the protein, other than small adjustments of some side chains, are observed when compared with glutathione adduct complexes. Our structure confirms that the role of Tyr-7 is to stabilize the thiolate by hydrogen bonding and to position it in the right orientation. A comparison of the enzyme-GSH structure reported here with previously described structures reveals rearrangements in a well-defined network of water molecules in the active site. One of these water molecules (W0), identified in the unliganded enzyme (carboxymethylated at Cys-47), is displaced by the binding of GSH, and a further water molecule (W4) is displaced following the binding of the electrophilic substrate and the formation of the glutathione conjugate. The possibility that one of these water molecules participates in the proton abstraction from the glutathione thiol is discussed.

Animals↗
PubMed1996

Glutathione, glutathione S-transferase and reactive oxygen species of human scalp sebaceous glands in male pattern baldness.

We investigated the contribution of reactive oxygen species to the development of sebaceous gland hyperplasia and the characteristics of the glutathione S-transferase/glutathione system in male pattern baldness. Glutathione S-transferase, glutathione, and thiobarbituric acid-reactive substances were determined in sebaceous gland-enriched scalp skin of men affected by male pattern baldness and were subjected to hair autotransplantation. In comparison with the hairy occipital-donor areas, the following results were obtained in alopecic frontoparietal samples: glutathione S-transferase-specific activity increased 7-fold (p < 0.001); enzyme affinity towards 1-chloro-2,4-dinitrobenzene decreased 2-fold (p = 0.009); glutathione content decreased 2.5-fold (p = 0.017); and thiobarbituric acid reactive substances increased 2-fold (p = 0.006). Chromatofocusing analysis, bromosulfophthalein IC50 values, enzyme-linked immunosorbent assay, and immunohistochemistry with polyclonal antibodies raised against glutathione S-transferases alpha, mu, and pi demonstrated the presence of alpha, pi, and probably the 5.8 alpha isoenzymes in the sebaceous gland. These results support the hypothesis that reactive oxygen species are involved in the pathogenesis of sebaceous gland hyperplasia in male pattern baldness.

Adult↗
PubMed1984

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies.

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.

Animals↗