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PubMed2002

The expression of glutathione reductase in the male reproductive system of rats supports the enzymatic basis of glutathione function in spermatogenesis.

Glutathione reductase (GR) recycles oxidized glutathione (GSSG) by converting it to the reduced form (GSH) using an NADPH as the electron source. The function of GR in the male genital tract of the rat was examined by measuring its enzymatic activity and examining the gene expression and localization of the protein. Levels of GR activity, the protein, and the corresponding mRNA were the highest in epididymis among testes, vas deferens, seminal vesicle, and prostate gland. The localization of GR, as evidenced by immunohistochemical techniques, reveals that it exists at high levels in the epithelia of the genital tract. In testis, GR is mainly localized in Sertoli cells. The enzymatic activity and protein expression of GR in primary cultured testicular cells confirmed its predominant expression in Sertoli cells. Intracellular GSH levels, expressed as mol per mg protein, was higher in spermatogenic cells than in Sertoli cells. As a result of these findings, the effects of buthionine sulfoximine (BSO), an inhibitor for GSH synthesis, and 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU), an inhibitor for GR, on cultured testicular cells were examined. Sertoli cells were prone to die as the result of BCNU, but not BSO treatment, although intracellular levels of GSH declined more severely with BSO treatment. Spermatogenic cells were less sensitive to these agents than Sertoli cells, which indicates that the contribution of these enzymes is less significant in spermatogenic cells. The results herein suggest that the GR system in Sertoli cells is involved in the supplementation of GSH to spermatogenic cells in which high levels of cysteine are required for protamine synthesis. In turn, the genital tract, the epithelia of which are rich in GR, functions in an antioxidative manner to protect sulfhydryl groups and unsaturated fatty acids in spermatozoa from oxidation during the maturation process and storage.

Animals↗
PubMed1975

Glutathione reductase activity in serum and liver tissue of human and rat with hepatic damage.

Glutathione reductase activity of both serum and liver tissue homogenates was measured in normal controls and in cases of hepatic parenchymatous diseases, and the results were compared with those from animal experiments in which hepatic damage was produced by CCl4 injection. Glutathione reductase showed a different attitude from those of transaminases and alkaline phosphatases under these clinical and experimental conditions. Glutathione reductase activity increased in both serum and liver in patients with hepatic damage, and this increase occurred earlier than the changes in alkaline phosphatase activity.

Acute Disease↗
PubMed1989

Steady-state kinetic studies of glutathione reductase.

The steady-state kinetic studies of yeast glutathione reductase, performed when [GSSG] = 10[NADPH] in the assay mixture, show that at concentrations of GSSG under 450 microM the enzymatic mechanism pathway is ping-pong. Furthermore, in the case of higher values, the enzymatic kinetics follows a sequential pathway. However when the glutathione reductase reaction passes to the ping-pong mechanism, the inhibition effect by excess of NADPH is stronger than when the reaction takes place over the sequential mechanism.

Fungal Proteins↗
PubMed1998

Role of active site tyrosine residues in catalysis by human glutathione reductase.

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.

Amino Acid Substitution↗
PubMed1985

Glutathione reductase is not required for maintenance of reduced glutathione in Escherichia coli K-12.

Seven independently isolated glutathione reductase-deficient (gor) Escherichia coli mutants were found to have an in vivo glutathione redox state that did not significantly differ from that of the parental strain, 98 to 99% reduced. Strains containing both a gor mutation and either a trxA mutation (thioredoxin deficient) or a trxB mutation (thioredoxin reductase deficient) were able to maintain a 94 to 96% reduced glutathione pool, suggesting that glutathione can be reduced independently of glutathione reductase and thioredoxin reductase.

Escherichia coli↗
PubMed2003

Nicotinamide adenine dinucleotide phosphate-regenerating system coupled to a glutathione-reductase microtiter method for determination of total glutathione concentrations in adherent growing cancer cell lines.

Improvements in the traditional glutathione (GSH)-reductase recycling method for determining total glutathione levels in adherent growing cells have been achieved by eliminating the direct use of expensive nicotinamide adenine dinucleotide phosphate (NADPH) and normalizing the levels of GSH to moles/liter instead of the more usual but more error-prone method of normalizing with cellular protein. A glucose-6-phosphate-dehydrogenase auxiliary reaction has been added to the microtiter-adapted enzyme method of Tietze; thus NADP(+) and glucose-6-phosphate replace NADPH in the method. This modification lowers the possibility for substrate inhibition of the reductase by high levels of NADPH during the initial phase of the reaction while at the same time reducing the assay costs by 75-85%. To calculate the cellular concentration of GSH, the number of cells used for the GSH determination, estimated by counting cell nuclei of benzalkonium chloride-lysed cells with a Coulter Counter Z2, and the average cell volume, also determined with the Coulter Counter, are multiplied to give the total sample volume. The quotient of the amount of GSH found in the cells and the total sample volume yields the GSH concentration in moles/liter. The assay has been validated with respect to precision (+/-2.6%), relative accuracy (-4.2 %), linearity (r(2)=0.98), linear range (0.5-10 microM), and limit of detection (80 pmol). Recovery was cell line dependent and ranged between 70 and 103% in the six cell lines. As an application of this method, the GSH concentrations in six human cancer cell lines were determined, without and with a 24-h preincubation with 200 microM D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of GSH biosynthesis. As expected, BSO lowered GSH levels on the average 85%.

Animals↗
PubMed1989

Titration studies on the active sites of pig heart lipoamide dehydrogenase and yeast glutathione reductase as monitored by the charge transfer absorbance.

Macroscopic pKa values associated with the influence of pH on the visible spectrum of 2-electron reduced pig heart lipoamide dehydrogenase and yeast glutathione reductase have been determined by monitoring changes in the principal flavin band near 460 nm and the charge transfer band at 540 nm. The ionization of at least three active site amino acid side chains can influence the spectra over the range of pH studied: the two nascent thiols (interchange thiol and electron transfer thiol) and the histidine residue which acts as the base catalyst in lipoamide dehydrogenase and the acid catalyst in glutathione reductase thiol-disulfide interchange reactions. These systems are analogous to, but more complex than, those in glyceraldehyde-3-phosphate dehydrogenase and papain where a single thiol and a histidine residue in a relatively apolar milieu form a thiolate-imidazolium ion pair which is favored over the thiol-imidazole prototropic tautomer. In an effort to more nearly mimic the papain titrations, the macroscopic pKa values were determined on reduced glutathione reductase which had been monoalkylated with iodoacetamide under conditions known to favor the reaction of the interchange thiol by at least 10 to 1 (Arscott, L. D., Thorpe, C., and Williams, C. H., Jr. (1981) Biochemistry 20, 1513-1520). Like papain and glyceraldehyde-3-phosphate dehydrogenase, alkylated glutathione reductase showed two macroscopic pKa values, at pH 3.7 and pH 9.1, and by analogy, these were associated primarily with the thiol and the imidazole, respectively. Results with the native enzymes depended on the wavelength monitored. Glutathione reductase had pKa values at 4.8, 7.1, and 9.2 when monitored at 540 nm and 5.1 and 8.2 when monitored at 462 nm. Lipoamide dehydrogenase had pKa values at 4.4 and 8.7 when monitored at 529 nm and 3.9, 7.0, and 9.3 when monitored at 455 nm.

Animals↗
PubMed1991

Thermal inactivation study of glutathione peroxidase and glutathione reductase activities in lenses of primates and non-primates.

Heat lability studies of glutathione peroxidase and glutathione reductase activities were conducted on rabbit, sheep, rat, human, galago, cat and rhesus monkey lens supernatants. These species represent five mammalian orders. Incubation periods were 10.0 minutes in duration, with temperatures ranging from 25-100 degrees C (depending on which enzyme was being investigated). Results obtained for glutathione peroxidase activity demonstrated nearly identical heat lability profiles for human and rhesus monkey lenses. Both species were extremely labile to heat, losing activity at 30 degrees C and becoming totally inactive at temperatures of 50 degrees C (rhesus monkey) and 55 degrees C (human). Their profiles were very dissimilar to those of the other five species investigated, providing evidence for the existence of an evolutionary break. Glutathione reductase activity was extremely stable under conditions of highly elevated temperature for all seven species investigated. The human lens enzyme, the most stable of the species, maintained nearly 100% of its original activity up to 65 degrees C. Lenticular glutathione reductase activity did not reach zero levels in any of the seven species until a temperature of at least 80 degrees C was attained.

Animals↗
PubMed1996

Molecular cloning and characterization of a putative glutathione reductase gene, the PfGR2 gene, from Plasmodium falciparum.

Recently, glutathione reductase (GR) has emerged as a promising target for antiparasitic drugs. The central role of GR in cellular antioxidant defence, the particular susceptibility of intracellular parasites like Plasmodium falciparum to oxidative stress, and successful inhibitor studies substantiate this approach. However, more information is required on the structural and functional characteristics of GR from malarial parasites and differences from the enzyme of host erythrocytes. We have identified a putative P. falciparum GR gene coding for a polypeptide (PfGR2) of 500 amino acids that exhibits 40-45% sequence identity with GR enzymes from other species. 18 out of 19 residues contributing to glutathione binding are identical in the putative PfGR2 and human GR. According to Southern blot analysis, the PfGR2 gene is present as a single-copy gene. It is expressed during the intraerythrocytic life cycle. Stage-specific Northern blot analysis demonstrates that the PfGR2 gene is only weakly transcribed in ring, early trophozoite, and segmenter stages; major transcription occurs in the late trophozoite/early schizont stage. This is consistent with the high glutathione reductase activity found in early schizonts. Other data also suggest that PfGR2 corresponds to the enzyme isolated from parasitized erythrocytes. These criteria include the subunit molecular mass (56.2 kDa), the N-terminal sequence (VYDLIVIGGGSGGMA), the presence of specific sequence motifs at ligand-binding sites, and, as demonstrated by Western blotting, the occurrence of a unique chain segment in the core of the central domain. In view of these data, the function(s) of PfGR2 as well as PfGR1, the product of another GR-like gene of P. falciparum (Müller et al., 1995) should be carefully assessed.

Amino Acid Sequence↗
PubMed1990

Purification and thermal dependence of glutathione reductase from two forage legume species.

Alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.) are forage legumes that differ in their responses to high and low temperature stresses. Thermal limitations on the function of glutathione reductase (EC 1.6.4.2) could adversely affect the ability of the plant to cope with adverse temperatures. Our objectives were to (a) purify glutathione reductase from ;Cimarron' alfalfa and ;PI 212241' sainfoin and (b) investigate the intraspecies variation in the thermal dependency of glutathione reductase from each of three cultivars of alfalfa and two cultivars and an introduction of sainfoin. Glutathione reductase was purified 1222-and 1948-fold to a specific activity of 281 and 273 units per milligram of protein, from one species each of alfalfa and sainfoin, respectively. The relative molecular mass of the protein was approximately 140 kilodaltons with subunits of 57 and 37 kilodaltons under denaturing conditions. The activation energies were approximately 50 kilojoules per mole for both species. Over a 5 to 45 degrees C temperature gradient, large variation among species and genotypes within species was found for: (a) the minimum apparent Michaelis constant (0.6-2.1 micromoles of NADPH), (b) the temperature at which the minimum apparent Michaelis constant was observed (10-25 degrees C), and (c) the thermal kinetic windows (6-19 degrees C width). Future studies will focus on relating the thermal dependence of the Michaelis constant of the glutathione reductases and plant growth rates and forage quality of these species throughout the growing season.

Journal Article↗
PubMed1992

Cooperativity induced by a single mutation at the subunit interface of a dimeric enzyme: glutathione reductase.

When glycine418 of Escherichia coli glutathione reductase, which is in a closely packed region of the dimer interface, is replaced with a bulky tryptophan residue, the enzyme becomes highly cooperative (Hill coefficient 1.76) for glutathione binding. The cooperativity is lost when the mutant subunit is hybridized with a wild-type subunit to create a heterodimer. The mutation appears to disrupt atomic packing at the dimer interface, which induces a change of kinetic mechanism. A single mutation in a region of the protein remote from the active site can thus act as a molecular switch to confer cooperativity on an enzyme.

Base Sequence↗
PubMed1990

The effect of haloalkene cysteine conjugates on rat renal glutathione reductase and lipoyl dehydrogenase activities.

An early event in the nephrotoxicity of haloalkene cysteine conjugates is their metabolism by cysteine conjugate beta-lyase to generate a reactive "thiol moiety" which binds to protein. This reactive metabolite(s) has been reported to cause mitochondrial dysfunction. We have examined the effect of three haloalkene cysteine conjugates on the activity of rat renal cortical cytosolic glutathione reductase and mitochondrial lipoyl dehydrogenase, two enzymes which have been reported to be inhibited by S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in the liver. N-Acetyl-S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L- cysteine (N-acetyl PCBC) produced a time- and concentration-dependent inhibition of glutathione reductase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 215 microM. The enzyme activity from male rat kidney was more sensitive to N-acetyl PCBC than that from female rat kidney. Aminooxyacetic acid, an inhibitor of cysteine conjugate beta-lyase, and bis-p-nitrophenyl phosphate, an amidase inhibitor, blocked the effect of N-acetyl PCBC on glutathione reductase, indicating that metabolism by the cytosol is required to produce enzyme inhibition. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC) and DCVC are also noncompetitive inhibitors of glutathione reductase but are less active than N-acetyl PCBC with Ki's of 2.6 and 6.2 mM for DCVC and TFEC, respectively, DCVC produced a time- and concentration-dependent inhibition of lipoyl dehydrogenase and kinetic studies showed that the inhibition was noncompetitive with a Ki of 762 microM. TFEC and PCBC also inhibit lipoyl dehydrogenase. Aminooxyacetic acid blocked the effect of DCVC, TFEC, and PCBC on lipoyl dehydrogenase, indicating that metabolism by the mitochondrial fraction is required to produce enzyme inhibition. Glutathione reductase activity in the renal cortex of male rats treated with 200 mg/kg hexachloro-1,3-butadiene (HCBD) was inhibited as early as 1 hour after dosing, before signs of marked morphological damage. The activity of lipoyl dehydrogenase was also reduced but was only statistically significant 8 hr after dosing when there was marked renal dysfunction. These findings indicate that the reactive thiol moiety formed by cysteine conjugate beta-lyase cleavage of PCBC can inhibit both glutathione reductase and lipoyl dehydrogenase activities in vivo following HCBD administration. We suggest that such inhibition is a general phenomenon, occurring with diverse and as yet unidentified renal proteins. The critical nature of mitochondrial function and the generation of reactive metabolites within this compartment make this organelle a prime target.

Animals↗
PubMed2003

Alternative start sites in the Saccharomyces cerevisiae GLR1 gene are responsible for mitochondrial and cytosolic isoforms of glutathione reductase.

To combat oxidative damage, eukaryotic cells have evolved with numerous anti-oxidant factors that are often distributed between cytosolic and mitochondrial pools. Glutathione reductase, which regenerates the reduced form of glutathione, represents one such anti-oxidant factor, yet nothing is known regarding the partitioning of this enzyme within the cell. Using the bakers' yeast Saccharomyces cerevisiae as a model, we provide evidence that a single gene, namely GLR1, encodes both the mitochondrial and cytosolic forms of glutathione reductase. A deletion in GLR1 drastically increases levels of oxidized glutathione in these two subcellular compartments. The GLR1 gene has two inframe start codons that are both used as translation initiation sites. Translation from the first codon generates the mitochondrial form that includes a mitochondrial targeting signal, whereas translation from the second codon produces the cytosolic form that lacks this sequence. Our results indicate that the sequence context of the two AUG codons influences the efficiency of translation initiation at each site, which in turn affects the relative levels of cytosolic and mitochondrial Glr1p. This method of subcellular distribution of glutathione reductase may be conserved in mammalian cells as well.

Amino Acid Sequence↗
PubMed1996

Glutathione reductase and glutamate dehydrogenase of Plasmodium falciparum, the causative agent of tropical malaria.

The use of glutathione reductase inhibitors in chemotherapy is the raison d'être for this study. Two enzymes were purified to homogeneity from the intraerythrocytic malarial parasite Plasmodium falciparum: glutathione disulfide reductase, an antioxidative enzyme, which appears to play an essential role for parasite growth and differentiation, and glutamate dehydrogenase, an enzyme not occurring in the host erythrocyte. The two proteins were copurified and separated by gel electrophoresis with yields of approximately 20%. Malarial glutathione reductase, a homodimer of 110 kDa with a pH optimum of 6.8 and a high preference for NADPH over NADH, was shown to contain FAD as its prosthetic group. The N-terminal sequence, VYDLIVIGGGSGGMA, which can be aligned with residues 20-34 of human glutathione reductase, represents the first beta strand and the diphosphate-fixing helix of the FAD domain. Glutamate dehydrogenase was confirmed as a hexamer with blocked N-termini; it is an enzyme that is highly specific for NADP and NADPH. The copurification of the proteins and the potential of P.falciparum glutathione reductase as a drug target are discussed.

Amino Acid Sequence↗
PubMed2000

In vitro effects of folic acid on gamma-glutamyltransferase and glutathione reductase activities in malignant lung and thymus tumors.

In vitro effects of folic acid (10(-5), 10(-4), and 10(-3)M) on activities of gamma-glutamyltransferase and glutathione reductase, the enzymes involved in glutathione metabolism, were studied in tissue samples obtained after surgical treatment of the lungs and thymus. Folic acid did not change gamma-glutamyltransferase activity in lung cancer tissue, but in thymoma tissue this substance in a concentration of 10(-3)M inhibited it by 16%. Folic acid had no effects on glutathione reductase activity in benign tumors and normal lung and thymus tissues, but increased this activity in thymoma and lung cancer tissues. Activation of glutathione reductase was probably related to binding of folic acid in the allosteric center of the enzyme, which probably induced conformational changes in the catalytic center, acceleration of electron transport from NADPH(2) to oxidized glutathione via flavin adenine nucleotide, and intense production of reduced glutathione.

Dose-Response Relationship, Drug↗
PubMed2003

Inhibition of glutathione reductase by cadmium ion in some rabbit tissues and the protective role of dietary selenium.

This study is aimed at investigating the inhibitory effect of cadmium ion on glutathione reductase activity of rabbit brain and liver and the relationship of this effect with dietary selenium. For this purpose, one group of New Zealand rabbits were fed a selenium-deficient diet, another group was fed a selenium-rich diet, and the control group was fed a normal diet. The brain and liver tissues of these groups were investigated for the in vitro inhibitory effects of Cd2+ on glutathione reductase activity. For liver, the percentage inhibition of glutathione reductase by 40 nmol/mg protein of Cd2+ was similar for selenium-deficient and control groups, but significantly lower in the selenium-rich group. For brain tissues, there was no difference with respect to cadmium inhibition of glutathione reductase in all three groups.

Animals↗
PubMed2002

Nuclear and nucleolar glutathione reductase, peroxidase, and transferase activities in livers of male and female Fischer-344 rats.

The present studies were to test the hypotheses that glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities are expressed in nuclei and nucleoli of rat liver cells, and that differences in activities of these enzymes would correlate with the greater resistance of female than of male Fischer-344 rats to hepatic necrosis in vivo, mediated by reactive oxygen species generated by redox-cycling metabolism of diquat. Adult male and female Fischer-344 rats were treated with comparably hepatotoxic doses of diquat (0.1 or 0.2 mmol/kg, respectively), or equal volumes of saline, ip. Six hours later, the livers were harvested, and purified nuclei and nucleoli were isolated by differential centrifugation. Nuclear GR activities in male and female rats were 12 and 15 mU/mg protein, and nucleolar activities were 30 and 51 mU/mg protein, respectively, p < 0.05. Some differences between male and female rats in nuclear and nucleolar activities of GPXs and GSTs were observed, as were some differences in the respective diquat-treated animals, but implications of these differences for susceptibility to diquat-induced oxidant stress effects are not apparent. Nuclear GR, GPX, and GST probably contribute to antioxidant defense mechanisms, but the functions served by localization of GR and GPX in nucleoli are less evident.

Animals↗