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PubMed1987

Induction of ascorbate peroxidase and glutathione reductase activities by interactions of mixtures of air pollutants.

The response of ascorbate peroxidase and glutathione reductase activities in peas (Pisum sativum var. Waverex) was investigated after three weeks of exposure to mixed fumigations with SO2, NO2 and O3 (0.050 parts per million each) and increasing concentrations of O3 (0-0.150 parts per million). The results show that plants respond similarly to a high concentration (0.150 parts per million) of a single air pollutant (ozone) and to mixtures of air pollutants (SO2, NO2 and O3) when individual concentrations are low (0.050 parts per million each). In both cases, levels of ascorbate peroxidase and glutathione reductase activities were approximately twice those to be found in plants grown in charcoal-filtered air (p less than 0.01).

Air Pollutants↗

PubMed2006

Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: role of glutathione reductase and glutaredoxin.

Macrophage death is a hallmark of advanced atherosclerotic plaque, and oxidized low-density lipoprotein (OxLDL) found in these lesions is believed to contribute to macrophage injury. However, the underlying mechanisms of this phenomenon are only poorly understood. Here we show that in human monocyte-derived macrophages, OxLDL depleted intracellular glutathione (GSH) and inhibited glutathione reductase, resulting in a marked diminution of the glutathione/glutathione disulfide ratio. In the absence of OxLDL, an 80% depletion of intracellular GSH levels did not affect cell viability, but glutathione depletion dramatically increased OxLDL-induced cell death. Conversely, supplementation of intracellular GSH stores with glutathione diethyl ester substantially diminished OxLDL toxicity. OxLDL also promoted protein-S-glutathionylation, which was increased in macrophages pretreated with the glutathione reductase inhibitor BCNU. Knockdown experiments with siRNA directed against glutathione reductase and glutaredoxin showed that both enzymes are essential for the protection of macrophages against OxLDL. Finally, the peroxyl-radical scavenger Trolox did not prevent GSH depletion but completely blocked OxLDL-induced protein-S-glutathionylation and cell death. These data suggest that OxLDL promotes ROS formation and protein-S-glutathionylation by a mechanism independent from its effect on GSH depletion. Neither mechanism was sufficient to induce macrophage injury, but when stimulated concurrently, these pathways promoted the accumulation of protein-glutathione mixed disulfides and cell death.

Cell Death↗

PubMed1989

A crystallographic study of the glutathione binding site of glutathione reductase at 0.3-nm resolution.

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X-ray crystallography at 0.3-nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione-I is bound in a V-shaped conformation, while glutathione-II is extended. The zwitterionic glutamyl end of glutathione-II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione-I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58-S gamma. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.

Binding Sites↗

PubMedDate not supplied

[Optimization of determining human erythrocyte glutathione reductase activity using a semiautomatic analyzer].

A relatively simple procedure was developed for estimation of the glutathione reductase activity using half-automatic analyzer. The procedure involved: preparation of hemolysates with minimal rate of erythrocyte destruction; substitution of buffers with high ionic strength for 0.05 M Tris-HCl buffer not containing EDTA. Most effective period of preincubation was found; dependence of the reaction rate on FAD was studied. The procedure developed was approved using two types of half-automatic analyzer and the model FP-901 was shown to be the most suitable for estimation of the glutathione reductase holoenzyme activity.

Automation↗

PubMed1981

Reevaluation of membrane binding of erythrocyte glutathione reductase (EC 1.6.4.2).

Data concerning membrane binding of glutathione reductase (GSSG-R) have been reexamined with respect to the following experimental conditions: 1. Haemolysis and membrane preparation; 2. influence of nonionic detergents (Triton X-100, Lubrol PX). Haemolysis and membrane preparations performed at physiological ionic strength, and reduced glutathione concentration do not give any evidence for binding of GSSG-R to the erythrocyte membrane. Under hypotonic conditions of ghost preparation about 2% of the total cellular GSSG-R activity are membrane bound, indicating weak enzyme association at low ionic strength only.

Adult↗

PubMed1991

Role of glutathione reductase system in disulfiram conversion to diethyldithiocarbamate.

Experiments were carried out to establish the role of glutathione reductase (GR), if any, in the metabolic conversion of disulfiram (DS) to diethyldithiocarbamate (DDC). It was observed that, under standard assay conditions, whereas DS was incorporated as a substrate instead of oxidised glutathione (GSSG), the enzymes from both human liver extract and yeast sources failed to reduce the parent compound, implying that glutathione reductase perse do not reduce disulfiram. However, the incorporation of disulfiram into an assay system comprising of GSSG, NADPH and reductase resulted in DS reduction to DDC. Further, the observation, that the GR assay system devoid of either GSSG or NADPH was found to lack DS reducing ability, implies that GSH as a reaction product of GR system is responsible for the reduction of DS to DDC. The results of in-vitro experiments indicated that GSH perse could reduce DS to DDC nonenzymatically, with a stoichiometric relationship of 2:1. Thus it is inferred that GR perse do not reduce DS, whereas GSH, as an intermediary metabolite of GR system, brings about non-enzymatic reduction of DS via a sulfhydral group exchange reaction.

Disulfiram↗

PubMed1981

Simultaneous inactivation of the catalytic activities of yeast glutathione reductase by N-alkylmaleimides.

A series of straight chain N-alkymaleimides was shown to simultaneously inactivate the reductase, transhydrogenase and diaphorase activities of yeast glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2.) at pH 7.5 and 25 degrees C. Apparent second-order rate constants for the inactivation of all enzyme activities exhibited parallel increases with increasing chainlength from C-2 through C-7 of the alkyl substituent of the enhanced binding of maleimides through nonpolar interactions with the enzyme. Reduction of the active site disulfide with NADPH was required prior to addition of maleimide for inactivation to occur. NADP, AcPyADP, SNADP, AADP, and oxidized glutathione all protected the enzyme from inactivation. 2'AMP, 3' AMP, 2'-phospho-5' AMP, 2'-phospho5'-ADP and 2'-phospho-ADP-ribose although all coenzyme-competitive inhibitors failed to protect the enzyme from N-ethylmaleimide inactivation. N-Phenyl and N-alkylmaleimides covalently modified two, of six available sulfhydryl groups per subunit. No other amino acid residues were modified. The reactivity of these sulfhydryl groups was at least two orders of magnitude higher than any reported for the N-ethylmaleimide reaction with many other 'essential sulfhydryl' enzymes. No change in the charge transfer band of the reduced enzyme was observed upon complete inactivation by N-ethyl, N-heptyl or N-phenylmaleimide. The retention of the charge transfer band after selective modification of two sulfhydryl groups suggests the involvement of a third reactive sulfhydryl group in the functioning of the yeast enzyme. No inactivation was observed when coenzymatically reduced enzyme was incubated with the site-specific sulfhydryl reagent, diazotized AADP.

Alkylation↗

PubMed2002

Electrostatic control of the isoalloxazine environment in the two-electron reduced states of yeast glutathione reductase.

The resonance Raman spectra of the oxidized and two-electron reduced forms of yeast glutathione reductase are reported. The spectra of the oxidized enzyme indicate a low electron density for the isoalloxazine ring. As far as the two-electron reduced species are concerned, the spectral comparison of the NADPH-reduced enzyme with the glutathione- or dithiothreitol-reduced enzyme shows significant frequency differences for the flavin bands II, III, and VII. The shift of band VII was correlated with a change in steric or electronic interaction of the hydroxyl group of a conserved Tyr with the N(10)-C(10a) portion of the isoalloxazine ring. Upward shifts of bands II and III observed for the glutathione- or dithiothreitol-reduced enzyme indicate both a slight change in isoalloxazine conformation and a hydrogen bond strengthening at the N(1) and/or N(5) site(s). The formation of a mixed disulfide intermediate tends to slightly decrease the frequency of bands II, III, X, XI, and XIV. To account for the different spectral features observed for the NADPH- and glutathione-reduced species, several possibilities have been examined. In particular, we propose a hydrogen bonding modulation at the N(5) site of FAD through a variable conformation of an ammonium group of a conserved Lys residue. Changes in N(5)(flavin)-protein interaction in the two-electron reduced forms of glutathione reductase are discussed in relation to a plausible mechanism of the regulation of the enzyme activity via a variable redox potential of FAD.

Binding Sites↗

PubMed1993

Lung glutathione reductase induction in aging catalase-depleted frogs correlates with early survival throughout the life span.

A comprehensive experimental study on free radical-related parameters was performed in the lung throughout the life span of 220 initially young or old frogs. No age related differences were found transversely or longitudinally for lung superoxide dismutase, catalase, Se-dependent and -independent glutathione peroxidases, glutathione reductase, GSH, GSSG, or GSSG/GSH ratio. Continuous catalase depletion with aminotriazole led to glutathione reductase induction in the lung after 14.5 months of experimentation. This was accompanied by a great increase in survival rate of treated animals in relation to controls (especially in the old group). After 26.5 months of experimentation, glutathione reductase induction was lost and GSSG/GSH values tended to increase. This was followed by a 3-month long period of acute decrease in survival rate of treated animals. It is suggested that a high antioxidant/prooxidant balance is of protective value against causes of early death and can possibly be used in the future (when appropriately controlled) to increase the number of healthy years of the normal life span.

Aging↗

PubMed1980

Glutathione reductase activity and pyridoxine (pyridoxamine) phosphate oxidase activity in the red cell.

The red-cell enzymes, glutathione reductase (FAD-dependent) and pyridoxine (pyridoxamine) phosphate oxidase (FMN-dependent), were studied in control subjects. The wide range in the glutathione reductase activity correlated inversely with the percentage stimulation by FAD added in vitro, and with pyridoxine (pyridoxamine) phosphate oxidase activity. Both enzymes were stimulated after ingestion of riboflavin. The results support the suggestion that the rate of metabolism of riboflavin in the red cell controls the activity of both enzymes, and the rate of red-cell metabolism of vitamin B-6.

Erythrocytes↗

PubMed1989

Elevated glutathione reductase activity coefficients in erythrocytes after treatment in vitro with inosine and adenine.

Normal erythrocytes incubated with inosine and adenine in an acid citrate/dextrose medium underwent a marked, but reversible, loss of glutathione reductase activity reflected by erythrocyte glutathione reductase activity coefficients in the range 3.21 +/- 0.39. The system therefore appears to simulate riboflavin deficiency in isolated red cells as assessed by this index. Deactivation was dependent on cell metabolism and was inhibited by treatment with methylene blue, ascorbate, and fluoride or by low temperature. The rate of deactivation was greatly increased by lowering the pH and by the presence of phosphate ions.

Adenine↗

PubMed1993

Glutathione reductase in human and murine lung tumors: high levels of mRNA and enzymatic activity.

The enzyme glutathione reductase (GR) (GSSG+NADPH+H+-->2 GSH+NADP+) plays a key role in the cellular defense against oxidative stress. High levels of GR activity are often associated with tumor growth and/or resistance mechanisms against drug and radiation therapy. In order to investigate the molecular basis of elevated glutathione reductase activities we studied the enzyme at the DNA, mRNA and protein levels in murine experimental tumor cell lines and in human lung tumors. A modified ultracentrifugation procedure was developed which allowed the simultaneous isolation of DNA and total cellular RNA. Out of 11 human bronchial carcinomas obtained from patients without prior chemotherapy, five tumors showed a GR activity which was 2.4 to 3.8 times higher than in the respective control tissues. In each case the elevated enzyme activity was accompanied by an elevated GRmRNA levels. For none of the tumors, GR gene rearrangement or amplification was observed by Southern blot analyses. The mouse tumor cell lines ASB XIV, Lewis lung carcinoma and EAT cells, also showed high levels of GRmRNA whereas this mRNA was hardly detectable in normal mouse lung tissue.

Animals↗

PubMed1990

Flavin analogs with antimalarial activity as glutathione reductase inhibitors.

10-(4'-Chlorophenyl)-3-methylflavin has antimalarial activity in vitro and in vivo (Cowden et al., J Med Chem 31: 799, 1988). This flavin analog and two of its derivatives were found to inhibit the antioxidant flavoenzyme glutathione reductase from human erythrocytes in its isolated form as well as in hemolysates. The mixed-type inhibition was completely reversible, the Ki-values being of the order of 1 microM. Surprisingly, the drugs were not competitive with FAD, but with GSSG, one of the enzyme's substrates. Malaria parasite glutathione reductase, extracted from Plasmodium falciparum, could also be inhibited by the compounds. Studies on the effects of the substances on P. falciparum in vitro, which were demonstrated morphologically and by growth inhibition, confirmed previous observations with 10-(4'-chlorophenyl)-3-methylflavin and showed similar parasiticidal characteristics for the two new derivatives. The activities of five other erythrocytic enzymes tested were not impaired by the drugs, nor was the nucleotide metabolism of erythrocytes and/or parasites significantly changed. Permeation into red blood cells was demonstrated for one compound by 19F-NMR-spectroscopy. Inhibition of glutathione reductase might contribute to, or account for, the antimalarial activity of this group of flavin analogs.

Animals↗

PubMed1988

Assay of glutathione reductase in crude tissue homogenates using 5,5'-dithiobis(2-nitrobenzoic acid).

A method for assaying glutathione reductase (GSH; EC 1.6.4.2) in crude plant extracts is described. The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is reduced by GSH. The effects of the following parameters on the assay were tested: various buffers, pH, buffer concentration, compounds commonly present in enzyme preparations, thiols, and the presence of another NADPH-dependent enzyme. The assay is more sensitive and less subject to interference than the widely used assay where NADPH oxidation is monitored. In particular, the specificity of DTNB allows assay of glutathione reductase in the presence of other NADPH-dependent enzymes and common protein extract contaminants.

Dithionitrobenzoic Acid↗

PubMed1995

Essential lysine residue in glutathione reductase: chemical modification by pyridoxal 5'-phosphate.

Yeast glutathione reductase was inactivated by pyridoxal 5'-phosphate. The inhibition was reversed by dilution. The enzyme-pyridoxal 5'-phosphate complex on reduction with sodium borohydride gave a characteristic absorption maximum at 325 nm and fluorescence maximum at 395 nm when exciated at 325 nm. These results were consistent with the reaction of epsilon-amino group of lysine residue of the enzyme with pyridoxal 5'-phosphate. The enzyme was protected against pyridoxal 5'-phosphate inhibition by NADP indicating thereby that the essential lysine residues are present during the NADP binding site.

Adenosine Monophosphate↗

PubMed1986

Similarities between rat liver mitochondrial and cytosolic glutathione reductases and their apoenzyme accumulation in riboflavin deficiency.

Glutathione reductase has been purified 5,500-fold from rat liver mitochondrial matrix in a yield of 30%. The mitochondrial enzyme was immunochemically indistinguishable from that of the cytosol and the subunit molecular weight was apparently similar to that of the cytosolic enzyme, that is, 50,000 daltons. The optimum pH and kinetic properties investigated were not significantly different from those of the cytosolic enzyme. When rats were fed a riboflavin-deficient diet, the enzyme activity in the mitochondria decreased to a greater extent than that in the cytosol, and greater accumulation of apo-enzyme in the former than that in the latter was confirmed by the amount of immunoprecipitable protein, activation by FAD addition in vitro, and the enzyme activity recovery after injection of riboflavin, into riboflavin-deficient rats.

Animals↗

PubMed1997

Modification of an essential amino group of glutathione reductase from yeast by pyridoxal 5'-phosphate.

Yeast glutathione reductase is inactivated by pyridoxal 5'-phosphate (PLP). The reactivation of the enzyme by dilution as well as a characteristic absorption peak at 325 nm exhibited by NaBH4-reduced-PLP modified enzyme show that the inactivation is due to the specific modification of the epsilon-amino group of lysine residue. The maximum of 70% inactivation was observed at 7mM PLP and the equilibrium was reached within 3 min. Kinetic and equilibrium analysis of inactivation data derived at different PLP concentrations showed that a noncovalent intermediate is formed prior to inactivation. From the studies on the effect of pH on the inactivation rate, the pKa of epsilon-amino group of the reactive lysine residue was calculated to be 7.3. Among various protecting agents tried, only NADP was found to be effective. The apparent stoichiometry of the reaction was one to one as the incorporation of 0.65 mole PLP/mole of enzyme led to 70% inactivation at saturating PLP concentration.

Crystallography, X-Ray↗

PubMed1989

Glutathione reductase in the south-western province of Saudi Arabia--genetic variation vs. acquired deficiency.

The activity of glutathione reductase (GR) is closely associated with the riboflavin level in diet. Dietary deficiency of this water-soluble vitamin causes glutathione reductase deficiency. Furthermore, a variable frequency of GR variants with reduced activity has been reported in several populations. In an attempt to determine GR deficiency due to genetic (GR variant) and acquired causes (riboflavin deficiency), red cell GR activity was estimated in 461 male and female Saudis from the South-Western province of Saudi Arabia. The frequency of genetic GR deficiency (GR variant) was 24.5% in Saudi males and 20.3% in females. The frequency of acquired GR deficiency (riboflavin deficiency) was 17.8% and 22.4%, respectively. Interaction between genetic GR deficiency and other genetic abnormalities, i.e. sickle cell gene and glucose-6-phosphate dehydrogenase deficiency were also estimated. No specific link could be demonstrated.

Female↗