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PubMed1992

Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence.

Time-resolved polarized fluorescence spectroscopy has been applied to the bound FAD in the structurally related flavoproteins lipoamide dehydrogenase from Azotobacter vinelandii (LipDH-AV) and glutathione reductase (GR) from human erythrocytes. The fluorescence parameters as obtained from the maximum entropy analysis differ considerably in both enzymes, reflecting the unique properties of the flavin microenvironment. Three conformational substates are revealed in LipDH-AV and five in GR. Almost 90% of the population of GR molecules has a fluorescence lifetime in the order of 30 ps which originates from efficient exciplex formation with Tyr197. Equilibrium fluctuations between conformational substates are observed for LipDH-AV on a nanosecond time scale in the temperature range 277-313 K. Interconversion between conformational substates in GR is slow, indicating that large activation barriers exist between the states. In agreement with these results, a model is postulated which ascribes a role in catalysis to equilibrium fluctuations between conformational substates in GR and LipDH-AV. From time-resolved fluorescence anisotropy as a function of temperature, distinction can be made between flavin reorientational motion and interflavin energy transfer. In both proteins intersubunit energy transfer between the prosthetic groups is observed. Furthermore, it is revealed that only the flavin in glutathione reductase exhibits rapid restricted reorientational motion. Geometric information concerning the relative orientation and distance of the flavins can be extracted from the parameters describing the energy-transfer process. The obtained spatial arrangement of the flavins is in excellent agreement with crystallographic data.

Azotobacter vinelandii↗
PubMed1997

Inhibition of glutathione reductase by dinitrosyl-iron-dithiolate complex.

The biological signal molecule nitric oxide (NO) exists in a free and carrier-bound form. Since the structure of the carrier is likely to influence the interaction of NO with macromolecular targets, we assessed the interaction of a dinitrosyl-iron-dithiolate complex carrying different thiol ligands with glutathione reductase. The enzyme was irreversibly inhibited by dinitrosyl-iron-di-L-cysteine and dinitrosyl-iron-di-glutathione in a concentration- and time-dependent manner (IC50 30 and 3 microM, respectively). Evaluation of the inhibition kinetics according to Kitz-Wilson yielded a Ki of 14 microM, and a k3 of 1.3 x 10(-3) s-1. A participation of catalytic site thiols in the inhibitory mechanism was indicated by the findings that only the NADPH-reduced enzyme was inhibited by dinitrosyl-iron complex and that blockade of these thiols by Hg2+ afforded protection against irreversible inhibition. This inhibition was not accompanied by formation of a protein-bound dinitrosyl-iron complex and/or S-nitrosation of active site thiols (Cys-58 and Cys-63). However, one NO moiety exhibiting an acid lability similar to a secondary N-nitrosamine was present per mol of inhibited monomeric enzyme. These findings suggest specifically N-nitrosation of glutathione reductase as a likely mechanism of inhibition elicited by dinitrosyl-iron complex and demonstrate in general that structural resemblance of an NO carrier with a natural ligand enhances NO+ transfer to the ligand-binding protein.

Animals↗
PubMed1996

Immunolocalization of glutathione reductase in the murine brain.

Free radical species arise from the univalent reduction of oxygen. The cytosolic agent H2O2, produced during enzymatic scavenging of the superoxide radical (O2-) is in turn removed predominantly via the oxidation of reduced glutathione (GSH) to the oxidized form (GSSG) by glutathione peroxidase. Subsequently GSSG is recycled back to GSH by glutathione reductase (GSH-red). Little is known about the distribution of this enzyme in the brain. The aim of this study was to determine the distribution of this enzyme in the brain of different murine species by means of immunocytochemical techniques, although most attention was given to the distribution of GSH-red in the forebrain. In most brain areas GSH-red positive neurons were detected, but the regional intracellular staining intensity differed markedly. The pre-piriform and piriform cortices, the pyramidal cell layers of the hippocampus, and the dentate gyrus were heavily stained. The caudate nucleus displayed a progressive increase in the intracellular staining intensity from the rostral to the caudolateral parts. Furthermore, in the thalamus, there was a gradual decrease in GSH-red staining from the medial to the lateral parts. The mesencephalon was poor in immunopositive cells, and in the substantia nigra pars reticulata, almost no labeling was detected. However, the substantia nigra pars compacta showed an intense GSH-red immunoreactivity. The results show a specific localization of glutathione reductase in distinct brain regions, suggesting a variable potency of different brain areas in dealing with the damaging oxidative actions of free radicals. Also, differential GSH-red expression patterns were found in the various murine species. Some species showed a pronounced GSH-red immunoreactivity in glial cells, specifically in regions that lacked neuronal GSH-red immunoreactivity.

Animals↗
PubMed1985

Elemental selenium and glutathione reductase.

Selenium is an essential trace element important to several metabolic processes, although selenium in the chemical form of elemental selenium (Se degree) is commonly believed to be biologically inert. Recent data shows that colloidal suspensions of red amorphous elemental selenium are more easily reduced than previously thought, and that such reductions can potentially take place under biological conditions. The enzyme glutathione reductase, already known to be involved in selenium metabolism, is a good candidate to mediate the reduction of colloidal selenium to hydrogen selenide (H2Se), a compound of established biological activity. The implications of this hypothesis include the possibility that elemental selenium may be biologically active at least under some conditions, and that glutathione reductase may function in selenium metabolism primarily by maintaining the glutathione concentration in a reduced state.

Colloids↗
PubMed1988

Glutathione reductase directly mediates the stimulation of yeast glucose-6-phosphate dehydrogenase by GSSG.

Yeast glucose-6-phosphate dehydrogenase was inhibited by low NADPH concentrations in cell-free extracts, and de-inhibited by GSSG; extensive dialysis of the crude extract did not diminish the GSSG effect. Immunoprecipitation of glutathione reductase abolished the de-inhibition of glucose-6-phosphate dehydrogenase by GSSG. Purified glucose-6-phosphate dehydrogenase was inhibited by NADPH but not de-inhibited by GSSG, and upon addition of pure glutathione reductase GSSG completely de-inhibited the glucose-6-phosphate dehydrogenase.

Antibodies↗
PubMedDate not supplied

The effect of reduced dietary protein level on the activity of transketolase and glutathione reductase in pig erythrocytes.

The effect was investigated of decreased protein level in a food ration on covering the requirements for vitamins B1 and B2. Two feeding experiments were conducted, each on 32 fattening pigs of Polish Landrace breed. The activity of transketolase and glutathione reductase in erythrocytes and vitamin B1 and B2 level in blood and liver were determined. It was shown that a reduction of the diatery protein level by 25% caused a decrease of thiamine utilization by pigs. It indicates the necessity of a dietary supplementation with a synthetic vitamin B1 preparation in such a condition. A reduced activity of glutathione reductase, indicating a riboflavin deficiency, was observed only when the low protein diet was additionally deprived of a vitamin-mineral premix containing a fodder B2 preparation. Reduction of the dietary protein level by 25% was not associated with the risk of riboflavin deficiency, provided that the ration was supplemented with a standard dose of synthetic vitamin B2.

Animals↗
PubMed2000

Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme.

The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H(+) <--> 2 GSH + NADP(+)). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E(ox)-EH(2) and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K(eq) from the Haldane relationship, 0.075, very similar to the K(eq) of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000) Biochemistry 39, 4711-4721). Enzyme-monitored turnover indicates that E(FADH(-))(S-S). NADP(+) and E(FAD)(SH)(2).NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.

Animals↗
PubMed1999

Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase.

We investigated whether endogenously or exogenously produced nitric oxide (NO) can inhibit cellular glutathione reductase (GR) via the formation of S-nitrosothiols to decrease cellular glutathione (GSH) and increase oxidative stress in RAW 264.7 cells. The specificity of this inhibition was demonstrated by addition of a NO-synthase inhibitor, and met- or oxyhemoglobin. Using isolated GR we found that only certain NO donors inhibit this enzyme via S-nitrosothiol. Furthermore, we found that cellular GSH decrease is paralleled by an increase of superoxide anion production. Our results show that the GR enzyme is a potential target of S-nitrosothiols to decrease cellular GSH levels and to induce oxidative stress in macrophages.

Animals↗
PubMed2006

Reduced cellular glutathione reductase activity and increased adhesion molecule expression in endothelial cells cultured with maternal plasma from women with preeclampsia.

OBJECTIVE: The purpose of the current study was to determine whether maternal circulating components could regulate oxidative status of glutathione redox cycle and adhesion molecule expression in endothelial cells (ECs). METHODS: Maternal plasma was extracted from venous blood obtained from normal term pregnant women and from women with preeclampsia (PE). Normal and PE pregnancies were defined as American College of Obstetricians and Gynecologists criteria. Confluent ECs were incubated with EC growth medium (EGM) containing 20% plasma from women with normal (n = 8) and PE (n = 8) pregnancies for 4 hours. ECs incubated with EGM only were used as control. EC oxidative status was assessed by measuring cellular glutathione reductase (GR) and glutathione peroxidase (GPx) activities. Adhesion molecule expressions for intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), P-selectin, and E-selectin were determined by colorimetric assays detected on EC surface by UV spectrophotometer at OD 450 nm. Data are presented as mean +/- SE and analyzed by analysis of variance (ANOVA). A P value < .05 was set as statistically significant. RESULTS: Cellular GR activity was reduced approximately 35% in ECs treated with normal plasma and 70% in ECs treated with PE plasma compared to that in untreated control cells: 0.072 +/- 0.014 (P < .05), 0.039 +/- 0.006 (P < .01), versus 0.117 +/- 0.010 U/mg cellular protein, respectively. In contrast, GPx activity was slightly increased in ECs treated with normal plasma and significantly increased in ECs treated with PE plasma compared to that in untreated control cells: 0.059 +/- 0.005, 0.075 +/- 0.012 (P < .05) versus 0.044 +/- 0.002 U/mg cellular protein, respectively. P-selectin, E-selectin, and VCAM expressions were elevated in cells treated with normal plasma but significantly increased in cells treated with PE plasma compared to those of untreated controls: P-selectin--0.18 +/- 0.03, 0.35 +/- 0.04 versus 0.04 +/- 0.01 OD 450 nm, P < .01; E--selectin-0.06 +/- 0.02, 0.10 +/- 0.02 (P < .05) versus 0.03 +/- 0.01 OD 450 nm; VCAM--0.12 +/- 0.02, 0.16 +/- 0.03 (P < .01) versus 0.08 +/- 0.02 OD 450 nm, respectively. There was no difference for ICAM expression in cells treated with normal or PE plasma compared to controls. CONCLUSIONS: These data suggest that endothelial pro- and anti-oxidative status could be directly affected by circulating components during pregnancy. Reduced cellular GR activity and increased GPx activity accompany increased inflammatory reactions in ECs responding to circulating "toxic" factors in preeclampsia.

Antibodies, Monoclonal↗
PubMed2003

Amperometric sensor for glutathione reductase activity determination in erythrocyte hemolysate.

The development of an amperometric sensor for glutathione reductase (GR) activity in erythrocyte hemolysate to contribute to oxidative stress evaluation is presented. In this assay, the reduced form of glutathione, the product of the GR reaction, reacts with 5,5(')-dithiobis(2-nitrobenzoic acid), producing GSTNB, which is easily reduced in the electrode surface. The current was recorded during 180 s after the sample addition, applying a potential of -300 mV. The sensor presented a suitable sensitivity, a good operational range, and precision. The effects of pH variations and specific uncompetitive inhibitor (safranin-O) in the enzyme activity were also evaluated. The GR activity determination in human erythrocyte hemolysate using this method has provided results that are statistically equal to those obtained by the classical spectrophotometric method, with 95% of confidence. The advantages of this method are the saved time, reagents, and samples and the possibility of its use in the field.

Cell Extracts↗
PubMed1998

Molecular cloning and characterization of the gene encoding glutathione reductase in Brassica campestris.

We have isolated the Brassica campestris cDNA encoding glutathione reductase of 502 amino acid residues with molecular mass of 54.5 kDa. The deduced amino acid sequences were 92.2%, and 79.5% identical to those of Arabidopsis thaliana, and pea, respectively. As expected, it exhibited a high degree of conservation within the region responsible for the redox reaction and for the binding of GSSG or NADPH. The gene was highly inducible by ozone fumigation or by paraquat treatment.

Amino Acid Sequence↗
PubMed1976

Response of plasma and red cell glutathione reductase of several mammalian species and domestic chickens to flavin adenine dinucleotide.

Glutathione reductase (GR) activity was determined in the plasma and red cells from a number of species (human, horse, cattle, sheep, chicken, rabbit, rat). The species with the highest plasma activity (horse and cattle) had the lowest red cell activity. Unlike red cell GR, plasma GR was not significantly stimulated by the addition of flavin adenine dinucleotide (FAD). In species with high plasma GR activity the determination of whole blood FAD activity coefficients may not be a good index of riboflavin status.

Animals↗
PubMed1981

Serum and erythrocyte glutathione reductase activity in chronic renal failure.

The activity of serum and erythrocyte glutathione reductase was studied in 66 undialysed uremic patients, 19 patients under treatment by chronic hemodialysis and 38 normal subjects. A statistically significant increase of the enzyme activity was found both in serum and erythrocytes in the uremic patients. This increase was in positive correlation with blood urea and creatinine. No difference has been observed in the enzyme activity before and after hemodialysis. By electrophoresis of the hemolysate in cellulose-acetate the pattern of the isoenzymes of the uremic patients was similar to that of the controls.

Adult↗
PubMed2005

Overexpression of a eukaryotic glutathione reductase gene from Brassica campestris improved resistance to oxidative stress in Escherichia coli.

Glutathione reductase (GR) plays an essential role in a cell's defense against reactive oxygen metabolites by sustaining the reduced status of an important antioxidant glutathione. We constructed a recombinant plasmid based on the expression vector pET-18a that overexpresses a eukaryotic GR from Brassica campestris (BcGR) in Escherichia coli. For comparative analyses, E. coli GR (EcGR) was also subcloned in the same manner. The transformed E. coli with the recombinant constructs accumulated a high level of GR transcripts upon IPTG induction. Also, Western blot analysis showed overproduction of the BcGR protein in a soluble fraction of the transformed E. coli extract. When treated with oxidative stress generating reagents such as paraquat, salicylic acid, and cadmium, the BcGR overproducing E. coli exhibited a higher level of growth and survival rate than the control E. coli strain, but it was not as high as the E. coli strain transformed with the inducible EcGR. The translated amino acid sequences of BcGR and EcGR share 37.3% identity but all the functionally known important residues are conserved. It appears that eukaryotic BcGR functions in a prokaryotic system by providing protection against oxidative damages in E. coli.

Bacteriophage T7↗
PubMed1999

Optimized heterologous expression of glutathione reductase from Cyanobacterium anabaena PCC 7120 and characterization of the recombinant protein.

Glutathione reductase (GR) from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli SG5. Silent random mutations were introduced in the 5' region of DNA encoding the enzyme in order to generate a high-level expression clone. To maximize protein expression, the culture conditions were also optimized. In the high-level expression clones selected, E. coli-preferred codons were selectively used at certain positions. Under the optimal expression conditions, a yield of 17 mg recombinant protein per liter was obtained, which is about 10-fold higher than that of the wild-type enzyme. A hexahistidine tag was added at the C-terminal of the protein in order to allow IMAC affinity purification. This strategy simplified the purification process and provided a homogeneous enzyme for functional characterization. Anabaena GR uses NADPH as a coenzyme, like most of the GRs from other sources, but the KM values for NADPH and GSSG are higher than those of enzymes previously studied. The Anabaena enzyme also shows significant activity when NADH is used as a reductant.

Amino Acid Sequence↗
PubMed1988

Glutathione reductase inhibitors as potential antimalarial drugs. Effects of nitrosoureas on Plasmodium falciparum in vitro.

Malarial parasites are believed to be more susceptible to oxidative stress than their hosts. BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) and HeCNU(1-(2-chloroethyl)-3-(2-hydroxythyl)-1-nitrosourea), inhibitors of the antioxidant enzyme glutathione reductase, were found to prevent the growth of Plasmodium falciparum in all intraerythrocytic stages. When exposing infected red blood cells to 38 microM BCNU or 62 microM HeCNU for one life cycle of synchronously growing parasites, the parasitemia decreased by 90%. During the formation of new ring forms, the parasites are even more susceptible to these drugs. The treatment with BCNU or HeCNU produced a rapid depletion of GSH in the parasites and their host cells; in addition, protection against lipid peroxidation was impaired in these cells. Possible mechanisms for the antimalarial action of the inhibitors are discussed. Our results suggest that erythrocyte glutathione reductase, an enzyme of known structure, might be considered as a target for the design of antimalarial drugs.

Animals↗
PubMedDate not supplied

Purification by affinity chromatography of glutathione reductase (EC 1.6.4.2) from Escherichia coli and characterization of such enzyme.

The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2'.5'-ADP-Sepharose affinity column from which it was specifically eluted by a 0-10 mM NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells, 6.9 mg of pure enzyme was obtained after a 2632-fold purification, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein, with an A272/A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 A hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH, respectively, as reductants. Apparent K'm values of 16, 377, and 66 microM were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD, NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing significantly only after 10 min at 70 degrees C. A marked activity loss was observed however, even at 0 degrees C, in the presence of 20 microM NADPH. The enzyme was inactivated by low concentrations of para-hydroximercuribenzoate; the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.

Chromatography, Affinity↗