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PubMed1987

Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli.

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.

Amino Acids↗

PubMed1986

Effect of garlic oil extract on glutathione reductase levels in rats fed on high sucrose and alcohol diets: a possible mechanism of the activity of the oil.

The effect on glutathione reductase activities of feeding garlic oil to white albino rats maintained on high sucrose and alcohol diets was studied. Whereas high sucrose and alcohol diets resulted in significant increases in the activity of glutathione reductase in liver, kidneys and serum, the presence of garlic oil restored the levels to near normal. It is proposed that the mechanism of this action of garlic oil involves the active principle, diallyl disulphide, which interacts in an exchange reaction with enzymes and substrates such as glutathione reductase and glutathione which contain thiol groups.

Animals↗

PubMed1985

Effect of dinitrophenyl modification on oxidation-reduction of glutathione reductase from yeast.

Covalent modification of glutathione reductase (GR) from yeast with 1-fluoro-2,4-dinitrobenzene (FDNB) inhibited the NADPH-GSSG reductase activity completely. This modification also decreased the NADPH-thio-NADP+ transhydrogenase activity, stimulated the NADPH-oxidase activity, and induced the NADPH-cytochrome c reductase activity. Spectrophotometric titration showed that one tyrosine residue per FAD was modified with a dinitrophenyl group. The modified enzyme showed conversion of the two-electron reduced form (EH2) to the four-electron reduced form (EH4) in anaerobic conditions and conversion of EH2 to the oxidized form (E) in aerobic conditions. These results indicate that the modification of one tyrosine residue of the active site induces the instability of EH2.

Binding Sites↗

PubMed1990

Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli.

Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned.

Alanine↗

PubMed1986

Purification and properties of glutathione reductases from bovine ciliary body.

Two glutathione reductases, designated GR-I and GR-II, were purified from bovine ciliary body to apparent homogeneity by ion-exchange chromatography on DEAE-agarose and gel filtration on Sephacryl S-200. The two enzymes were isolated whether protease inhibitors were included or not in the medium during tissue homogenization. GR-I was found to have a molecular weight of about 140,000 by gel filtration and be composed of two identical subunits. On the other hand, GR-II was shown to exist as aggregates with molecular weights larger than 670,000. The subunit molecular weight of GR-II was estimated to be about 45,000 by SDS-polyacrylamide gel electrophoresis. The high molecular aggregate dissociated into smaller species by treatment with mercaptoethanol. The isoelectric points of GR-I and GR-II were determined to be 5.8 and 6.4, respectively. The K'm values for NADPH and oxidized glutathione (GSSG) were 22- and 84 microM for GR-I and 11- and 98 microM for GR-II. Coenzyme A-glutathione disulfide served as a substrate, though it was much less reactive than oxidized glutathione, for both enzymes. S-alkyl glutathiones were inactive as substrates. Nitrofurantoin was a non-competitive inhibitor of the enzymes. 1,3-bis(2 chloroethyl)-1-nitrosourea (BCNU) inhibited GR-I and GR-II irreversibly. The two enzymes are similar, if not identical, in their amino-acid composition. The two enzymes are also very similar on the basis of the relative position of tryptic peptides separated in two-dimensional peptide maps. From the tissue content and turnover numbers of these enzymes it is concluded that bovine ciliary body has a sufficient capability for rapid reduction of oxidized glutathione produced in the tissue during glutathione-dependent peroxide detoxification.

Amino Acids↗

PubMed1976

Mapping of the gene for glutathione reductase on chromosome 8.

Red cell glutathione reductase (E-GSR) activity in 3 patients with mos46,XY/47,XY,+8 was higher than the mean value in controls, confirming the previous assignment of the E-GSR locus to chromosome 8. In a infant with a terminal deletion of the short arm of chromosome 8, 46,XX,del(8)(:p21 leads to qter), E-GSR activity was markedly lower than in infant controls. The authors suggest that the E-GSR locus is in the region 8p21 leads to pter.

Chromosome Mapping↗

PubMed1992

Glutathione reductase from Saccharomyces cerevisiae undergoes redox interconversion in situ and in vivo.

Redox interconversion of glutathione reductase was studied in situ with S. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 microM NADPH or 100 microM NADH caused 50% inactivation in normal cells in 30 min, 0.75 microM NADPH or 50 microM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP+ concentrations. The inactivation of yeast glutathione reductase has also been studied in vivo. The activity decreased to 75% after 2 hours of growth with glucono-delta-lactone as carbon source, while NADPH rose to 144% and NADPH+ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 microM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD+ and NADPH/NADP+ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-delta-lactone is oxidized promotes in vivo inactivation of glutathione reductase.

Cell Membrane Permeability↗

PubMed1994

Low glutathione reductase and peroxidase activity in age-related macular degeneration.

Age-related macular degeneration (ARMD) may result from events initiated by reactive oxygen species. Blood samples from 18 patients with ARMD and 18 similarly aged controls were analysed for activities of important antioxidants. Blood glutathione reductase activity was lower in patients with ARMD compared with controls (p = 0.035). The activities of glutathione peroxidase (p = 0.18) and erythrocyte superoxide dismutase (p = 0.29) were similar between the two groups by a Student's two sample t test. Logistic regression was used to determine which enzyme activities were associated with ARMD after adjusting for possible confounding variables: smoking history, age, multivitamin use, and cardiovascular disease. Glutathione reductase activity (p = 0.05) and glutathione peroxidase activity (p = 0.065) were significantly associated with ARMD by this analysis. The relation of glutathione reductase and glutathione peroxidase activity to ARMD merits further study.

Aged↗

PubMed1986

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima.

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.

Amino Acids↗

PubMed2004

Processing of the dual targeted precursor protein of glutathione reductase in mitochondria and chloroplasts.

Pea glutathione reductase (GR) is dually targeted to mitochondria and chloroplasts by means of an N-terminal signal peptide of 60 amino acid residues. After import, the signal peptide is cleaved off by the mitochondrial processing peptidase (MPP) in mitochondria and by the stromal processing peptidase (SPP) in chloroplasts. Here, we have investigated determinants for processing of the dual targeting signal peptide of GR by MPP and SPP to examine if there is separate or universal information recognised by both processing peptidases. Removal of 30 N-terminal amino acid residues of the signal peptide (GRDelta1-30) greatly stimulated processing activity by both MPP and SPP, whereas constructs with a deletion of an additional ten amino acid residues (GRDelta1-40) and deletion of 22 amino acid residues in the middle of the GR signal sequence (GRDelta30-52) could be cleaved by SPP but not by MPP. Numerous single mutations of amino acid residues in proximity of the cleavage site did not affect processing by SPP, whereas mutations within two amino acid residues on either side of the processing site had inhibitory effect on processing by MPP with a nearly complete inhibition for mutations at position -1. Mutation of positively charged residues in the C-terminal half of the GR targeting peptide inhibited processing by MPP but not by SPP. An inhibitory effect on SPP was detected only when double and triple mutations were introduced upstream of the cleavage site. These results indicate that: (i) recognition of processing site on a dual targeted GR precursor differs between MPP and SPP; (ii) the GR targeting signal has similar determinants for processing by MPP as signals targeting only to mitochondria; and (iii) processing by SPP shows a low level of sensitivity to single mutations on targeting peptide and likely involves recognition of the physiochemical properties of the sequence in the vicinity of cleavage rather than a requirement for specific amino acid residues.

Amino Acid Sequence↗

PubMed1992

Antioxidant-influenced alterations in glutathione reductase activity in different age groups of male mice.

The glutathione reductase activity was estimated in brain and liver of male mice of different age groups fed on butylated hydroxy anisole (BHA), propyl gallate (PG) and alpha-tocopherol (TP). A significant increase in glutathione reductase activity was observed with TP during all the phases (pre-reproductive, reproductive and post-reproductive). Antioxidants enhanced the enzyme activity in both the tissues with the exception of BHA in brain during reproductive and PG in liver during pre-reproductive phases respectively. The enhanced reducing potential of the cell in terms of added bioconversion of GSSG to GSH may account for increase in longevity on antioxidant treatment.

Aging↗

PubMed1991

Protein-chemical standardization of the erythrocyte glutathione reductase activation test (EGRAC test). Application to hypothyroidism.

The erythrocyte glutathione reductase activation coefficient (EGRAC) is an index of riboflavin deficiency or, more exactly, of FAD deficiency in man. In this report a sensitive version of the EGRAC test is introduced that is based on the molecular properties of glutathione reductase and its FAD-free apoenzyme. The hemoglobin concentration of the blood sample can be estimated simultaneously using the spectrophotometric absorption at 340 nm. - The method was tested for 33 thyroidectomized patients in comparison with a euthyroid control group. From the average EGRAC values (1.40 vs. 1.22) it was deduced that the average free FAD level was approximately 2 times lower for the patients' than for the control group. Discussed is the role of the EGRAC test in hormonal and nutritional disorders.

Adult↗

PubMed1984

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

Animals↗

PubMed1994

No evidence for inhibition of human glutathione reductase by valproic acid.

The human red blood cell enzyme glutathione reductase (GR) was reported to be inhibited by the anticonvulsant drug valproic acid (VPA) [Cotariu et al., Biochem Pharmacol 43: 425-429, 1992]. When attempting to reproduce and extend these experiments, we could not detect any significant effect of VPA on glutathione reductase in haemolysates from 20 healthy children and 10 children under VPA therapy, no matter which concentration of the drug (0.9 or 1.8 mM in a haemolysate diluted by a factor of 50 or 1.8 mM directly in the assay), which incubation time (0-60 min) and which assay system were chosen. An influence of VPA on FAD-free apoglutathione reductase was also excluded. GR-activities of 10 children under VPA therapy (1.08 +/- 0.14 U/mL blood or 7.57 +/- 0.94 U/g Hb) were almost identical with the activities of age- and sex-matched controls (1.04 +/- 0.17 U/mL or 7.79 +/- 1.32 U/g Hb). No correlation between erythrocyte GR activity and serum levels of VPA was observed. Finally, incubation of crystalline human GR with VPA did not lead to enzyme inhibition; rather, in most experiments the enzyme was stabilized by incubation with VPA. Possible explanations for the discrepancies between the results of Cotariu et al. and our data are discussed.

Adolescent↗

PubMed1986

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer.

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.

Cytochrome c Group↗

PubMed2002

Serum glutathione-S-transferase and glutathione reductase activity in head and neck cancer patients.

Glutathione, an antioxidant plays an important role in phase-II detoxification of carcinogens. The levels of reduced glutathione are maintained by glutathione-depleting as well as replenishing enzymes such as glutathione-s-transferase (GST) and glutathione reductase (GR), respectively. Pre and post treatment changes in GST and GR activities in head and neck cancer patients were analysed. Serum GST and GR were analysed from untreated head and neck cancer patients (PT) (n=146), controls with habit of tobacco (VHT) (n=25) as well as without (no) habit of tobacco (NHT) (n=25) and patients with oral precancerous conditions (OPC) (n=50). The cancer patients were followed-up after initiation of anticancer therapy. Follow-up blood samples were collected. Serum GST and GR activities were estimated by highly sensitive and specific spectrophotometric methods. Untreated cancer patients showed elevated mean serum GST and GR activities as compared to NHT. Patients with OPC had declined mean GST activity as compared to WHT and untreated cancer patients. Paired t-test revealed that complete responders (CR) showed significantly elevated GST levels and declined GR activities (p < 0.001) as compared to those in PT. No correlation was found between stage of the disease and GST, GR activity. Paired t-test showed significant decreased in GR activity in nonresponders (NR) treated with radiotherapy (p=0.01). The study suggested that analysis of glutathione and glutathione-depleting enzymes can be helpful for treatment monitoring of head and neck cancer patients.

Adult↗

PubMed1976

Clinical significance of serum glutathione reductase in various clinical conditions, especially in liver diseases.

Serum glutathione reductase activity was measured in various conditions including acute hepatitis, chronic hepatitis, liver cirrhosis, malignant neoplastic diseases, and obstructive jaundice. A statistically significant elevation of the enzyme activity was found in all of these clinical conditions above normal value, especially in patients with acute hepatitis, some liver cancer, and malignant biliary obstruction. Comparison with other liver function tests showed the existence of statistically significant correlations of serum glutathione reductase with SGOT, SGPT and alkaline phosphatase in acute hepatitis, and with alkaline phosphatase in cirrhosis. In parenchymatous liver disease, serial determination was found to be important. High values in obstructive jaundice suggest the malignant obstruction.

Adult↗

PubMed1981

Glutathione reductase from yeast. Differential reactivity of the nascent thiols in two-electron reduced enzyme and properties of a monoalkylated derivative.

Two-electron reduced glutathione reductase from yeast reacted with iodoacetamide is alkylated almost exclusively in the nascent thiol nearer the amino terminus of the protein. The charge-transfer absorbance, maximal at 530 nm, characteristic of the two-electron reduced enzyme is not lost as the alkylation proceeds, and the product has a spectrum virtually identical with that of the two-electron reduced enzyme. This observation demonstrates that the thiol alkylated is not the charge-transfer-donor thiolate which interacts with the FAD. The spectrum of the monoalkylated derivative is stable in the presence of oxidized glutathione, indicating that the charge-transfer-donor thiol is not involved in interchange with the substrate in the native enzyme. Thus, the nascent thiols produced upon two-electron reduction of glutathione reductase have distinct functions, interchange with the substrate and interaction with the FAD. Treatment of the monoalkylated derivative with the apolar phenylmercuric acetate eliminates the charge-transfer interaction. The spectrum of the resulting species is similar to that of the oxidized enzyme but less resolved and blue shifted by 10 nm. The dependence on pH of the absorbance associated with the thiolate to FAD charge-transfer interaction in native two-electron reduced glutathione reductase is biphasic, with pK values at approximately 4.8 and 7.4. By analogy with glyceraldehyde-3-phosphate dehydrogenase and papain, these data indicate that the thiolate is stabilized by an adjacent basic residue. The pK 7.4 is associated with the titration of the base to give the ion pair, and the pK of 4.8 is associated with the titration of the thiolate. Unlike lipoamide dehydrogenase, glutathione reductase is sufficiently stable to allow titration with dithionite at pH 3.7. The spectrum at this pH is essentially the same as that of the monoalkylated derivative treated with phenylmercuric acetate. The changes with pH are completely reversible.

Alkylation↗