Mercuric reductase: homology to glutathione reductase and lipoamide dehydrogenase. Iodoacetamide alkylation and sequence of the active site peptide.
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Alkylation↗
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Results for “GLUTATHIONE REDUCTASE”
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[Glutathione reductase and glutathione-insulin-transhydrogenase and their participation in the oxydation-reduction transformation of glutathione].
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Animals↗
Plant Morphological and Biochemical Responses to Field Water Deficits: III. Effect of Foliage Temperature on the Potential Activity of Glutathione Reductase.
Activity of glutathione reductase has been related to stress tolerance; however, these enzyme assays are generally conducted at 25 degrees C. Foliage temperature varies greatly in the field in response to soil water availability and ambient conditions and this may affect enzyme response. This study was conducted to determine the effect of changing foliage temperature on glutathione reductase activity of wheat under field conditions. Wheat leaf glutathione reductase was purified and the temperature response of the enzyme was determined at 2.5 degrees C intervals between 12.5 and 45 degrees C. These data, in conjunction with continuous measurements of field-grown wheat foliage temperatures, were used to compare the temperature-related changes in potential glutathione reductase activities in water stressed and control plants. Assuming saturating substrate levels, the results indicate that early in the season the daily potential enzyme activity of the irrigated and stressed plants could never have reached the daily activity predicted from the 25 degrees C (room temperature) measurements. Later in the season, the daily potential activity of the irrigated plants was lower, and the daily potential activity of the stressed plants was higher, than the activities predicted from the 25 degrees C determinations. These results suggest that a better understanding of the regulation of plant metabolism will be obtained by linking continuous temperature measurements of plant foliage with enzyme responses to temperature.
Journal Article↗
Effect of heparin and dextran sulfate on the activity of glutathione reductase from yeast.
Glutathione reductase (EC 1.6.4.2) is a pivotal enzyme of the glutathione antioxidant system in a cell. The kinetic studies of the interaction of glutathione reductase with unfractionated and low molecular weight heparin and dextran sulfate can contribute to explanation of polyanions effect on the conformation changes of glutathione reductase. The tested polyanions inhibit this enzyme and the inhibition effect depends on the ionic strengths and pH value. The most potent inhibitor is dextran sulfate (ID50 is 4.1 micrograms/ml, pH = 6.8, without NaCl). The ionic strength (> 100 mM) allows the reactivating of GR if the concentration of DS is not higher than 80 micrograms/ml. The inhibition effect of tested polyanions is caused by electrostatic interactions with enzyme; the kinetic analyses indicate that it is a mixed inhibition with respect to oxidized glutathione or NADPH.
Anions↗
Glutathione reductase, selenium-dependent glutathione peroxidase, glutathione levels, and lipid peroxidation in freshwater bivalves, Unio tumidus, as biomarkers of aquatic contamination in field studies.
The aim of this study was to evaluate the usefulness of antioxidant parameters in the freshwater bivalve, Unio tumidus, as biomarkers of exposure to pollutants and to study their potential interest in predicting toxicity. Selenium-dependent glutathione peroxidase (Se-GPx), non-selenium-dependent glutathione peroxidase (non-Se-GPx), glutathione reductase (GRd), catalase, and superoxide dismutase (SOD) activities; reduced (GSH) and oxidized (GSSG) glutathione levels; and lipid peroxidation were measured in the gills and digestive glands of Unio. Control mussels were encaged and transplanted for 15 and 30 days to sites where the contamination of sediments was analyzed, along a river receiving domestic and industrial sources of pollution. After 15 days of exposure, all antioxidant parameters of the bivalves transferred to the most polluted sites had strongly decreased compared with control values. This was particularly true for Se-GPx and GRd activities, which were inhibited by 60 and 80% in the two tissues, and for GSH levels (80% reduction in the gills and 60% in digestive glands). These decreases were associated in the gills with lipid peroxidation (measured by malondialdehyde content) and with a high level of contamination of sediments by polycyclic aromatic hydrocarbons and polychlorinated biphenyls. In the mussels exposed at the least polluted sites, the same parameters decreased in the gills, but to a lesser extent: 50% for Se-GPx and 32% for GRd activities, and 45% for GSH levels. The gills appeared more sensitive than the digestive glands. After 30 days of exposure, while Se-GPx, GRd, and GSH remained reduced, a significant induction of non-Se-GPx and catalase activities was recorded in the gills, which reflected an adaptation of the transplanted species to their unsafe environment. All the results indicated that antioxidant defense components, namely, Se-GPx, GRd, and GSH, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The relationship between the degree of deficiency of antioxidant defenses and lipid peroxidation suggests that these parameters could also be biomarkers for toxicity.
Animals↗
Inhibition of thioredoxin reductase but not of glutathione reductase by the major classes of alkylating and platinum-containing anticancer compounds.
Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.
Animals↗
Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity.
The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured.
Amino Acid Sequence↗
Effect of cystamine on rat tissue GSH level and glutathione reductase activity.
Reduced glutathione (GSH) level and glutathione reductase activity were determined by means of the spectrophotometric method in various rat tissues after i.p. administration of cystamine (50 mg/kg and 20 mg/kg). GSH amount dropped in the spleen and kidney at 10 and 20 min; following this interval, an increase of GSH level was observed in the liver at 20--30 min, in the spleen and kidney at 60 min after the treatment with a radioprotective cystamine dose (50 mg/kg). The changes in GSH level induced by a non-radioprotective cystamine dose (20 mg/kg) had an opposite tendency. The activity of glutathione reductase was decreased in all tissues studied. As to the mechanism of the radioprotective action, both the inactivation of glutathione reductase activity and the changes in GSH level seem to be the factors contributing to the radioprotective effect of cystamine by strengthening the cellular radioresistance.
Animals↗
Influence of riboflavin and of erythrocyte stroma on glutathione reductase activity in normal, glucose 6 phosphate dehydrogenase deficient and low glutathione reductase individuals.
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Cell Membrane↗
Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids.
Glutathione reductase from trypanosomes and leishmanias, unlike glutathione reductase from other organisms, requires an unusual low molecular weight cofactor for activity. The cofactor was purified from the insect trypanosomatid Crithidia fasciculata and identified as a novel glutathione-spermidine conjugate, N1,N8-bis(L-gamma-glutamyl-L-hemicystinyl-glycyl)spermidine, for which the trivial name trypanothione is proposed. This discovery may open a new chemotherapeutic approach to trypanosomiasis and leishmaniasis.
Animals↗
Use of transferred nuclear-Overhauser-effect spectroscopy to measure the bound conformation of a disulphide-replaced analogue of glutathione disulphide as an inhibitor of yeast glutathione reductase.
The analogue of glutathione disulphide (GSSG) in which the disulphide bridge of GSSG is replaced by -CH2-S- was synthesised from L-cystathionine using t-butoxycarbonyl and t-butyl ester protection with triethylsilane-promoted deprotection. This analogue (GCSG) was found to be a linear, competitive inhibitor of yeast glutathione reductase (Ki value 981 microM at pH 7.0), a very poor substrate and not to act as an irreversible inhibitor of glutathione reductase. The weak binding of GCSG to glutathione reductase permitted the use of transferred nuclear Overhauser effect spectroscopy (TRNOESY) to investigate the bound conformation of GCSG in its complex with glutathione reductase. The solution structure of free GCSG was investigated by NMR spectroscopy using a range of NMR techniques. The TRNOESY experiment allowed a range of conformations to be determined for the central bridge region (containing the -CH2-S- replacement) of GCSG bound to yeast glutathione reductase. Using the nuclear Overhauser effect constraints thus derived, in combination with molecular graphics and energy minimisation based on the known crystal coordinates of glutathione disulphide (GSSG) bound to human erythrocyte glutathione reductase, allowed an explanation of the lack of substrate activity of GCSG, its inactivity as a suicide inactivator and its relatively weak binding in terms of the enforced mislocation of the -CH2-S- bridge with respect to the catalytic residues (relative to GSSG). Thus, the simple replacement of -S- by -CH2-, common in medicinal chemistry, can lead to poor receptor binding if the replacement occurs in a central, rather than peripheral, part of the ligand under modification.
Binding Sites↗
Pea chloroplast glutathione reductase: purification and characterization.
Glutathione reductase (EC 1.6.4.2) was purified from intact pea (Pisum sativum) chloroplasts by a method which includes affinity chromatography on ADP-agarose. Fractions from the affinity column which had glutathione reductase activity consisted of polypeptides of 60 and 32 kilodaltons. Separation of the proteins by electrophoresis on native gels showed that glutathione reductase activity was associated with 60 kilodalton polypeptides and not with the 32 kilodalton polypeptides. Antibodies to spinach whole leaf glutathione reductase (60 kilodaltons) cross-react with the chloroplast 60 kilodalton glutathione reductase but not the 32 kilodalton polypeptides. In the absence of dithiothreitol the 60 kilodalton polypeptides showed a shift in apparent molecular weight on sodium dodecyl sulfate gels to 72 kilodaltons. Dithiothreitol did not alter the activity of the chloroplast enzyme. Chloroplast glutathione reductase is relatively insensitive to NADPH.
Journal Article↗
Molecular organization of the glutathione reductase gene in Drosophila melanogaster.
Glutathione reductase catalyzes the conversion of the oxidized form of glutathione to regenerate reduced glutathione, which acts as a versatile intracellular reductant. The present study provides initial characterization of the glutathione reductase gene in Drosophila melanogaster and its response to experimentally induced oxidative stress. Drosophila cDNA clones were isolated, based on cross-hybridization to the Musca domestica glutathione reductase cDNA. Genomic clones were isolated by cross-hybridization with the Drosophila cDNA as hybridization probe. Northern analysis of adult Drosophila poly(A)+ RNA, utilizing the Drosophila cDNA probe, revealed a hybridization signal in the 2-kb range. The entire sequence of one cDNA was determined. In addition to a coding domain of 1431 bases, the sequence included 206 bases upstream of a putative start codon and 355 bases downstream of a putative stop codon. Based on the cDNA sequence, the 476 amino acid sequence of the Drosophila glutathione reductase gene was deduced and was found to have extensive similarities with the glutathione reductase gene from other species. Gene mapping of a 13-kb genomic fragment revealed that the glutathione reductase gene consists of at least two exons spanning approximately 5 kb. A first exon contains sequence for only the first 5 amino acids and the first base of the sixth and appears to be separated by a ca. 2.5-kb intron from the remainder of the coding region, which is confined to <2 kb. The Drosophila glutathione reductase is single copy and its cytogenetic position, as determined by in situ hybridization, is 7D-E on the X chromosome. mRNA levels of glutathione reductase, measured by RT-PCR, increased in response to exposure to 100% ambient oxygen by almost twofold and administration of paraquat by greater than threefold. Exposure of flies to hyperoxia also induced a 60% increase in the activity of glutathione reductase and augmented the concentration of total glutathione by ca. 40% following an initial drop. The present study, besides providing an initial molecular characterization of the glutathione reductase gene in Drosophila, demonstrates its dynamic involvement in response to experimentally induced oxidative stress.
Amino Acid Sequence↗
Stimulatory effect of histone on glutathione reductase of human erythrocytes.
Glutathione reductase of hemolyzates, as well as intact erythrocytes from normal individuals can be activated by the addition of human leukocyte nuclear histones and histone subfractions f1 and f3. Intact erythrocytes incubated with histone show also in their hemolyzates a high glutathione reductase activity as compared with such not treated with histone cells. The degree of stimulation of glutathione reductase by histones is comparable to that caused by flavin adenosine diphosphate (FAD). Histones and FAD show a competitive stimulatory effect on glutathione reductase activity. ATP shows an inhibitory effect on glutathione reductase activity, which can be reversed by addition of histone.
Adenosine Triphosphate↗
Effects of nicotine and vitamin E on glutathione reductase activity in some rat tissues in vivo and in vitro.
Effects of nicotine, and nicotine+vitamin E on glutathione reductase (Glutathione: NADP(+) oxidoreductase, EC 1.8.1.7) activity in the muscle, heart, lungs, testicles, kidney, stomach, brain and liver tissues were investigated in vivo and also in vitro. The groups were: nicotine [0.5 mg/kg/day, intraperitoneal (i.p.)]; nicotine+vitamin E [75 mg/kg/day, intragastric (i.g.)]; and control group (receiving only vehicles). There were eight rats per group and supplementation period was 3 weeks. The results showed that nicotine (0.5 mg/kg, i.p.) inhibited glutathione reductase activity significantly in the liver, lungs, heart, stomach, kidney, and testicles by approximately 61.5%, approximately 65%, approximately 70.5%, approximately 72.5%, approximately 64% and approximately 71.5%, respectively, while it had activated glutathione reductase activity in the brain by approximately 11.8%, and had no effect on the muscle glutathione reductase activity. Vitamin E supplementation prevented this nicotine-induced decrease in glutathione reductase activity in liver, lungs, heart, stomach, and kidney. However, it did not prevent this nicotine-induced decrease in testicles. In vitro studies were also carried out to elucidate the effects of nicotine and vitamin E on glutathione reductase activity. In vitro results correlated well with in vivo experimental results in liver, lungs, heart, stomach, and testicular tissues. These results show that vitamin E administration generally restores the inactivation of glutathione reductase activity due to nicotine administration in various rat tissues in vivo, and also in vitro.
Animals↗
Glutathione reductase deficiency in red cells in aplastic anemia. II. Effects of flavine compounds on red cell glutathione reductase.
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Anemia, Aplastic↗
Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase.
Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 microM EDTA or 10 microM DETAPAC yielding full protection. Ag+, Zn2+ and Cd2+ potentiated the redox inactivation promoted by NADPH alone, while Cr3+, Fe2+, Fe3+, Cu+, and Cu2+ protected the enzyme. The Zn2+ and Cd2+ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn2+ or Cd2+. The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP+ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.
Anaerobiosis↗
The regulation of gelation of Phloem exudate from cucurbita fruit by dilution, glutathione, and glutathione reductase.
The average glutathione equivalent concentration in phloem exudate collected from squash fruit (Cucurbita moschata [Duchesne] Poir. var Butternut) and pumpkin fruit (Cucurbita pepo [L.] var Jack-o-lattern) was 1.02 and 0.60 millimolar, respectively. Glutathione reductase (EC 1.6.4.2) activity in phloem exudate from squash and pumpkin fruit averaged 0.48 and 1.74 micromole NADPH oxidized per minute per milliliter, respectively. Protein concentrations in fruit phloem exudates averaged 67 milligrams per milliliter for squash and 57 milligrams per milliliter for pumpkin. The phloem-specific P-proteins account for most of the protein content of exudate. Pure exudate from fruit does not gel for hours or days, but when diluted with neutral or alkaline aqueous solutions, exudate gels rapidly. Exudate solutions undergo biphasic pH changes with dilution. We suggest that P-protein undergoes conformational change upon dilution, exposing titratable groups and sulfhydryl residues. Oxidation of the latter forms the intermolecular disulfide bridges of the gel. The gelation of diluted exudate is regulated by factors (oxygen, pH, glutathione, NADPH) which affect the maintenance of reduced sulfhydryl residues and the activity of glutathione reductase. While these factors may also act in vivo to regulate redox conditions in phloem, their relationship to hypothetical sol/gel transitions or motile and nonmotile phases in the transport conduit is unknown.
Journal Article↗