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PubMedDate not supplied

[The activity of glutathione-reductase and FAD-effect as indicators of the riboflavin level in experiments and in patients with phenylketonuria].

Subjects of discussion in the paper are various methods of determining the riboflavin allowances and the results achieved by the application of two of them that concern determining the activity of the glutathione-reductase and the FAD-effect in the study of an experimental B2-deficiency in rats and the riboflavin allowances in children suffering from phenylketonuria. Exclusion of vitamin B2 from the ration of rats resulted in that in 8 weeks they developed B2-deficiency ascertained with the help of the above cited enzymatic tests. In a certain percentage of children with phenylketonuria after discontinued administration of group "B" vitamins (D1 diet) there could be observed an intensification of the FAD-effect and, accordingly, a reduced glutathione-reductase activity. Resumed taking of the vitamins (D2 diet) normalized the riboflavin allowance values in at least, one week's time.

Animals↗

PubMed2004

Native specific activity of glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (PHGPx) and glutathione reductase (GR) does not differ between normo- and hypomotile human sperm samples.

Glutathione-dependent selenoenzymes in human spermatozoa are responsible for a generalized protection against reactive oxygen species (ROS) as well as some other metabolic and structural regulation during spermiogenesis and sperm cell maturation. Glutathione peroxidase (GPx-1), phospholipid hydroperoxide glutathione peroxidase (GPx-4 or PHGPx) and glutathione reductase (GR) native specific activities have been studied in human Percoll-purified spermatozoa from healthy fertile subjects and asthenozoospermic patients. The mean values obtained for the three enzymes in normal specimens are 1.52 +/- 0.90 mU/10(6) sperm cells (PHGPx), 4.26 +/- 1.73 mU/10(6) sperm cells (GPx-1) and 1.95 mU/10(6) sperm cells (GR). No statistically significant differences for any of the three enzymes were encountered between these values and those of asthenozoospermic patients. These results are discussed and compared with recent literature data on both rescued and native PHGPx specific activity in human spermatozoa, as well as with data obtained for GPx in human seminal plasma.

Adult↗

PubMed1993

Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant.

NAD and NADP are ubiquitous coenzymes in biological redox reactions. They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP. The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme. Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases. Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial. We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH. A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme.

Amino Acid Sequence↗

PubMed1996

High-performance liquid chromatographic resolution of NADP+ after induction of fluorescence and its application to assay for an NADPH-dependent enzyme: application to the determination of glutathione reductase activity in plant leaf extracts.

A method is described for the determination of glutathione reductase activity (GR; EC 1.6.4.2) in plant extracts utilizing HPLC quantitation of NADP+ following the reduction of glutathione disulfides. After protein incubation, fluorescence of NADP+ was induced under strongly basic conditions, and the product was directly resolved from the reaction medium by isocratic reversed-phase elution on a silica-coated alumina support which took 2 min. The mobile phase was acetonitrile-water (50:50) delivered at a flow rate of 1.5 ml/min. The adduct (stable for at least 7 days) was detected fluorometrically and quantitated by direct integration of peak area.

Chromatography, High Pressure Liquid↗

PubMed1992

Glycation (non-enzymic glycosylation) inactivates glutathione reductase.

Non-enzymic binding of sugars to proteins (glycation) is a common biological phenomenon that is increased in diabetes. Most work has been directed towards structural proteins which may be present for many years and would continue to accumulate sugar residues. As glycation is a non-specific reaction, other proteins such as enzymes will also be susceptible to glycation and could well display altered activity. We investigated the effect of various sugars whose concentrations increase in diabetes in insulin-independent tissues on glutathione reductase, an enzyme that maintains the GSH level in cells. Glucose, glucose 6-phosphate and fructose all displayed a time-dependent inhibition of glutathione reductase activity, suggesting that these sugars glycate this enzyme. Aspirin gave some protection against the loss of activity induced by glucose.

Acetaminophen↗

PubMed1994

[Establishment of Chinese hamster ovary cell lines with reduced expression of glutathione reductase after antisense-oriented gene transfection and assessment of the sensitivity to oxidant injury].

Glutathione reductase (GR) protects tissues from oxidant injury by catalysing the reduction of glutathione disulfide (GSSG) to glutathione (GSH). In order to study the effect of GR in protecting cells from oxidant injury, we generated Chinese hamster ovary (CHO) cell lines stably transformed after antisense-oriented gene transfection. The coding region of the human GR was cloned using revere transcription PCR method and selected by transient expression study in mammalian cells. A clone HGR135 showed overexpression of GR in CHO cells and was proved to have no base substitution. This clone, then, was ligated into MEP4 expression vector in an antisense orientation to the human metallothionein promoter and transfected to CHO cells with polybrene. Among 12 cell lines isolated, G17 showed to have the least GR activity (48% of the control), while another four were mildly GR deficient. Southern hybridization of genomic DNA digests and transformation experiment on E. coli revealed that the promoter-antisense coding region component was integrated. Northern hybridization detected reduced amount of GR transcript but no antisense message. Baseline cellular GSH concentrations were lower in G17 than in control (25.7 +/- 2.5 vs. 36.1 +/- 1.9 nmole/mg protein, P < 0.05), while cellular GSSG concentrations were higher (0.61 +/- 0.19 vs. 0.39 +/- 0.09 nmole/mg protein, P < 0.05). After four hours of treatment of G17 and control cells with increasing doses (1 to 10 mM) of t-butylhydroperoxide (t-BuOOH), cellular GSH concentrations in G17 decreased with an elevation of GSSG concentration at 1 mM followed by no further increase at higher t-BuOOH concentration, while GSSG concentrations increased in the control cells without reduction of GSH concentrations at 1-5 mM t-BuOOH treatment. The concentrations of GSH were lower in G17 than in controls at all doses of t-BuOOH. Four hours of exposure to 10 mM t-BuOOH resulted in greater LDH release in G17 than in control (57.3 +/- 4.7 vs. 32.1 +/- 6.5%, P < 0.05). Similarly, G17 cells released more of their LDH to the media than did CHO cells in response to exposure to 95% O2 for 72 hours (19.3 +/- 5.9 vs. 11.9 +/- 5.4%, P < 0.05). The partial GR deficiency in G17 cells impairs their ability to recycle GSSG and this deficiency offers the best explanation for the increased sensitivity of these cells to injury by t-BuOOH or hyperoxia.

Animals↗

PubMedDate not supplied

Biochemical parameters as biomarkers for the early recognition of environmental pollution on Scots pine trees. II. The antioxidative metabolites ascorbic acid, glutathione, alpha-tocopherol and the enzymes superoxide dismutase and glutathione reductase.

Field investigations with Scots pine trees (Pinus sylvestris L.) were performed in eastern Germany, where ambient SO2, NOx and O3 concentrations differed significantly in 1992-99 at three sites, namely Neuglobsow (yearly mean SO2 in 1992: 9 microg m(-3)), Taura (yearly mean SO2 in 1992: 54 microg m(-3)) and Rösa (yearly mean SO2 in 1992: 73 microg m(-3)). To investigate the effects of SO2, NOx and O3 on antioxidants (superoxide dismutase, ascorbic acid, glutathione, glutathione reductase, alpha-tocopherol) and pigments including chlorophyll fluorescence as well as visible damage symptoms in the form of needle yellowing and tip necroses, needles of the 1st and 2nd age class from young and mature trees were collected at the sites every October. Eight years after the start of the field study in 1992, the ambient SO2 concentrations had decreased significantly at Neuglobsow (yearly mean SO2 in 1999: 4 microg m(-3)), Taura (yearly mean SO2 in 1999: 5 microg m(-3)) and Rösa (yearly mean SO2 in 1999: 5 microg m(-3)). NOx and O3 differed less at the three sites and showed no temporal variations. Whole needle glutathione continuously decreased, although concentrations were higher in needles of the 1st and 2nd age class from the polluted sites Taura and Rösa than the unpolluted site Neuglobsow. The activities of glutathione reductase exhibited the same site-related differences and temporal variations and were correlated with concentrations of oxidized glutathione (GSSG). In contrast, the activities of the enzyme superoxide dismutase and the concentrations of whole needle ascorbic acid remained unchanged over the period. Only at the end of the investigation period did the concentrations of oxidized ascorbic acid (dehydroascorbate) increase in six-month-old needles at the polluted sites Taura and Rösa. Despite the clear decreases in SO2, the visible symptoms of needle tip necroses remained unchanged, especially at the polluted sites Taura and Rösa, although the needles contained higher pigment concentrations than needles from the unpolluted sites. The results of measurements with antioxidants as biomarkers for SO2-mediated stress in pine needles show that the adult Scots pine trees at the polluted sites suffered from greater oxidative stress than the needles from the less polluted site.

Ascorbic Acid↗

PubMed1996

Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in Pinus sylvestris L. needles.

Glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) are two well-known enzymes involved in the scavenging of reactive oxygen intermediates. However, little is known about the regulation of Gor and Sod genes in plant cells. To obtain information about hypothetical redox regulatory mechanisms controlling Gor and Sod gene expression we artificially enhanced the levels of reduced and oxidized forms of glutathione (GSH and GSSG) in Pinus sylvestris L. needles. Scots pine shoots were placed for 12 h in beakers containing 5 mM GSH, 5 mM GSSG or water. Increased levels of both GSSG and GSH were observed in the GSSG-treated needles after 3 h. In contrast, only the GSH level was increased by the GSH treatment. Thus, the GSH/GSSG ratio increased up to 15-fold during the GSH treatment and decreased approximately two-fold during the GSSG treatment. The GR activity was significantly higher (60%) when GSSG was applied, without any apparent change in the amount and isoform population of GR or accumulation of Gor gene transcripts. This indicates that the GR activity increased per se in the GSSG treatment. The level of cytosolic CuZn-Sod transcripts was decreased significantly by the GSH treatment without any change in enzyme activity. The chloroplastic CuZn-Sod gene generally showed a more stable transcript level in the different treatments. However, a similarity between the cytosolic and chloroplastic levels of CuZn-Sod transcripts could be observed in different treatments. This suggests that the redox state of glutathione plays an important role in the in vivo regulation of CuZn-Sod gene expression in plants.

Amino Acid Sequence↗

PubMed1984

Glutathione reductase and nitroblue tetrazolium reduction deficiencies in neutrophils of patients with primary idiopathic myelofibrosis.

Latex ingestion and nitroblue tetrazolium reduction were measured in neutrophils of 40 patients with primary idiopathic myelofibrosis. The percentage of neutrophils that ingested latex, in the presence of either autologous or control sera, was lower (P less than 0.001) than that of the controls. The percentage of the ingesting neutrophils that reduced nitroblue tetrazolium was also lower (P less than 0.001) in the patients than in the controls. Activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione peroxidase and NAD(P)H oxidases were not different from those of the controls. In contrast, glutathione reductase activity was significantly lower (P less than 0.001) in the patients than in the controls either with or without the addition of flavin adenine dinucleotide. Glutathione reductase and nitroblue tetrazolium reduction activities were correlated (r = 0.913). These results are discussed within the framework of the acquired enzymopathies and the increased susceptibility to infection observed in these patients.

Adult↗

PubMed1976

Trisomy 8 in the bone marrow associated with high red cell glutathione reductase activity.

In a series of 841 patients with hematologic disorders, 10 individuals were found to have an extra C group chromosome in their bone marrow cells. In two the extra chromosome was not identified, but in the remaining eight it was No. 8. Four of these ten patients had leukemia, and the others had cytopenias or other probably preleukemic conditions. The mean value for glutathione reductase activity in the red cells of four patients with trisomy 8 was significantly higher (2980 +/- 940 mumoles/min/liter of erythrocytes) than in normal controls (1930 +/- 360) or in any of five different control groups of patients with hematologic disorders. The extent of enzyme activation as a result of preincubation with exogenous flavin adenine dinucleotide was similar in the erythrocytes of all groups. The reasons for the high values of red cell glutathione reductase activity in patients with trisomy 8 are discussed in the light of the proposed assignment of the gene for that enzyme to chromosome 8.

Anemia, Aplastic↗

PubMed1989

Effect of vitamin E on survival, glutathione reductase and formation of chromium (V) in Chinese hamster V-79 cells treated with sodium chromate (VI).

The effect of vitamin E on cytotoxicity induced by Na2CrO4 was evaluated by colony-forming assay using Chinese hamster V-79 cells. Pre-treatment with alpha-tocopherol succinate (vitamin E) for 24 h prior to exposure to Na2CrO4 resulted in a marked decrease in the cytotoxicity caused by this compound. The reduction of chromate-induced cytotoxicity was observed at all concentrations of Na2CrO4 (5-15 microM), and the protective effect increased with higher concentrations of vitamin E (5-25 microM). The level of glutathione reductase activity, which is capable of reducing chromate, was not affected by cellular pre-treatment with vitamin E. However, Na2CrO4 decreased glutathione reductase activity in a concentration-dependent fashion (5-15 microM) and pretreatment with vitamin E resulted in a significant recovery of enzyme activity suppressed by Na2CrO4, suggesting that this enzyme inhibition is linked to the cytotoxicity of this metal. Electron spin resonance studies showed that a paramagnetic chromium (V) complex was formed in cells treated with Na2CrO4, and that cellular pre-treatment with vitamin E reduced the formation of this chromium (V) intermediate. These results indicate that vitamin E protects cells from chromate-induced cytotoxicity as well as from enzyme inhibition, and also suggest that Na2CrO4-induced cytotoxicity is mediated by the generation of a reactive intermediate.

Animals↗

PubMed1999

Purification of glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells.

Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.

Animals↗

PubMed1980

Glutathione-peroxidase and glutathione-reductase activities of normal and pathologic human liver: relationship with age.

Liver glutathione-peroxidase (L-GSH-Px) and glutathione-reductase (GSSG-Red) activities were measured in supernatants of liver tissues obtained from a total of 36 subjects. Sixteen of these patients had a functionally normal liver (control group), whereas of the remaining 20 patients, 10 were cirrhotic and 10 had a liver disease other than cirrhosis. The mean value of L-GSH-Px of the control group was 33.12 +/- 12.66 U/g protein, a value similar to that found in patients with liver disease. The L-GSH-Px of the control group was positively correlated with the age of the subjects (r = 0.620; p less than 0.02). In contrast, in patients with liver disease an opposite behaviour of the two parameters was noted (r = -0.497; p less than 0.05). L-GSH-Px activity tended to be higher in males than in females, whereas the erythrocyte glutathione-peroxidase (E-GSH-Px) of the same patients was higher in females, albeit not significantly. L-GSH-Px and E-GSH-Px were not correlated either in normal or in liver disease. The mean GSSG-Red of the control group was 40.63 +/- 11.10 U/g protein, which is not different from that of the group of liver patients. GSSG-Red was not correlated with L-GSH-Px or with the age of patients. In two patients with hepatoma, the GSH-Px activity of the cancer tissue was low and the GSSG-Red activity high.

Adult↗

PubMed1999

Glutathione reductase activity deficiency in homozygous Gr1a1Neu mice does not cause haemolytic anaemia.

A glutathione reductase (GR) mutant with approximately 50% residual enzyme activity in blood compared with wild-type was detected amongst offspring of isopropyl methanesulphonate-treated male mice. Homozygous mutants with only 2% residual enzyme activity were recovered in progeny of inter se matings of heterozygotes. Results of linkage studies indicate a mutation at the Gr1 structural locus on chromosome 8. The loss of GR activity was evident both in blood and in other tissue extracts. Erythrocyte and organo-somatic indices did not show differences between wild-types and homozygous mutants, indicating no association between the GR deficiency and haemolytic anaemia in this potential animal model.

Anemia, Hemolytic↗

PubMed1985

Glutathione reductase deficiency in association with sickle cell and thalassaemia genes in Saudi populations.

Glutathione reductase (GR) deficiency is reported to occur with a variable frequency in some populations of the world. In this study, the populations of two regions of Saudi Arabia which have a high frequency of sickle cell, thalassaemia and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, were screened for GR deficiency. Studies were also carried out to investigate the frequency of GR deficiency with other genetic blood disorders. The frequencies of complete GR deficiency were 0.0065 and 0.006, while those of partial deficiency were 0.146 and 0.074 in Al-Hafouf and Khaiber, respectively. GR deficiency was encountered in combination with the sickle gene, the G-6-PD deficiency gene and the thalassaemia gene in both regions. Individuals with GR deficiency showed slightly reduced haematological parameters. In thalassaemic/GR-deficient subjects, mean cell volume and mean cell haemoglobin were low, while in sickle cell anaemia patients with GR deficiency the haematological parameters were higher than in sickle cell anaemia patients without GR deficiency.

Anemia, Sickle Cell↗

PubMed1998

Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.).

We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.). The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform. The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo. Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol. mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues. Southern blot analysis showed that the RGRC2 gene exists as a single copy gene. Here, we have also isolated a genomic clone completely corresponding to RGRC2. The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon. From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity. The expression of RGRC2 was strongly induced by all these treatments. We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses.

5' Untranslated Regions↗

PubMed1981

[Determination of serum activity of glucose phosphate isomerase and glutathione reductase in intra and extra hepatic cholestasis.(author's transl)].

Serum activity of glutathione reductase (GR), glucose phosphate isomerase (GPI), aspartate aminotransferase (AST), alanine aminotransferase (ALT) phosphate alkaline (PAL), and gamma-glutamyl transferase (GGT) was studied in 142 patients, in all serum bilirubin was more than 2 mg/dl. Distribution was as follows; 68 cirrhosis of the liver; 27 acute hepatitis; 31 benign extra-hepatic biliary obstruction; and 16 neoplastic obstruction of the biliary tract without liver metastasis. Fifty-three healthy volunteer blood donors were used as the control group. Mean values for GR activity in our patients were significantly higher than those for the control group, although less so in benign obstruction (p less than 0.01) than in those with acute hepatitis (p less than 0.001), cirrhosis (p less than 0.01) and neoplasic biliary obstruction (p less than 0.001). The GPI values were higher than the control groups in patients with acute hepatitis (p less than 0.001) and obstructive neoplastic jaundice (p less than 0.02). In cases with cirrhosis, 87% presented slightly higher values of GR, while GPI was within normal levels in 93 % of all cases. In patients with acute hepatitis, 92% showed a definite increase in GPI and GR values. In 71% of those with benign biliary obstruction levels for both enzymes were normal, as they were in only 6% of those with obstructive neoplastic jaundice. These findings are statistically significant in all cases and of diagnostic value in establishing a differential enzymatic diagnosis in patients presenting with clinical and biological patterns of cholestasis.

Adult↗

PubMed1997

Isolation, expression, and regulation of the pgr1(+) gene encoding glutathione reductase absolutely required for the growth of Schizosaccharomyces pombe.

The pgr1(+) gene encoding glutathione reductase (GR, EC 1.6.4.2) was isolated from Schizosaccharomyces pombe using a polymerase chain reaction fragment as a probe. The gene consists of two exons and an intron of 55 nucleotides, encoding a polypeptide of 465 amino acids (50,238 Da) with conserved residues characteristic of GR. The transcriptional start site was localized at 239 nucleotides upstream from the ATG initiation codon. The level of transcript as well as the GR enzyme activity increased more than 11-fold when the cloned pgr1(+) gene was expressed on a multicopy plasmid. This overexpression conferred on S. pombe cells more resistance against menadione, a redox cycling agent, but not against H2O2. The level of pgr1(+) transcripts increased by treatment with oxidants such as menadione, cumene hydroperoxide, and diamide. It also increased by treatment with high osmolarity, heat shock, or at the stationary growth phase. The deletion of the pap1(+) gene encoding an AP-1 homolog in S. pombe caused reduction in the pgr1(+) gene expression. Furthermore, Deltapap1 cells lost the inducibility of pgr1(+) gene expression by the above stresses, implying that Pap1 is involved in general stress-inducible gene expression. When the pgr1(+) gene was disrupted, the haploid spores were not viable. Repression of nmt1 promoter-driven pgr1(+) expression by thiamine caused cessation of growth, which was rescued by the episomal pgr1(+) gene. These results indicate that GR activity, which efficiently reduces GSSG, is essentially required for the growth of S. pombe, unlike in Saccharomyces cerevisiae or Escherichia coli.

Aerobiosis↗