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Phenyl glyoxal in borate buffer specifically inactivates the NADPH-linked activity of yeast glutathione reductase. While NAD+ is ineffective, NADP+ prevents the enzyme from inactivation, and in the process, protects two arginine residues from glyoxalation. These two arginine residues presumably function as the recognition site for the anionic 2'-phosphate group of NADP+.
A new method was devised for the direct measurement of glutathione reductase activity in crude tissue samples using high-performance liquid chromatography and electrochemical detection with a mercury/gold electrode. This assay depends on the high sensitivity of the electrode to detect SH groups produced from a substrate, oxidized glutathione, the interference of which can be excluded by setting the oxidation voltage as low as 100 mV. The sensitivity was high enough to detect the activity in crude samples containing less than 1 microg of protein. This method proved to be sensitive and simple for the measurement of this biologically important reductant activity without the need for a further purification procedure.
Kinetic characterization of the inhibition effect of nickel on glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G-6-PD) and glutathione reductase (GR; EC 1.6.4.2) from Saccharomyces cerevisiae was made. The effect of nickel on G-6-PD activity is consistent with a mixed-type inhibition pattern, with a competitive character, since the inequality ki,int greater than ki,slope shows an inverse relation between varied substrate concentrations and fractional inhibition. An inhibition effect of nickel on GR activity, when NADPH is the varied substrate, is also consistent with a mixed-type inhibition pattern. However, pure competitive inhibition is found on GR reaction when oxidized glutathione is the varied substrate. This investigation shows the highest sensibility of GR before the inhibitory effect of nickel, in agreement with the experimental values of inhibition constants found in this study, where constants related to the GR system are lower than the ones of the G-6-PD system.
The effects of water deficits on plant morphology and biochemistry were analyzed in two photoperiodic strains of field-grown cotton (Gossypium hirsutum L.). Plants grown under dryland conditions exhibited a 40 to 85% decrease in leaf number, leaf area index, leaf size, plant height, and total weight per plant. Gross photosynthesis decreased from 0.81 to 0.47 milligram CO(2) fixed per meter per second and the average midday water, osmotic, and turgor potentials decreased to -2.1, -2.4, and 0.3 megapascals, respectively.There was a progressive increase in glutathione reductase activity and in the cellular antioxidant system in the leaves of stressed plants compared to the irrigated controls. The stress-induced increases in enzyme activity occurred at all canopy positions analyzed.Irrigation of the dryland plots following severe water stress resulted in a 50% increase in leaf area per gram fresh weight in newly expanded leaves of both strains over the leaves which had expanded under the dryland conditions. Paraquat resistance (a relative measure of the cellular antioxidant system) decreased in the strain T25 following irrigation. Glutathione reductase activities remained elevated in the T25 and T185 leaves which were expanded fully prior to irrigation and in the leaves which expanded following the irrigation treatment.
An automated AutoAnalyzer method using 5:5'-dithiobis-2-nitrobenzoic acid is described for determining whole blood glutathione reductase (BGR) activity and for measuring in vitro activation of BGR with flavin adenine dinucleotide (FAD). BGR activity is expressed as mumoles glutathione regenerated from oxidized glutathione per ml of whole blood (WB) or per g of hemoglobin. The stimulatory effect of FAD on BGR activity divided by the activity without FAD determined the activity coefficient (AC). We found that NADPH and oxidized glutathione assay concentrations of 0.100 mmole/liter and 0.250 mmole/liter, respectively, in 0.1 mole/liter phosphate buffer, pH 7.4, gave consistent results when WB, before assay, was diluted 20-fold. WB samples to be stored are initially diluted 10-fold with distilled water and frozen. Prior to assay, two aliquots of the sample are diluted 2-fold, one aliquot with distilled water and another with 46 mumole/liter FAD. With sample and manifold dilutions the assay FAD concentrations is 1.0 mumole/liter: assay concentrations greater than 5.0 mumole FAD/liter were shown to be inhibitory. We examined blood samples from 617 children in the age range 6 to 60 months and determined the normal AC range to be between 1.00 and 1.35. Six weaned rats (23 days of age), maintained on a riboflavin-deficient diet, showed a mean AC of 1.23, 1.54, 2.02, and 2.41 at 23, 26, 30, and 36 days of age, respectively. Six control rats maintained an AC of 1.23 +/- 0.05 (SD) during the same period.
The mode of binding of NADPH and oxidized glutathione to the flavoenzyme glutathione reductase has been determined by x-ray crystallography. Furthermore, two intermediates of the reaction have been produced in the crystal and have been structurally elucidated. All these analyses were done at 0.3 nm resolution. The results allow the stereochemical description of the mechanism of the enzyme. The dinucleotide NADPH is bound in an extended conformation with the nicotinamide ring stacking onto the re-face of the flavin part of FAD, and adenine located at the protein surface. The binding of NADPH results in the 2-electron reduced form of the enzyme, EH2. This form has also been analyzed without any ligand bound. In EH2 the redoxactive disulfide bridge of the protein, which lies at the si-face of the flavin ring, is opened and the sulfur of Cys-58 moves by about 0.1 nm into a position where it can attack one of the sulfurs of the substrate oxidized glutathione. This interchange leads to a mixed glutathione-protein disulfide, which can be stabilized in crystals and has been analyzed. By selectively reacting Cys-58 with iodoacetamide the crystalline enzyme can be blocked in its EH2 state. The imidazole of His-467' is near to all sulfurs taking part in the disulfide bridge exchange and is therefore certainly crucial for catalysis. The crystallographic results establish that electrons flow from NADPH to the substrate GSSG via flavin and the redoxactive protein disulfide bridge. This is consistent with the scheme that has been postulated from biochemical, spectroscopic, and model studies.
We have isolated 4 cDNA clones (GRT1-4) encoding glutathione reductase (GR) from a tobacco (Nicotiana tabacum L.) leaf cDNA library. The cDNAs were almost identical: GRT1, GRT3 and GRT4 represented the same gene, differing only in that GRT4 contained an intron within the C-terminal part of the coding sequence. Failure to splice out this intron resulted in a substitution of the final 13 amino acids of the deduced amino acid sequence. A second gene was represented by GRT2. Southern blots indicated that there were two related GR genes in tobacco. The presence of multiple isoforms of GR in tobacco may be explained in part by the expression of a small gene family. In addition, alternative isoforms may result from translation of different mRNAs derived from the same gene by intron skipping during the splicing of nascent GR mRNAs.
Two patients with an inverted duplication of bands 8p21-p23 are described. The gene for glutathione reductase (GSR; E.C.1.6.4.2) has previously been localized to band 8p21. In one of the patients subband 8p21.1 was included in the duplication; GSR activity in the red blood cells was increased. In the other patient, subband 8p21.1 was not included in the duplication and GSR activity was normal. This allows GSR to be assigned to subband 8p21.1. Including the present 2 patients, at least 13 cases of this abnormality have been published. We have obtained data on at least 8 further cases (unpublished). We conclude that inv dup (8p) is a non-randomly occurring de novo structural aberration in man. The GSR results in our cases prove that breakpoints can be different in different patients. Clinical symptoms and signs include some common features but show marked interpatient variation which should, at least in part, be caused by the differences in break-points. A detailed collaborative study to determine the clinical and epidemiological features of this entity is recommended.
Intracellular glutathione (GSH) concentrations have been implicated recently as a regulatory determinant of multidrug resistance protein (MRP)-mediated drug efflux. Inhibition of glutathione reductase (GR) activity of N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) has been employed extensively to investigate the role of GSH redox cycle in cellular function. The present study examined the effect of BCNU on the MRP-mediated efflux of doxorubicin in the multidrug-resistant human fibrosarcoma cell line HT1080/DR4 overexpressing MRP. No significant difference in GR activity between HT1080 (parental) and multidrug-resistant HT1080/DR4 cells was detected (38.6 +/- 2.2 and 37.8 +/- 5.28 nmol/min/10(6) cells, respectively). Exposure of HT1080 and HT1080/DR4 cells to 100-500 microM BCNU decreased GR activity concentration dependently with subsequent reduction in cellular GSH pools in both cell lines. Inhibition of GSH biosynthesis by D,L-buthionine-(S,R)-sulfoximine (D,L-BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced MRP-mediated drug efflux and potentiated the cytotoxicity of doxorubicin in MRP-expressing HT1080/DR4 cells (dose modifying factor 20.8). While equally effective inhibition of GR activity by BCNU was observed in parental and resistant cells, a significant increase in intracellular retention of doxorubicin was only achieved in MRP-expressing HT1080/DR4 cells. Furthermore, inhibition of MRP function following treatment with BCNU or D,L-BSO was directly related to the degree of GSH depletion in MRP-expressing tumor cells [r = 0.94 (P < 0.001) and 0.99 (P < 0.001), respectively]. Based on northern blot analysis of MRP mRNA levels, exposure of HT1080/DR4 cells to BCNU did not produce down-regulation of MRP gene expression. The results reported herein indicate that derivatives of nitrosourea with carbamoylating properties are potent inhibitors of MRP function. Depletion of intracellular GSH pools by inhibition of the GSH redox cycle or GSH de novo biosynthesis significantly inhibited MRP-mediated doxorubicin transport and restored intracellular drug concentrations in vitro.
PURPOSE: To determine the mechanism that leads to the UVA light-dependent loss of glutathione reductase (GR) activity in human lens (HL). METHODS: Both the HL water-soluble (WS) fraction and yeast GR were irradiated with UVA light (200 mW/(cm(2). h) for 1 hour at +20 degrees C, and the specific activity (SA) was observed. GR apoenzyme (apo-GR) was prepared from either HL-WS fractions or yeast GR by treatment with a cold solution of acidic ammonium sulfate. Reconstitution of apo-GR was conducted by mixing enzyme with an excess of flavine adenine dinucleotide (FAD) and purification of GR on a size-exclusion separation column. RESULTS: One hour of UVA photolysis of an HL-WS fraction resulted in a 96% decrease in the SA of GR (6.32 +/- 0.22 vs. 0.39 +/- 0.01 mU/mg lens protein). Action spectra of GR SA in the WS fraction from HL within the range 320 to 500 nm showed that the enzyme was most vulnerable to the wavelengths in the UVA region with the highest decrease in the SA at 320 to 350 nm ( approximately 23%-28% activity loss within 1 hour of irradiation), and lowest with the wavelengths beyond 400 nm (7%-8% SA loss). UVA irradiation of apo-GR in the crude HL-WS fraction, followed by reconstitution with FAD, showed that 90% of the original SA was recovered. The original GR activity either in HL or yeast GR, however, was not recovered by (NH(4))(2)SO(4) (pH 2.25) treatment followed by reconstitution with FAD after UVA photolysis. Experiments with UVA-photolyzed yeast GR revealed that UVA photolysis caused the formation of additional SH groups within the enzyme, as shown by the incorporation of an SH-specific fluorescent probe, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Similar results were obtained on the photolyzed iodoacetamide-alkylated yeast GR, which was evaluated by matrix-assisted desorption ionization-time of flight (MALDI-TOF) mass spectrometry. CONCLUSIONS: The results show that the reduction of HL GR activity by UVA light was directly linked to the presence of FAD within the enzyme. That the irradiated GR showed de novo formed SH groups argues that UVA photolysis of GR leads to the reduction of the redox-active disulfide within the reaction center of the enzyme, making it inactive.
Kinetic characterization of the inhibition effects of Cd(II), Cu(II) and Zn(II) on glutathione reductase (GSSGR; EC 1.6.4.2.) and glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) from Saccharomyces cerevisiae was made. No inhibition effect was found with Zn(II) on these enzymatic systems. The effect of Cd(II) (up to 0.16 mM) and Cu(II) (up to 0.008 mM) on GSSGR activity is consistent with a pure competitive inhibition or an uncompetitive pattern, when the varied substrate is the oxidized glutathione or the NADPH, respectively. Only Cd(II) (up to 1.0 mM) showed an inhibition effect on G6PD system, which is consistent with a mixed-type inhibition pattern. When NADP+ changes over an extensive range of concentration, the inhibition effect shows an uncompetitive character. Furthermore, when G-6-P is the varied substrate, the character of the mixed-type inhibition is competitive.
The urinary and blood levels of riboflavin and erythrocyte glutathione reductase (EGR) activity and its stimulation with FAD (EGR-AC) were examined in preschool children suffering from either measles or other upper respiratory infections and matched controls. Patients showed significantly higher levels of urinary riboflavin (per unit creatinine), erythrocyte riboflavin and EGR activity and lower EGR-AC values. This trend reversed after treatment. Mobilization of riboflavin from a labile tissue pool during infections may produce artefactual changes in the biochemical indices of riboflavin status. It would also prevent tissue saturation despite supplementation.
The X-ray structure analyses of four glutathione reductase complexes and derivatives have been extended to 2 A resolution and refined. The results are discussed in conjunction with the structure of the oxidized native enzyme known at 1.54 A resolution. While the residual co-ordinate errors are around 0.2 A, some significant shifts even in this range could be established. Points of particular interest are the 3.2 A approach of C4N of nicotinamide to N5F of flavin in hydride transfer geometry, the hydrogen bond geometries of the 2'-phosphate of NADPH as compared to inferior geometries for an inorganic phosphate binding together with NADH, the differential mobilities of parts of the substrates as derived from refined atomic temperature factors, and the stabilization of the thiolate of the proximal Cys63 by conformational changes of neighboring residues as well as by flavin. In addition, catalytically competent His467' is seen to interact more optimally with the sulfur of glutathione-I than with the distal sulfur of Cys58. The observed participation of water molecules for both NADPH and glutathione binding is so extensive that a prediction of the binding mode merely from the polypeptide structure would be very difficult. The accurately known geometries allowed us to draw some conclusions on the enzyme mechanism and suggest a possible scenario of the catalysis.
The effect of different abiotic stresses (extreme temperatures and osmotic stress) on the synthesis of glutathione and hydroxymethylglutathione, on the ratio of the reduced to oxidised forms of these thiols (GSH/GSSG, hmGSH/hmGSSG), and on the glutathione reductase (GR) activity was studied in maize and wheat genotypes having different sensitivity to low temperature stress. Cold treatment induced a greater increase in total glutathione (TG) content and in GR activity in tolerant genotypes of both species than in sensitive ones. The GSH/GSSG and hmGSH/hmGSSG ratios were increased by this treatment only in the frost-tolerant wheat variety. High-temperature stress increased the TG content and the GSH/GSSG ratio only in the chilling-sensitive maize genotype, but GR activity was greater after this treatment in both maize genotypes. Osmotic stress resulted in a great increase in the TG content in wheat and the GR activity in maize. The amount of total hydroxymethylglutathione increased following all stress treatments. These results indicate the involvement of these antioxidants in the stress responses of wheat and maize.
The objective of this study was to investigate the possible role of oxygen free radicals in the aging process by studying the pattern of age-related changes in a broad spectrum of antioxidant defenses and indicators of oxidative stress in adult male Drosophila melanogaster. There were selective, but not universal, changes in the antioxidant defenses and indicators of oxidative stress. Activities of catalase and glutathione reductase and concentration of reduced glutathione decreased during the latter part of life. Superoxide dismutase activity tended to increase with age whereas glutathione peroxidase activity was undetectable. The ratios of reduced/oxidized glutathione and NADPH/NADP+, which are widely regarded as indicators of oxidative stress, decreased in the terminal phase of life. NADH/NAD+ ratio increased in the latter part of life. Concentration of inorganic peroxides increased during the first trimester of life and remained unchanged thereafter while that of thiobarbituric acid-reactants tended to decrease during aging. The main conclusion of this study is that age-related changes in antioxidant defenses and in levels of oxygen free radical reaction products are selective in nature and are quite variable in different species and tissues; however, the level of oxidative stress tends to increase during aging.
The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.
The aim of the study was to estimate the activity of glutathione peroxydase, glutathione reductase and superoxide dysmutase in the brain tumours. The activity of GSH-Px was evaluated with the use of spectrophotometry, GSSG-R was evaluated based on the Mize and Langdon method, and SOD-1 with Sykes et al. method. The 55 specimens (45 specimens of neoplasm and 10 specimens of healthy brain tissue) were studied. The statistical analysis revealed significant increase of enzymes within the brain tumours in comparison to the healthy brain tissue.
Blood lead, erythrocyte glutathione reductase (GSSG-R), 5-aminolevulinic acid dehydratase (ALA-D), and free sulfhydryl (SH) groups were measured in normal subjects and in those with occupational exposure to lead. With increasing blood lead concentration the activity of GSSG-R rises and that of ALA-D decreases. There is also a fall in the level of free SH with rising blood lead concentrations. There is a high degree of correlation between these parameters, and it is suggested that the changes represent part of a biological control mechanism to compensate for the reduction of available sulfhydryl groups by lead ions.