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Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees.

A poplar hybrid, Populus tremula x Populus alba, was transformed with the bacterial genes for either glutathione reductase (GR) (gor) or glutathione synthetase (GS) (gshII). When the gor gene was targeted to the chloroplasts, leaf GR activities were up to 1000 times greater than in all other lines. In contrast, targeting to the cytosol resulted in 2 to 10 times the GR activity. GR mRNA, protein, and activity levels suggest that bacterial GR is more stable in the chloroplast. When the gshII gene was expressed in the cytosol, GS activities were up to 100 times greater than in other lines. Overexpression of GR or GS in the cytosol had no effect on glutathione levels, but chloroplastic-GR expression caused a doubling of leaf glutathione and an increase in reduction state. The high-chloroplastic-GR expressors showed increased resistance to photoinhibition. The herbicide methyl viologen inhibited CO2 assimilation in all lines, but the increased leaf levels of glutathione and ascorbate in the high-chloroplastic-GR expressors persisted despite this treatment. These results suggest that overexpression of GR in the chloroplast increases the antioxidant capacity of the leaves and that this improves the capacity to withstand oxidative stress.

Antioxidants↗

Dinitrosyl-dithiol-iron complexes, nitric oxide (NO) carriers in vivo, as potent inhibitors of human glutathione reductase and glutathione-S-transferase.

Human glutathione reductase (GR) and rat liver glutathione-S-transferases (GSTs) had been shown to be inhibited by the nitric oxide (NO) carrier S-nitroso-glutathione (GSNO). We have now extended these studies by measuring the effects of dinitrosyl-iron complexed thiols (DNIC-[RSH]2) on human GR, GST and glutathione peroxidase. DNIC-[RSH]2 represent important transport forms of NO but also of iron ions and glutathione in vivo. Human GR was found to be inhibited by dinitrosyl-iron-di-glutathione (DNIC-[GSH]2) and dinitrosyl-iron-di-L-cysteine (DNIC-Cys2) in two ways: both compounds were competitive with glutathione disulfide (GSSG), the inhibition constant (Ki) for reversible competition of DNIC-[GSH]2 with GSSG being approximately 5 microM; preincubating GR for 10 min with 4 microM DNIC-[GSH]2 and 40 microM DNIC-Cys2, respectively, led to 50% irreversible enzyme inactivation. More than 95% GR inactivation was achieved by incubation with 36 microM DNIC-[GSH]2 for 30 min. This inhibition depended on the presence of NADPH. Absorption spectra of inhibited GR showed that the charge-transfer interaction between the isoalloxazine moiety of the prosthetic group flavin adenine dinucleotide (FAD) and the active site thiol Cys63 is disturbed by the modification. Cys2 and FAD could be ruled out as sites of the modification. Isolated human placenta glutathione-S-transferase and GST activity measured in hemolysates were also inhibited by DNIC-[GSH]2. This inhibition, however, was reversible and competitive with reduced glutathione, the Ki being 20 nM. The inhibition of GST induced by GSNO was competitive with reduced glutathione (GSH) (Ki = 180 microM) and with the second substrate of the reaction, 1-chloro-2,4,-dinitrobenzene (Ki = 170 microM). An inhibition of human glutathione peroxidase by GSNO or DNIC-[RSH]2 was not detectable. Inactivation of GR by DNIC-[GSH]2 is by two orders of magnitude more effective than modification by GSNO; this result and the very efficient inhibition of GST point to a role of DNIC-[RSH]2 in glutathione metabolism.

Animals↗

Changes in glutathione, glutathione reductase and glutathione-S-transferase as a function of cell concentration and age.

The activities of glutathione-S-transferase (GST) and glutathione reductase (GSR) in mouse lymphocytes as a function of cell concentration and age were determined. Lymphocytes from 2-, 9- and 24-month-old mice were isolated and the activity of GST was determined spectrophotometrically using 1-chloro-2,4-dinitrobenzene as the substrate. Lymphocyte concentrations of 0.44, 0.75 and 0.55 X 10(6) cells/ml were found to be optimal for assaying GST in 2-, 9- and 24-month-old mice, respectively. Determination of GSR activity was based on NADPH reduction of oxidized glutathione and cell concentrations of 0.29, 0.30 and 0.22 X 10(6) cells/ml were chosen for assaying the enzyme activity for the three age groups, respectively. Glutathione levels and GSR activity of mice lymphocytes were higher in 2- and 24-month-old mice as compared to 9-month-old animals. However, GST activity of mouse lymphocytes was low in 2- and 9-month-old mice and increased significantly in 24-month-old animals.

Aging↗

One- and two-electron reduction of quinones by glutathione reductase.

Yeast glutathione reductase (E.C. 1.6.4.2) catalyzes the oxidation of NADPH by p-quinones and ferricyanide with a maximal turnover number (TNmax) of 4-5 s-1.NADP+ stimulates the reaction and the TNmax/Km value of acceptors is reached at NADP+/NADPH greater than or equal to 100. TNmax is increased up to 30-33 s-1. The stimulatory effect of NADP+ may be associated with its complexation with the NADPH-binding site in the reduced enzyme (Kd = 40-60 microM). It is suggested that NADP+ shifts the electron density towards FAD in the two-electron-reduced enzyme and, evidently, changes its one-electron-reduction potentials, while quinones oxidize an equilibrium form of glutathione reductase containing reduced FAD. In the absence of NADP+ the reduction of quinones by glutathione reductase proceeds mainly in a two-electron manner. At NADP+/NADPH = 100 a one-electron reduction makes up 44% of the total process. At pH 6.0-7.0 the reduced forms of naphthoquinones undergo cyclic redox conversions. A hyperbolic dependence exists of the log TN/Km of quinones on their one-electron-reduction potentials.

Catalysis↗

Differential expression of glutathione reductase and cytosolic glutathione peroxidase, GPX1, in developing rat lungs and kidneys.

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.

Animals↗

Multifunctional activities of yeast glutathione reductase.

Yeast glutathione reductase exists in a single molecular form which exhibits preferred NADPH and weak NADH linked multifunctional activities. Kinetic parameters for the NADPH and NADH linked reductase, transhydrogenase, electron transferase and diaphorase reactions have been determined. The functional preference for the NADPH linked reductase reaction is kinetically related to the high catalytic efficiency and low dissociation constants for substrates. NADP+ and NAD+ may interact with two different sites or different kinetic forms of the enzyme. The active site disulfide and histidine are required for the reductase activity but are not essential to the transhydrogenase, electron transferase and diaphorase activities. Amidation of carboxyl groups and Co(II) chelation of glutathione reductase facilitate the electron transferase reaction presumably by encouraging the formation of an anionic flavosemiquinone.

Binding Sites↗

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant.

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.

Amino Acid Sequence↗

Electron transfer between reduced methyl viologen and oxidized glutathione: a new assay of Saccharomyces cerevisiae glutathione reductase.

Pure glutathione reductase from Saccharomyces cerevisiae catalyzed under anaerobic conditions the enzymatic reduction of GSSG using electrochemically reduced methyl viologen as electron donor. The new assay was completely dependent on the amount of active enzyme present, and involved the formation of 1 mol GSH per mole of reduced methyl viologen consumed. The enzyme followed a standard Michaelis-Menten kinetics; a Km = 230 microM for reduced methyl viologen and a turnover number of 969 mumol GSSG reduced per minute per micromole enzyme were determined. The enzymatic activity seemed to depend on the redox potential, showing half-maximal activity at -0.407 V. The enzyme was quite specific: the activity using reduced benzyl viologen as electron donor was just 1.5% of that obtained with reduced methyl viologen at the same concentration and potential. Glutathione reductase was totally inactivated after a brief anaerobic exposure with reduced methyl viologen in the absence of GSSG; a partial reactivation was observed following addition of glutathione disulfide. No inhibition of the methyl viologen-dependent activity was observed in the presence of 2',5'-ADP or 2'-P-5'-ADP-ribose, two NADP(H) analogs, at concentrations which drastically inhibited the NADPH-dependent activity, thus suggesting that the reduced viologen does not interact with the pyridine nucleotide-binding site.

Adenosine Diphosphate↗

[Multiple forms of NADPH-dependent glutathione reductase in serum. Studies on the NADPH-dependent glutathione-reductase in serum II. (author's transl)].

Sera with elevated activities of glutathione reductase were investigated by gel electrophoresis in agar or polyacrylamide, and by gel filtration. In both separation methods the glutathione reductase activity in individual samples was resolved, showing up to three fractions differing in rate of migration and molecular size. The fractions with the lowest and highest molecular weight, corresponded respectively to the slowest and fastest migrating bands in agar gel electrophoresis. Preincubation of the serum samples with FAD or neuraminidase had no effect on the rate of migration of the three fractions. After the addition of beta-mercaptoethanol to the serum, gel electrophoresis and gel filtration showed only the enzyme fraction with the slowest rate of migration and the lowest molecular weight (140,000). The other two fractions reappeared after removal of the thiol from the serum. Further studies on the isolated (agar gel electrophoresis) fractions showed the existence of oligomeric forms of the enzyme, which are reversibly interconvertible.

Electrophoresis, Agar Gel↗

2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)- phenylcarbamoylsulfanyl]propionic acid and its derivatives as a novel class of glutathione reductase inhibitors.

Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione to reduced glutathione. The enzyme is an attractive target for the development of antimalarial agents, agents to decrease malarial drug resistance and anticancer agents. In addition, inhibition of the enzyme has been employed as a tool in research for various purposes. In this paper, we present a rational design of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid and its derivatives as irreversible GR inhibitors. The K(i) and k(inact) values of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid, the most potent derivative of the series, are 88 muM and 0.1 min(-1), respectively. Although the K(i) value of the inhibitor is in the micromolar range, it is more potent than N,N-bis(2-chloroethyl)-N-nitrosourea, which is currently the most commonly employed irreversible GR inhibitor with a reported IC(50) value of 646 microM. Additional attractive features of the inhibitor include its ready availability through a one-step synthesis and good solubility in both organic and aqueous solutions.

Carmustine↗

Effects of Elevated Cytosolic Glutathione Reductase Activity on the Cellular Glutathione Pool and Photosynthesis in Leaves under Normal and Stress Conditions.

Tobacco (Nicotiana tabacum var Samsun) was transformed using the bacterial gor gene coding for the enzyme glutathione reductase. Transgenic plants were selected by their kanamycin resistence and expression of the bacterial gor gene. After separation by isoelectric focusing techniques, leaf extracts from transgenic plants having both native and bacterial glutathione reductase activity gave, in addition to the six bands of the native enzyme, two further closely running isoenzymes. These additional bands originating from the expression of the bacterial gor gene were nonchloroplastic. Leaves from transgenic plants had two- to 10-fold higher glutathione reductase activity than non-transgenic controls. The amount of extractable glutathione reductase activity obtained in transgenic plants was dependent on leaf age and the conditions to which leaves were exposed. Both light and exposure to methylviologen increased leaf glutathione reductase activity. Elevated levels of cytosolic glutathione reductase activity in transgenic plants had no effect on the amount or reduction state of the reduced glutathione/oxidized glutathione pool under optimal conditions or oxidative conditions induced by methylviologen. The glutathione pool was unaltered despite the oxidation-dependent loss of CO(2) assimilation and oxidation of enzymes involved in photosynthesis. However, the reduction state of the ascorbate pool was greater in transgenic plants relative to nontransgenic controls following illumination of methylviologen-treated leaf discs. Therefore, we conclude that in the natural state glutathione reductase is present in tobacco at levels above those required for maximal operation of the ascorbate-glutathione pathway.

Journal Article↗

Modification of arginine residues at the substrate binding site of yeast glutathione reductase.

Yeast glutathione reductase (GR) was inactivated by phenylglyoxal (PG), which specifically modifies arginine residues of the enzyme. Inactivation followed psuedo-first order rate kinetics. There was no reversible complex formation prior to inactivation. Analysis of the kinetic data showed the order of reaction to be unity with respect to the modifier. Inactivation of GR was completely prevented by the presence of oxidised glutathione (GSSG), whereas NADP gave only partial protection. Stoichiometric studies showed that around four arginine residues per subunit were modified by PG in the absence of GSSG, whereas only one was modified in its presence. From these observations, it is concluded that essential arginine residues are present at the substrate binding site.

Arginine↗

Kinetics and mechanism of the oxidation of the glutathione dimer by hypochlorous Acid and catalytic reduction of the chloroamine product by glutathione reductase.

Oxidized glutathione (GSSG) reacts with two molar equivalents of HOCl/OCl- (a neutrophil-derived oxidant and a common biocide) to form the dichloro (bis-N-chloro-gamma-l-glutamyl) derivative (NDG). The reaction of less than two molar equivalents of HOCl with GSSG does not yield the unsymmetrical monochloro derivative (NCG) but rather a stoichiometric amount of NDG and GSSG. This result is explained by a faster reaction of the second equivalent of HOCl with NCG than that of the first equivalent of HOCl with GSSG. The rates of reaction of GSSG2-, GSSG3-, and GSSG4- (successive deprotonation of the ammonium groups) have been investigated, and it is clear that GSSG2- is unreactive, whereas GSSG4- is about twice as reactive as GSSG3-. Accordingly, the following mechanism is proposed (constants for 5 degrees C): H+ + OCl- = HOCl, pK1 = -7.47; GSSG2- = GSSG3- + H+, pK2 = 8.5; GSSG3- = GSSG4- + H+, pK3 = 9.5; GSSG3- + HOCl --> NCG3- + H2O, k4 = 2.7(2) x 106 M-1 s-1; GSSG4- + HOCl --> NCG4- + H2O, k5 = 3.5(3) x 107 M-1 s-1; NCG3- --> NDG4- + H+, k6 = fast; and NCG4- + HOCl --> NDG4- + H2O, k7 = fast. At physiologic pH, the k4 pathway dominates. NDG decomposes at pH 7.4 in a first-order process with kdec = 4.22(1) x 10-4 s-1 (t1/2 = 27 min). Glutathione reductase (EC 1.6.4.2) is capable of catalyzing the reduction of NDG by NADPH. The only NDG-derived product that is observed (by NMR) after the reduction by NADPH is GSH. Thus, in the presence of the GOR/NADPH system, GSH is capable of redox buffering a 3/2 mol equiv of HOCl rather than a 1/2 mol equiv as previously assumed.

Catalysis↗

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase.

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.

Chromatography, Gas↗

Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, catalase, glutathione reductase, inorganic peroxides and glutathione in the adult housefly.

The effects of oxidative stress in the adult male housefly were examined by the administration of 1 mM paraquat. Houseflies exhibit NADH and NADPH-diaphorase activity. Paraquat caused a significant decrease in life span, metabolic rate and the concentration of thiobarbituric acid-reactants. Concentrations of reduced glutathione and inorganic peroxides were increased by paraquat. Paraquat stimulated the activity of catalase but did not affect activities of superoxide dismutase and glutathione reductase. The levels of oxidized glutathione and the rate of fluorescent age pigment accumulation were unaffected by paraquat. Results indicate that paraquat toxicity does not result from lipid peroxidation.

Animals↗

Glutathione peroxidase and glutathione reductase activities towards glutathione-derived antioxidants.

A new class of glutathione derivatives with antioxidant properties has been prepared by transformation of the NH2 group into a pyrrole ring with various substitutions at the 2 and 5 positions. Due to steric hindrance and/or hydrophobicity of the 2-5-disubstituted pyrrole ring, the reduced glutathione derivatives are poor substrates of the glutathione peroxidase and do not effectively compete with GSH. The oxidized glutathione derivatives are, in turn, relatively good substrates (Km = 1.5 mM) of the glutathione reductase as compared to natural oxidized glutathione (Km = 0.51 mM) but are not effective competitors of the enzyme. It can be considered that the new glutathione derivatives do not strongly interfere with the natural enzymatic defence against fatty acid hydroperoxides formed during an oxidative stress.

Animals↗

Rat and calf thioredoxin reductase are homologous to glutathione reductase with a carboxyl-terminal elongation containing a conserved catalytically active penultimate selenocysteine residue.

We have determined the sequence of 23 peptides from bovine thioredoxin reductase covering 364 amino acid residues. The result was used to identify a rat cDNA clone (2.19 kilobase pairs), which contained an open reading frame of 1496 base pairs encoding a protein with 498 residues. The bovine and rat thioredoxin reductase sequences revealed a close homology to glutathione reductase including the conserved active site sequence (Cys-Val-Asn-Val-Gly-Cys). This also confirmed the identity of a previously published putative human thioredoxin reductase cDNA clone. Moreover, one peptide of the bovine enzyme contained a selenocysteine residue in the motif Gly-Cys-SeCys-Gly (where SeCys represents selenocysteine). This motif was conserved at the carboxyl terminus of the rat and human enzymes, provided that TGA in the sequence GGC TGC TGA GGT TAA, being identical in both cDNA clones, is translated as selenocysteine and that TAA confers termination of translation. The 3'-untranslated region of both cDNA clones contained a selenocysteine insertion sequence that may form potential stem loop structures typical of eukaryotic selenocysteine insertion sequence elements required for the decoding of UGA as selenocysteine. Carboxypeptidase Y treatment of bovine thioredoxin reductase after reduction by NADPH released selenocysteine from the enzyme with a concomitant loss of enzyme activity measured as reduction of thioredoxin or 5,5'-dithiobis(2-nitrobenzoic acid). This showed that the carboxyl-terminal motif was essential for the catalytic activity of the enzyme.

Amino Acid Sequence↗