Purification and immunological studies of glutathione reductase from rat liver evidence for an antigenic determinant at the nucleotide-binding domain of the enzyme

Glutathione reductase has been purified to homogeneity by a method which is an improvement of an earlier procedure (Carlberg, I. and Mannervik, B. (1975) J. Biol. Chem. 250, 5475–5480). The new steps in the purification scheme include affinity chromatography on 2′,5′ ADP-Sepharose 4B. Antibodies to glutathione reductase from rat liver were raised in rabbits and used for analysis of the enzyme by quantitative ‘rocket’ immunoelectrophoresis. Glutathione reductase from human erythrocytes, porcine erythrocytes, and calf-liver gave precipitin lines showing partial identity with the rat liver enzyme in Ouchterlony double diffusion experiments. Enzyme from spinach, yeast (Saccharomyces cerevisiae), and the photosynthetic bacterium Rhodospirillum rubrum did not give precipitates with the antibodies to the enzyme from rat liver. Titration of glutathione reductase from the different sources with antibodies confirmed the cross-reactivity of the mammalian enzymes; the human enzyme giving the strongest heterologous reaction. No reaction was observed with the enzyme from spinach, yeast, and Rhodospirillum rubrum. NADPH, NADP⁺, and 2′,5′ ADP were found to inhibit the interaction between antibodies and glutathione reductase from rat liver and human erythrocytes. NADH, glutathione, or glutathione disulfide did not protect the enzyme from reacting with the antibodies. It is concluded that glutathione reductase has an antigenic binding site for the antibodies at the pyridine nucleotide-binding site of the enzyme molecule.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

Purification by affinity chromatography of yeast glutathione reductase, the enzyme responsible for the NADPH-dependent reduction of the mixed disulfide of coenzyme A and glutathione.

Glutathione reductase (NAD(P)H : oxidised-glutathione oxidoreductase, EC 1.6.4.2) was purified from baker's yeast by a new procedure involving affinity chromatography on 2',5'-ADP-Sepharose 4B. The yield was 65% of essentially homogeneous enzyme. The activity was assayed with both glutathione disulfide (GSSG) and the mixed disulfide of coenzyme A and glutathione (CoAssg). The two disulfide substrates gave coinciding activity profiles and a constant ratio of the activities in different chromatographic and electrophoretic systems. No evidence was obtained for the existence of a reductase specific for CoASSG distinct from glutathione reductase. It is concluded that normal baker's yeast contains a single reductase active with both GSSG and CoASSG.     

Immunological comparison of glyoxalase I from yeast and mammals and quantitative determination of the enzyme in human tissues by radioimmunoassay

Antibodies to glyoxalase I from yeast, rat liver, porcine erythrocytes and human erythrocytes were raised in rabbits. Gel precipitation and immunotitration experiments demonstrated that the mammalian enzymes were immunologically related, but distinct from the yeast enzyme. Fab fragments of the antibodies to human glyoxalase I did not inhibit the catalytic activity, indicating that the antigen binding sites were not directed towards the active site of the enzyme. A radioimmunoassay for glyoxalase I was developed. Quantitative analysis of human adult as well as fetal organs demonstrated that glyoxalase I was present in a concentration of approximately 0.2 micrograms/mg protein in most human tissues.     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

An essential histidine residue in the catalytic mechanism of mammalian glutathione reductase

Glutathione reductase from human erythrocytes was inactivated by ethoxyformic anhydride, and > 95% activity was lost by modification of about 1–1.5 histidine residues per flavin (or subunit), as measured by the increased absorbance at 240 nm. Full reactivation was obtained with hydroxylamine. The rate of inactivation increased with pH and an apparent pK = 5.9 was obtained for the protolytic dissociation. The modified enzyme was inactive with NADPH and GSSG as substrates, but almost fully active in catalysis of a transhydrogenase reaction involving pyridine nucleotides. The visible absorption spectrum of oxidized or two-electron-reduced enzyme was not changed, but the flavin fluorescence of oxidized enzyme increased 2-fold after the modification. NADPH or NADP⁺ did not protect the enzyme against inactivation. It is concluded that the modification affects a histidine involved in the second half-reaction of the catalysis, i.e. reduction of GSSG by the dithiol of reduced enzyme. Glutathione reductase from three additional mammalian sources was similarly inactivated, but enzyme from yeast was much less inactivated by the corresponding treatment with ethoxyformic anhydride.     

Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.     

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.     

Immunological Properties of Spinach Glutathione Reductase and Inductive Biosynthesis of the Enzyme with Ozone

Glutathione reductase (GR) was purified from spinach leavesto the homogeneous state, based on native- and SDS-PAGE bindings.The GR had a polypeptide of 60 kilodalton and its absorptionspectrum was similar to that of GR from human erythrocytes.Antibody against spinach GR, prepared from rabbit, inhibitedGR activity, while the non-immune serum had no effect on theenzyme activity. Purified enzyme and crude extracts from spinachleaves produced fused precipitin lines with anti-GR on the Ouchterlonydouble diffusion tests. Although crude extracts from tobaccoand petunia leaves reacted with anti-GR, these precipitin linesfused only partially with the line between purified spinachGR and anti-spinach GR. Moss, fern and Chlorella crude extractsand purified yeast GR produced no precipitin lines with anti-spinachGR. The extractable GR activity increased significantly in 0.07ppm O₃-fumigated spinach leaves, whereas they suffered no visibleinjuries. The results from the immunoblotting method confirmedthat the O₃-induced increase in extractable GR activity is dueto an increase in the protein level of GR. (Received December 17, 1987; Accepted March 16, 1988)     

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.     

Inhibition of glutathione reductase by oncomodulin

Evidence for a specific interaction between oncomodulin and glutathione reductase is presented. Glutathione reductase (EC 1.6.4.2) isolated from either the bovine intestinal mucosa or the rat liver was bound in a Ca2(+)-dependent manner to oncomodulin which was covalently attached to Sepharose. In addition, glutathione reductase was able to catalyze the reduction of the disulfide-linked dimer of oncomodulin. The interaction of these proteins could also be indirectly demonstrated by monitoring glutathione reductase activity since oncomodulin was shown to inhibit the enzyme in a dose-dependent manner with an apparent IC50 of approximately 5 microM. The kinetic analysis of the oncomodulin-dependent effects on glutathione reductase activity indicates that oncomodulin interacts at a site other than the active site as the oncomodulin-induced inhibition was of the noncompetitive type. The in vivo inhibition of glutathione reductase appears to be an oncomodulin-specific effect as closely related members of the troponin C superfamily such as rabbit (pI 5.5) or carp (pI 4.25) parvalbumins, as well as calmodulin, failed to affect the activity of this enzyme. The present in vitro study indicating that oncomodulin can regulate the activity of glutathione reductase could be very significant with respect to the elucidation of a physiological role for oncomodulin.     

Carbon isotope effect on carboxylation of ribulose bisphosphate catalyzed by ribulosebisphosphate carboxylase from Rhodospirillum rubrum

The carbon isotope effect at CO2 has been measured in the carboxylation of ribulose 1,5-bisphosphate by the ribulosebisphosphate carboxylase from Rhodospirillum rubrum. The isotope effect is obtained by comparing the isotopic composition of carbon 1 of the 3-phosphoglyceric acid formed in the reaction with that of the carbon dioxide source. A correction is made for carbon 1 of 3-phosphoglyceric acid which arises from carbon 3 of the starting ribulose bisphosphate. The isotope effect is k12/k13 = 1.0178 +/- 0.0008 at 25 degrees C, pH 7.8. This value is smaller than the corresponding value for the spinach enzyme. It appears that substrate addition with the R. rubrum enzyme is principally ordered, with ribulose bisphosphate binding first, whereas substrate addition is random with the spinach enzyme. The carboxylation step is partially rate limiting with both enzymes.     

Glutathione reductases from a variety of sources are inhibited by physiological levels of glutathione.

1. Glutathione reductase from human platelets, bovine intestinal mucosa, yeast and E. coli were inhibited in vitro by physiological levels of reduced glutathione with IC50s of 6.61 mM, 2.92 mM, 2.40 mM and 12.11 mM, respectively. 2. A steady-state kinetic examination revealed that glutathione inhibited the NADPH oxidation (at constant [glutathione-disulphide]) catalysed by the eucaryotic enzymes uncompetitively, whereas the E. coli enzyme appeared unaffected by glutathione concentrations of up to 10 mM. 3. With respect to glutathione inhibition of glutathione-disulphide reduction (at constant [NADPH]), the human enzyme was inhibited uncompetitively; the bovine and yeast enzymes displayed apparent mixed hyperbolic inhibition; the E. coli enzyme was inhibited competitively.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH. The FAD of the reductase is reduced by NADPH, and reducing equivalents are passed to a redox-active disulfide to complete the first half-reaction. The nascent dithiol of two-electron reduced enzyme (EH(2)) interchanges with glutathione disulfide forming two molecules of glutathione in the second half-reaction. It has long been assumed that a mixed disulfide (MDS) between one of the nascent thiols and glutathione is an intermediate in this reaction. In addition to the nascent dithiol composed of Cys(45) and Cys(50), the enzyme contains an acid catalyst, His(456), having a pK(a) of 9.2 that protonates the first glutathione (residue numbers refer to the yeast enzyme sequence). Reduction of yeast glutathione reductase by glutathione and reoxidation of EH(2) by glutathione disulfide indicate that the mixed disulfide accumulates, in particular, at low pH. The reaction of glutathione disulfide with EH(2) is stoichiometric in the absence of an excess of glutathione. The equilibrium position among E(ox), MDS, and EH(2) is determined by the glutathione concentration and is not markedly influenced by pH between 6.2 and 8.5. The mixed disulfide is the principal product in the reaction of glutathione with oxidized enzyme (E(ox)) at pH 6. 2. Its spectrum can be distinguished from that of EH(2) by a slightly lower thiolate (Cys(50))-FAD charge-transfer absorbance at 540 nm. The high GSH/GSSG ratio in the cytoplasm dictates that the mixed disulfide will be the major enzyme species.     

Evolution of antioxidant mechanisms: Thiol-Dependent peroxidases and thioltransferase among procaryotes

Summary Glutathione peroxidase and glutathione S-transferase both utilize glutathione (GSH) to destroy organic hydroperoxides, and these enzymes are thought to serve an antioxidant function in mammalian cells by catalyzing the destruction of lipid hydroperoxides. Only two groups of procaryotes, the purple bacteria and the cyanobacteria, produce GSH, and we show in the present work that representatives from these two groups (Escherichia coli, Beneckea alginolytica, Rhodospirillum rubrum, Chromatium vinosum, andAnabaena sp. strain 7119) lack significant glutathione peroxidase and glutathione S-transferase activities. This finding, coupled with the general absence of polyunsaturated fatty acids in procaryotes, suggests that GSH-dependent peroxidases evolved in eucaryotes in response to the need to protect against polyunsaturated fatty acid oxidation. A second antioxidant function of GSH is mediated by glutathione thiol-transferase, which catalyzes the reduction of various cellular disulfides by GSH. Two of the five GSH-producing bacteria studied (E. coli andB. alginolytica) produced higher levels of glutathione thiol-transferase than found in rat liver, whereas the activity was absent in the other three species studied. The halobacteria produced γ-glutamylcysteine rather than GSH, and assays for γ-glutamylcysteine-dependent enzymes demonstrated an absence of peroxidase and S-transferase activities but the presence of significant thioltransferase activity. Based upon these results it appears that GSH and γ-glutamylcysteine do not function in bactera as antioxidants directed against organic hydroperoxides but do play a significant, although not universal, role in main-taining disulfides in a reduced state. The function of GSH in the photosynthetic bacteria, aside from providing a form of cysteine resistant toward autoxidation, remains a puzzle, as none of the GSH-dependent enzymes tested other than glutathione reductase were present in these organisms.     

Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase

The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.     

Purification and properties of glutathione peroxidase from human placenta.

Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.     

Photo-induced activation of cytochrome P450/reductase fusion enzyme coupled with spinach chloroplasts

Summary Photoactivation of cytochrome P450 monooxygenase was studied using a combination of spinach chloroplasts and yeast microsomes containing rat P4501A1/yeast reductase fusion enzyme. Under illumination, in the reaction mixture, NADP was reduced, transferring electrons to the P450/reductase fusion enzyme to convert 7-ethoxycoumarin to 7-hydroxycoumarin.     

Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2′,5′-ADP-Sepharose 4B

Glutathione reductase has been purified to at least 98% homogeneity from calf liver. An essential part in the procedure involves affinity chromatography on 2′,5′-ADP-Sepharose 4B to which the enzyme remains bound in the presence of 0.4 m phosphate. This step separates glutathione reductase from the closely related thioredoxin reductase. Some of the physical and catalytic properties as well as the amino acid composition of the enzyme are reported.