Purification of glutathione reductase from chicken liver and investigation of kinetic properties.
ABSTRACT Glutathione reductase was purified from chicken liver and some characteristics of the enzyme were investigated. The purification procedure was composed of four steps: preparation of homogenate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Owing to the four consecutive procedures, the enzyme was purified 1714-fold, with a yield of 38%. Specific activity at the final step was 120 enzyme unit (EU)/mg of protein. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was found to be 100 kDa by Sephadex G-200 gel filtration chromatography, and the subunit molecular weight was found to be 43 kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, and optimum temperature were 7.0, 7.4, 0.75 M Tris-HCl buffer including 1 mM EDTA, and 50 degrees C, respectively. KM and Vmax values for NADPH and glutathione disulfide (GSSG) substrates were also determined for the enzyme.
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ABSTRACT: Glutathione reductase (GR) carries out the enzymatic reduction of glutathione disulfide (GSSG) to its reduced form (GSH) at the expense of the reducing power of NADPH. Previous studies have shown that GR from several species is progressively inactivated in the presence of NADPH, but that the mechanism of inactivation (especially in the presence of metals) has not been fully elucidated. We have investigated the involvement of iron ions in the inactivation of yeast (Saccharomyces cerevisiae) GR in the presence of NADPH. Even in the absence of added iron, inactivation of GR was partly blocked by the iron chelators, deferoxamine and ortho-phenanthroline, suggesting the involvement of trace amounts of contaminating iron in the mechanism of inhibition. Exogenously added antioxidants including ethanol, dimethylsulfoxide and 2-deoxyribose did not protect GR against NADPH-induced inactivation, whilst addition of exogenous Fe(II) (but not Fe(III)) potentiated the inactivation. Moreover, removal of oxygen from the medium led to increased inhibition of GR, whereas pre-incubation of the Fe(II)-containing medium for 30 min under normoxic conditions prior to the addition of GR abolished the enzyme inactivation by NADPH. Under these pre-incubation conditions, Fe(II) is fully oxidized to Fe(III) within 1 min. Furthermore, GR that had been previously inactivated in the presence of Fe(II) plus NADPH could be partially reactivated by treatment with ortho-phenanthroline and deferoxamine. In contrast, Fe(III) had no effect on GR reactivation. Together, these results indicate that yeast GR is inactivated by a reductive mechanism mediated by NADPH and Fe(II). According to this mechanism, GR is diverted from its normal redox cycling by the generation of an inactive reduced enzyme form in which both the FAD and thiol groups at the active site are likely in a reduced state.Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 01/2008;
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ABSTRACT: Glutathione reductase (E.C.188.8.131.52) was purified from Phaeodactylum tricornutum cells grown axenically in an autotrophic medium. The overall procedure started with preparation of the cell extract and addition of ammonium sulfate to 20% saturation, followed by anion exchange and affinity interaction chromatography (Blue-A- and 2',5'-ADP-Sepharose). Complete purification required native polyacrylamide gel electrophoresis as the final step. The enzyme was purified to homogeneity and functionally characterized. Its native molecular mass was estimated to be 118 kDa; which corresponds to a dimer. The enzyme exhibited a specific activity of 190 U mg(-1) with an optimal activity at pH 8.0 and 32 degrees C. We determined K(m) values of 14 microM and 60 microM for NADPH and oxidized glutathione, respectively. Products inhibited the enzyme according to a hybrid ping-pong reaction mechanism. After MALDI-TOF analysis, the purified enzyme was unambiguously identified as one of the two proteins annotated as glutathione reductases in the genome of the diatom. The properties of the enzyme help to understand redox metabolic scenarios in P. tricornutum.Protist 09/2009; 161(1):91-101. · 4.14 Impact Factor