Horse-liver glutathione reductase: purification and characterization.
1. Purification of horse-liver glutathione reductase was obtained by affinity chromatography on N6-(6-aminohexyl)-adenosine-1'5'-bisphosphate Sepharose (N6-2'5'-ADP-Sepharose) and Reactive Red-120-Agarose, and chromatography on DEAE-Sephadex and Sephacryl S-300. 2. The final preparation had 248 U/mg specific activity after 11,174-fold purification with 47% final recovery, and was homogeneous by SDS-electrophoresis. It showed charge heterogeneity in non-denaturing electrophoresis and chromatofocusing, with several peaks of pI between 5.7 and 6.7. 3. The enzyme was homodimeric (107,000 native MW), with S20w = 6.31 S, and 41.22 A of hydrodynamic radius. It showed absorption peaks at 270, 370 and 462 nm, a characteristic of flavoproteins. 4. When NADPH was substituted by deamino-NADPH or NADH the enzyme showed 69 and 8.5% activity, respectively, while with glutathione-CoA mixed disulfide the enzyme had 23% of the activity shown with GSSG. Apparent Km values of 8.8, 680, 59, and 560 microM were measured for NADPH, NADH, GSSG and ferricyanide, respectively.
García-Alfonso
C
C
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
Martínez-Galisteo
E
E
Llobell
A
A
Bárcena
J A
JA
López-Barea
J
J
D000818:Animals; D002846:Chromatography, Affinity; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005980:Glutathione Reductase / Q000302:isolation & purification / Q000378:metabolism*; D006736:Horses; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction
D009243:NAD; D009249:NADP; D005980:Glutathione Reductase