Characterization of glutathione reductase from porcine erythrocytes.
Glutathione reductase (NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2) was purified to homogeneity from porcine erythrocytes by use of affinity chromatography on 2',5'-ADP-Sepharose 4-B. Analytical ultracentrifugation experiments were analysed to give the following physical parameters for the enzyme: s20,w = 5.7 S, D20,w = 50 microgram2/s, and Mw = 103 000 (protein concentration, 0.5 mg/ml). The frictional ratio was 1.37 and the Stokes radius was 4.3 nm. The enzyme molecule is a dimer composed of subunits of equal size each containing a FAD molecule. The amino acid compositions and circular dichroism spectra of the porcine and human enzymes indicated extensive structural similarities. The isoelectric point was at pH 6.85 (at 4 degrees C). The absorption spectrum of the oxidized enzyme had maxima at 377 and 462 nm. In vivo the enzyme appears to be partially reduced. At a physiological concentration of reduced glutathione the apparent Michaelis constants for glutathione disulfide and NADPH were higher than in the absence of reduced glutathione. At 0.15 M ionic strength the catalytic activity obtained with NADPH as reductant was optimal at pH 7 and more than 200 times higher than that obtained with NADH. S-sulfoglutathione and some mixed disulfides of glutathione were poor substrates with the exception of the mixed disulfide of coenzyme A and reduced glutathione. The purified enzyme displayed low transhydrogenase activity with oxidized pyridine nucleotide analogs and diaphorase activity with 2,6-dichlorophenolindophenol as acceptor substrates; both NADPH and NADH served as donors.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D008058:Dihydrolipoamide Dehydrogenase / Q000097:blood; D004912:Erythrocytes / Q000201:enzymology*; D005980:Glutathione Reductase / Q000097:blood*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D046911:Macromolecular Substances; D008058:Dihydrolipoamide Dehydrogenase; D005980:Glutathione Reductase