Mouse-liver glutathione reductase. Purification, kinetics, and regulation.
Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004789:Enzyme Activation; D005978:Glutathione / Q000494:pharmacology; D005980:Glutathione Reductase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D000596:Amino Acids; D009243:NAD; D009249:NADP; D005980:Glutathione Reductase; D005978:Glutathione