Highly purified detergent-solubilized NADPH-cytochrome P-450 reductase from phenobarbital-induced rat liver microsomes

Abstract

NADPH-cytochrome P-450 reductase was highly purified from liver microsomes of phenobarbital-induced rats by column chromatography on DEAE-cellulose, DEAE-Sephadex A-50, and hydroxylapatite in the presence of deoxycholate or Renex 690, a nonionic detergent. The purified enzyme gave a single major band with a molecular weight of 79,000 daltons on SDS-polyacrylamide gel electrophoresis. FMN and FAD were present in about equal amounts. The most active reductase preparation catalyzed the reduction of 40.9 [mu]moles of cytochrome per min per mg of protein and, as an indirect measure of cytochrome P-450 reduction, the oxidation of 2.0 [mu]moles of NADPH per min per mg of protein in a reconstituted hydroxylation system containing benzphetamine as the substrate.