Preparation of Partially Purified, Lipid-depleted Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome c Reductase from Rat Liver Microsomes

Abstract

Abstract Partially purified cytochromes P-450 and P-448 were obtained from hepatic microsomes of rats treated with either phenobarbital or 3-methylcholanthrene, using the ionic detergent sodium cholate and the nonionic detergent emulgen 911. The preparations contained 9 to 11 nmoles of cytochrome P-450 or P-448 per mg of protein and were free of NADPH-cytochrome c reductase and cytochrome b5. The total phospholipid content of the partially purified cytochrome P-450, per mg of protein, was reduced by more than 98% relative to microsomes. A partially purified NADPH-cytochrome c reductase preparation, essentially free of cytochrome b5 and phospholipid, is also described. Reconstitution of the cytochrome P-450 and reductase fractions with phospholipid resulted in restoration of enzymatic activity for the oxidative metabolism of a variety of substrates. Since the ratio of cytochrome P-450 to cytochrome b5 in this reconstituted system was greater than 200 to 1, it is suggested that cytochrome b5 is not an obligatory component of the mixed function oxidase system.