Properties of Reconstituted Cholesterol 7α-Hydroxylase System from Rat and Rabbit Liver Microsomes

7α-Hydroxylation of cholesterol was studied in reconstituted systems containing cytochrome p-450 and NADPH-cytochrome P-450 reductase from rat and rabbit liver microsomes. NADPH-cytochrome P-450 reductase was prepared with a biospecific affinity chromatographic method. Cytochrome P-450 was prepared by solubilization with non-ionic detergents and various chromatographic procedures. After the last purification step two fractions of cytochrome P-450 from rat liver were analyzed for cholesterol 7α-hydroxylase activity. One fraction had low or no detectable 7α-hydroxylase activity but was able to catalyze 7α-hydroxylation of taurodeoxycholic acid, and 11-hydroxylation and 12-hydroxylation of lauric acid. The other fraction catalyzed an efficient 7α-hydroxylation of cholesterol as well as 7α-hydroxylation of cholestanol. This fraction also catalyzed 12α-hydroxylation and 26-hydroxylation of 5β-cholestane-3α,7α-diol, 7α-hydroxylation of taurodeoxycholic acid and, 11-hydroxylation and 12-hydroxylation of lauric acid. Three cytochrome P-450 fractions from rabbit liver, obtained after the final purification step, were analyzed for cholesterol 7α-hydroxylase activity. Two fractions had no detectable 7α-hydroxylase activity but were able to catalyze 12α-hydroxylation and 25-hydroxylation of 5β-cholestane-3α,7α-diol, 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol and 11-hydroxylation and 12-hydroxylation of lauric acid. The third fraction catalyzed in addition 7α-hydroxylation of cholesterol and cholestanol. The cholesterol 7α-hydroxylase activity showed an absolute requirement for cytochome P-450 and NADPH-cytochrome P-450 reductase. Phospholipid had no significant effect on cholesterol 7α-hydroxylation.