PROPERTIES OF RECONSTITUTED CHOLESTEROL 7 ALPHA-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. Copyright © 1979, Wiley Blackwell. All rights reserved