Partial Purification and Characterization of Cholesterol 7α-Hydroxylase from Guinea Pig

Cholesterol 7α-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7α-xhydroxylating, EC 1.14.13.17), was partially purified from liver microsomes of guinea pig. The purified sample showed a specific activity of 1. 76 nmol/min/mg of protein and a turnover number of 2.3 nmol/min/nmol of cytochrome P-450, which were 100 times as high as respective values of microsomes. Cholesterol 7α-hydroxy lase activity was reconstituted from the partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and the NADPH generating system. The reconstituted system showed an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH. The apparent Km value for cholesterol in the reconstituted system was 33 μM and Vmax was 3.4 nmol/min/mg of protein. Cholesterol 7α-hydroxylase activity was significantly inactivated by iodoacetamide and p-chloromercuribenzoate, but not either by aminoglutethimide or by metyrapone.