Cholesterol 7α‐Hydroxylase of Rat Liver

Open Access
Publisher Website
Google Scholar
Abstract
1 Cytochrome P-450 was prepared from the liver microsomes of cholestryamine-fed rats by solubilisation with the non-ionic detergent Nonidet P42 followed by chromatography on a DEAE-cellulose column and on hydroxyapatite. NADPH-cytochrome P-450 reductase was preapred by a technique of affinity column chromatography using 2′,5′ ADP Sepharose. 2 The activity of cholesterol 7α-hydroxylase was measured in a reconstituted system of microsomal mixed function oxidase containing cytochrome P-450 and NADPH-cytochrome P-450 reductase from rat liver plus cholesterol and NADPH. Endogenous cholesterol was largely depleted from the enzyme preparations by the treatment of the microsomes with cold n-butanol/acetone. 3 The reconstituted system of mixed-function oxidase catalysed a highly effective and specific 7α-hydroxylation of cholesterol. The reconstituted system showed higher activity of cholesterol 7α-hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH. 4 The apparent Km for cholesterol in the reconstituted system was 15 μm and the V was 1.4 nmol 7α-hydroxycholesterol formed min−1 (nmol cytochrome P-450)−1 5 The reconstituted system also catalysed the 7α-hydroxylation of taurodeoxycholic acid, the 7α-hydroxylation of 26-norcholesterol and to a limited degree the 12α-hydroxylation reactions and the 12α-hydroxylation reaction was significantly less than the ability of the system to 7α-hydroxylate cholesterol.

This publication has 32 references indexed in Scilit: