Cholesterol 7α‐Hydroxylase of Rat Liver

Abstract

1. Cytochrome P-450 was prepared from the liver microsomes of cholestyramine-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 prepared by a technique of affinity column chromatography using 2',5'ADP Sepharose.2. The activity of cholesterol 7a-hydroxylase was measured in a reconstituted system of microsomal mixedfunction 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-butmollacetone.3. The reconstituted system of mixed-function oxidase catalysed a highly effective and specific 7a-hydroxylation of cholesterol. The reconstituted system showed a higher activity of cholesterol 7a-hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P-450, NADPHcytochrome P-450 reductase and NADPH.4. The apparent K,,, for cholesterol in the reconstituted system was 15pM and the V was 1.4nmol 7a-hydroxycholesterol formed min-(nmol cytochrome P-450)-5. The reconstituted system also catalysed the 7a-hydroxylation of taurodeoxycholic acid, the 7a-hydroxylation of 26-norcholesterol and to a limited degree the 12a-hydroxylation of cholest-4-ene-3-one-7a-ol. The ability of this reconstituted system to effect these two 7a-hydroxylation reactions and the 12a-hydroxylation reaction was significantly less than the ability of the system to 7a-hydroxylate cholesterol.

Competitive inhibition of bile acid synthesis by endogenous cholestanol and sitosterol in sitosterolemia with xanthomatosis. Effect on cholesterol 7 alpha-hydroxylase.

“…1 C). The apparent Km value of 46 ,uM observed with acetone-treated microsomes was lower than the range of 80-225 itM reported for untreated microsomes (20)(21)(22) and closer to the value of 15 AM observed with butanol/acetonetreated microsomes (23).…”

Inhibition of cholesterol 7α-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC

“…Adult male or female Wistar rats (100-200 g) were fed a diet containing either 4 (w/w) cholestyramine (Bristol-Myers Pharmaceuticals, Uxbridge, Middlesex, U.K.) in Purina PRD powdered rat chow, or a soft diet consisting of 7500 whole wheat flour, 25 powdered milk and 5 yeast for 7 days prior to the experiments. Livers were perfused with 0.105 M-KCI/50 mM-NaF and microsomes were prepared by fractional centrifugation [3].…”

“…Cholesterol 7az-hydroxylase was originally identified as a cytochrome P-450 because of the requirement for molecular oxygen and NADPH as cofactors and the inhibitory effects of carbon monoxide [2,3]. These early findings have been substantiated by the isolation of cytochrome P-450 with this activity [4][5][6]. The isolation of this protein in an active form has proved to be very difficult, and studies on properties of the purified enzyme are still limited, due to the lack of a suitable antibody for immunochemical analysis [7] and molecular cloning.…”