Rat hepatic cholesterol 7α-hydroxylase: Biochemical properties and comparison to constitutive and xenobiotic-inducible cytochrome P-450 enzymes

Abstract

Cytochrome P-450 Ch7 α (cholesterol 7α-hydroxylase) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids in mammalian liver. The properties of this cytochrome P-450 ( P-450) form were studied in rat hepatic microsomal preparations in comparison to those of several well characterized constitutive and xenobiotic-inducible rat hepatic P-450s. Administration of the bile acid-sequestering resin cholestyramine [4% ( w w ) in the diet] to male or female rats maintained on a reverse light cycle led to a 10- to 15-fold induction of P-450 Ch7 α activity relative to untreated, standard light cycle controls. By contrast, the levels of four hepatic steroid hormone hydroxylating P-450 enzymes, designated 2a, 2c, 3, and PB-4 [Waxman, D. J. (1984) J. Biol. Chem. 259, 15481–15490] , were not significantly affected by cholestyramine treatment. Antibody inhibition experiments established that P-450 Ch7 α is immunochemically distinct from nine other rat hepatic P-450s, including P-450 3, a highly regio- and stereoselective steroid hormone 7α-hydroxylase. P-450 Ch7 α was shown to be selectively inactivated by micromolar concentrations of the disulfide-containing reagents disulfiram (Antabuse ®) and 2,2′-dithiopyridine. This inactivation was readily reversed upon incubation with 2-mercaptoethylamine, suggesting the presence of a highly reactive thiol group at the active site of P-450 Ch7 α . These findings demonstrate that P-450 Ch7 α corresponds to a unique P-450 enzyme exhibiting inductive, biochemical, immunochemical, and regulatory properties distinct from those of nine well-characterized rat hepatic P-450 forms.