Biochemical characterization of the 7α-hydroxylase activities towards 27-hydroxycholesterol and dehydroepiandrosterone in pig liver microsomes

Abstract

Microsomal cytochrome P450 catalyzing the 7α-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone was partially purified from pig liver. This enzyme fraction also catalyzed 7α-hydroxylation of 25-hydroxycholesterol and pregnenolone but did not 7α-hydroxylate cholesterol or testosterone. Studies with extrahepatic tissues have suggested the possibility of one common enzyme responsible for the 7α-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone. A series of experiments was performed to study if there are one or several enzymes 7α-hydroxylating these steroids in the liver. The activities towards the two substrates copurified but the ratio between 27-hydroxycholesterol and dehydroepiandrosterone 7α-hydroxylation varied considerably in different purification steps and between different preparations. The enzyme inhibitors disulfiram, N-bromosuccinimide, ketoconazole, metyrapone and α-naphthoflavone affected the activities in a similar way. Dehydroepiandrosterone inhibited 27-hydroxycholesterol 7α-hydroxylation whereas 27-hydroxycholesterol had almost no inhibitory effect on dehydroepiandrosterone 7α-hydroxylation. Experiments to examine the nature of inhibition by dehydroepiandrosterone indicated that the two steroids did not compete for the same active site. The results of this study do not rule out the possibility of one single enzyme catalyzing 7α-hydroxylation of the two steroids. However, taken together the data suggest that hepatic microsomal 7α-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two, probably closely related, enzymes.