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

Microsomal cytochrome P450 catalyzing the 7 alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone was partially purified from pig liver. This enzyme fraction also catalyzed 7 alpha-hydroxylation of 25-hydroxycholesterol and pregnenolone but did not 7 alpha-hydroxylate cholesterol or testosterone. Studies with extrahepatic tissues have suggested the possibility of one common enzyme responsible for the 7 alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone. A series of experiments was performed to study if there are one or several enzymes 7 alpha-hydroxylating these steroids in the liver, The activities towards the two substrates copurified but the ratio between 27-hydroxycholesterol and dehydroepiandrosterone 7 alpha-hydroxylation varied considerably in different purification steps and between different preparations. The enzyme inhibitors disulfiram, N-bromosuccinimide, ketoconazole, metyrapone and alpha-naphthoflavone affected the activities in a similar way. Dehydroepiandrosterone inhibited 27-hydroxycholesterol 7 alpha-hydroxylation whereas 27-hydroxycholesterol had almost no inhibitory effect on dehydroepiandrosterone 7 alpha-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 7 alpha-hydroxylation of the two steroids. However, taken together the data suggest that hepatic microsomal 7 alpha-hydroxylation of 27-hydroxycholesterol and dehydroepiandrosterone involves at least two, probably closely related, enzymes. (C) 1998 Elsevier Science B.V.