Pregnenolone-7 beta-hydroxylating activities of yeast-expressed mouse cytochrome P450-1A1 and mouse-tissue microsomes.

ABSTRACT

In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7 alpha position by a cytochrome P450 (P450)-containing microsomal enzyme complex. In addition, 7-hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7 beta-hydroxypregnenolone and 7 beta-hydroxy-DHEA was not supported by formal identification, and the P450 responsible for 7 alpha-hydroxylation and 7 beta-hydroxylation of pregnenolone and DHEA have not been identified. Based on results of analyses by crystallization to constant specific activity and gas chromatography/mass spectrometry, we report that mouse-liver and mouse-brain microsomes carried out 7 beta-hydroxylation of pregnenolone and DHEA, and that yeast-expressed mouse cytochrome P450-1A1 (P450 1A1) transformed pregnenolone into 7 beta-hydroxypregnenolone (Km = 25.1 +/- 0.4 microM, turnover number = 979 +/- 30 pmol.min-1.nmol-1 mouse P450 1A1). Neither 7-hydroxy derivatives of DHEA nor 7 alpha-hydroxypregnenolone was produced by P450 1A1. The presence of P450 1A1 in liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1A1 by beta-naphthoflavone resulted in increased 7 beta-hydroxylation of pregnenolone in liver microsomes. Studies of the brain-microsome 7 beta-hydroxylating enzyme with pregnenolone or DHEA gave Km of 5.0 microM and 4.9 microM, respectively, and Vmax of 4.5 pmol.min-1.mg-1 and 6.1 pmol.min-1.mg-1, respectively, and showed the absence of cross-inhibitions between the two steroids. These findings indicate that, in addition to unidentified P450, P450 1A1 is involved in 7 beta-hydroxylation of pregnenolone and may contribute in part to the production of the 7-hydroxylated steroids necessary for activation of immune defenses.