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In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7α 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 7β-hydroxypregnenolone and 7β-hydroxy-DHEA was not Supported by formal identification, and the P450 responsible for 7α-hydroxylation and 7β-hydroxylation of pregnenolone and DttEA have not been identified. Based on results of analyses by cryslallizatin to constant specific activity and gas chromatography/mass spectrometry, we report that mouse-liver and mouse-brain microsomes carried out 7β-hydroxylation of pregnenolone and DHEA, and that yeast-expressed mouse cytochrome P4501A1 (P450 1A) transformed pregnenolone into 7β-hydroxypregnenolone [Km = 25.1 ± 0.4 µM, turnover number = 979±30 pmol · min-1· nmol-1 mouse P450 1A1 ). Neither 7-hydroxy derivatives of DHEA nor 7α-hydroxypregnenolone was produced by P450 1A1. The presence of P450 IAI m liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1Al by β-naphthoflavone resulted in increased 7β-hydroxylation of pregnenolobe in liver microsomes. Studies of the brain-microsome 7β-hydroxylating enzyme with pregnenolone or DHEA gave Km of 5.0 µM and 4.9 µM, 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 IAI is involved in 7β-hydroxylation of pregnenolone and may contribute in part to the production of the 7-hydroxylated steroids necessary for activation of immune defenses. [ABSTRACT FROM AUTHOR]