Pregnenolone-7 beta-hydroxylating activities of yeast-expressed mouse cytochrome P450-1A1 and mouse-tissue microsomes.
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.
Doostzadeh
J
J
Laboratoire de Biotechnologie, Conservatorie National des Arts et Métiers, Paris, France.
Urban
P
P
Pompon
D
D
Morfin
R
R
D000818:Animals; D001921:Brain / Q000201:enzymology; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D003687:Dehydroepiandrosterone / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D011284:Pregnenolone / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D003687:Dehydroepiandrosterone; D011284:Pregnenolone; D019363:Cytochrome P-450 CYP1A1