Studies of the enzyme complex responsible for pregnenolone and dehydroepiandrosterone 7 alpha-hydroxylation in mouse tissues

7 alpha-Hydroxylation of pregnenolone (PREG) and dehydroepiandrosterone (DHEA) is known to take place in numerous tissues of mouse and rat. The responsible cytochrome P450 species has not yet been identified Interest in the production of 7 alpha-hydroxylated steroid derivatives results from their ability to increase the immune response in mice. Using crystallizations to constant specific activity and gas chromatography-mass spectrometry, 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA metabolites produced by microsomes of liver, brain, thymus, and spleen were identified. Study of the 7 alpha-hydroxylating enzyme in these tissues indicated that microsomes contained most of the activity, except for brain, where it was primarily mitochondrial. Production yields of 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA by microsomes from heart, spleen, thymus, brain, and liver of 7-week-old mice were higher than those of 1-week-old and (except for liver) 41-week-old animals. Ar the optimal pH (7.4) and in all tested tissues bur liver, microsomal 7 alpha-hydroxylation was more extensive for PREG than for DHEA. With brain and thymus microsomes, K-M were lower for PREG than for DHEA and decreased when phosphate was used instead of Tris buffer. With brain microsomes, the use of 1 mM EDTA increased 7 alpha-hydroxylating activity. Complete inhibition was obtained with 0.1 mM Zn2+ or Cu2+ and with 1 mM Fe2+ or Fe3+. 7 alpha-Hydroxylation of PREG was activated only by 0.5 mM Ca2+ and that of DHEA only by 0.25 mM Mg2+. Since the production rates of 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA in tissues may be a key to the triggering of immune defenses, and since both 7 alpha-hydroxylation and immunity decrease with aging, these data will prove to be useful in studies of the enzyme responsible and of the mechanisms that control its activity. (C) 1996 by Elsevier Science Inc.