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

Abstract

7α-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 7α-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, 7α-hydroxy-PREG and 7α-hydroxy-DHEA metabolites produced by microsomes of liver, brain, thymus, and spleen were identified. Study of the 7α-hydroxylating enzyme in these tissues indicated that microsomes contained most of the activity, except for brain, where it was primarily mitochondrial. Production yields of 7α-hydroxy-PREG and 7α-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. At the optimal pH (7.4) and in all tested tissues but liver, microsomal 7α-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 7α-hydroxylating activity. Complete inhibition was obtained with 0.1 mM Zn 2+ or Cu 2+ and with 1 mM Fe 2+ or Fe 3+. 7α-Hydroxylation of PREG was activated only by 0.5 mM Ca 2+ and that of DHEA only by 0.25 mM Mg 2+. Since the production rates of 7α-hydroxy-PREG and 7α-hydroxy-DHEA in tissues may be a key to the triggering of immune defenses, and since both 7α-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.