Relationship between the pregnenolone-induced difference spectrum and cytochrome b5 reduction in guinea-pig adrenal microsomes.

Abstract

Addition of pregnenolone to guinea-pig adrenal microsomes produces a slowly developing difference spectrum with peaks at about 425 and 557 nm and a trough at about 410 nm. The spectral change is similar to that resulting from the reduction of cytochrome b5 by NADH or NADPH. In the presence of sufficient quantities of NADH to fully reduce cytochrome b5, pregnenolone produces a typical type I difference spectrum (ΔA385–420 nm). Pregnenolone is converted to progesterone by adrenal microsomes without addition of cofactor (NAD+) for the 3β-hydroxysteroid dehydrogenase (HSD) reaction. The rate of conversion is increased 2–3 fold by NAD+ and inhibited by NADH. Accompanying the metabolism of pregnenolone (with or without added NAD+) is the production of NADH and reduction of cytochrome b5. Addition of pregnenolone alone to adrenal microsomes results in 60–80% reduction of cytochrome b5. The reduction of cytochrome b5 is maintained for at least as long as pregnenolone is being metabolized. Inhibition of pregnenolone metabolism changes the pregnenolone-induced spectral change to a type I and prevents the reduction of cytochrome b5. The results suggest that the oxidation-reduction state of cytochrome b5 in adrenal microsomes is controlled in part by pregnenolone metabolism which in turn influences the pregnenolone-induced difference spectrum. Oxidation of NADH by cytochrome b5 may serve to prevent NADH inhibition of HSD activity and to generate additional NAD+ as cofactor for the reaction.