TOPOLOGY OF 3-BETA-HYDROXY-5-ENE-STEROID DEHYDROGENASE/DELTA(5)-DELTA(4)-ISOMERASE IN ADRENAL-CORTEX MITOCHONDRIA AND MICROSOMES

3 beta-Hydroxy-5-ene-steroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD) is a NAD(+)-dependent membrane-bound enzyme that catalyzes the oxidation of Delta(5)-3 beta-hydroxysteroids to Delta(4)-3-keto structures during adrenal, gonadal, and placental steroidogenesis. Enzyme activity is located in both microsomes and mitochondria. In these experiments we examined the membrane topologies of 3 beta HSD in rat and calf adrenal microsomes and mitochondria by comparing access to the active sites of coenzyme and the inhibitor mersalyl, a nonpenetrant organic mercurial anion. Microsomal activity required exogenous NAD(+) and was inhibited by mersalyl, indicating that the active site faced the medium in vitro and the cytoplasm in vivo. In contrast, mitochondrial 3 beta HSD used matrix space NAD(+), was inhibited by reduction of intramitochondrial NAD(P)(+), and was insensitive to mersalyl. Mitochondrial activity was decreased by exogenous NADH (apparent K-i, 2.8 mu M) and increased by added NAD(+) (apparent K-a, 2.4 mu M). However, mersalyl blocked the effects of exogenous NADH and NAD(+) and returned the activity to that observed before coenzyme addition. The membrane-sidedness of the NAD(+) activation was examined further in submitochondrial particles prepared by sonication of pyridine nucleotide-depleted calf adrenal cortex mitochondria. Particles were prepared in the absence or presence of 10 mM NAD(+) and contained none or 2.9-7.3 nmol NAD(+)/mg protein, respectively. Both groups of submitochondrial particles required exogenous NAD(+) for 3 beta HSD activity, indicating that the active site faced the medium (the particles were everted), and the contained NAD(+) was inside the particles. However, 3 beta HSD activity was increased 12-140% in particles that contained NAD(+). The results suggest that mitochondrial 3 beta HSD is an integral inner membrane protein, that the active site faces the matrix space and is influenced by coenzyme availability, and that a regulatory site(s) faces the intermembrane space. Binding of NAD(+) or NADH to this external site increases or decreases, respectively, the rate of catalysis at the active site. Mitochondrial 3 beta HSD activity may be enhanced by oxidation of intermembrane space NADH via an active rotenone- and antimycin-a-insensitive NADH oxidase.