Endocrinology, Vol 134, 751-759, Copyright © 1994 by Endocrine Society

ARTICLES

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4- isomerase in adrenal cortex mitochondria and microsomes

LA Sauer, JC Chapman and RT Dauchy
Institute for Medical Research, Mary Imogene Bassett Hospital, Cooperstown, New York 13326.

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 Ki, 2.8 microM) and increased by added NAD+ (apparent Ka, 2.4 microM). 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.

This article has been cited by other articles:

				P. E. Micevych, V. Chaban, J. Ogi, P. Dewing, J. K. H. Lu, and K. Sinchak Estradiol Stimulates Progesterone Synthesis in Hypothalamic Astrocyte Cultures Endocrinology, February 1, 2007; 148(2): 782 - 789. [Abstract] [Full Text] [PDF]

				J. Simard, M.-L. Ricketts, S. Gingras, P. Soucy, F. A. Feltus, and M. H. Melner Molecular Biology of the 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Gene Family Endocr. Rev., June 1, 2005; 26(4): 525 - 582. [Abstract] [Full Text] [PDF]

				D. Boerboom and J. Sirois Equine P450 Cholesterol Side-Chain Cleavage and 3{beta}-Hydroxysteroid Dehydrogenase/{{Delta}}5-{{Delta}}4 Isomerase: Molecular Cloning and Regulation of Their Messenger Ribonucleic Acids in Equine Follicles During the Ovulatory Process Biol Reprod, January 1, 2001; 64(1): 206 - 215. [Abstract] [Full Text]

				New Insight into the Molecular Basis of 3{beta}-Hydroxysteroid Dehydrogenase Deficiency: Identification of Eight Mutations in the HSD3B2 Gene in Eleven Patients from Seven New Families and Comparison of the Functional Properties of Twenty-Five Mutant Enzymes J. Clin. Endocrinol. Metab., December 1, 1999; 84(12): 4410 - 4425. [Abstract] [Full Text]