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 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.
Sauer
L A
LA
Institute for Medical Research, Mary Imogene Bassett Hospital, Cooperstown, New York 13326.
Chapman
J C
JC
Dauchy
R T
RT
D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D008634:Mersalyl / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D011375:Progesterone Reductase / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013251:Steroid Isomerases / Q000737:chemistry* / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology
C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D009097:Multienzyme Complexes; D009243:NAD; D008634:Mersalyl; D011375:Progesterone Reductase; D013251:Steroid Isomerases