PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE

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3beta-Hydroxy-delta 4-5-steroid dehydrogenase (3-HSD) and steroid delta 4-5-isomerase (isomerase) complex is a rate limiting (due to 3-HSD) regulator of steroid biosynthesis in all mammalian tissues. In human placenta, it metabolizes fetal androgens to estrogen precursors and maternal pregnenolone to progesterone. Since estrogens stimulate myometrial contractions via prostaglandins and progesterone relaxes the myometrium, 3-HSD/isomerase may regulate the initiation of human labor. 3-HSD and isomerase activities for C-21 (pregnene) and C-19 (androstene) substrates have been copurified from the microsomes and mitochondria of human term placenta as a tetramer with monomeric Mr=19,000. Pregnenolone and dehydroepiandrosterone competitively inhibit the oxidation of each other to suggest that the C-21, C-19 3-HSD activities reside at the same active site. A pilot affinity alkylation study using the C-21 product analog 16 alpha-bromoacetoxy-progesterone and microsomal enzyme suggest that the C19 and C-21 substrates are modified identically at 2 catalytic sites, one for 3-HSD and one for isomerase. This proposal will characterize the microsomal and the mitochondrial enzymes: are they the same enzyme localized in different cell organelles? Since the enzyme is membrane associated in vivo, is enzyme in solution identical to enzyme bound to artificial phospholipid vesicles? What is the primary structure determine by a human placental cDNA lambda gt11 library, and validated by comparison with amino acid sequences analyzed by Edman degradation? To understand how the C-19 and C-21 activities of both 3-HSD and isomerase coexist on one protein, the active site(s) will be probed with affinity alkylating steroids and cofactors. Bromoacetoxy analogs of androgen and progestin substrates and products along with analogs of estrogen inhibitors will be synthesized to compared inactivation of the 4 enzyme activities, radioalkylate active site amino acids, and identify peptide regions which proximate the catalytic center(s). These studies will demonstrate the number of catalytic sites responsible for the activities and how "sister C-19 and C-21 analogs" align within the active sites(s). Complementary studies using enzyme generated alkylators of isomerase (estryne) and 3-HSD (2-methylene-5alpha- androstan-3 beta-01) affinity alkylators cofactor analogs will confirm whether the isomerase and dehydrogenase activities reside at one or separate sites, and the mechanism(s) of this multiactivity will be explored. All results will be insights on the physiological control of the initiation of labor.