3Beta-Hydroxysteroid dehydrogenase/delta 4-5 isomerase (3-HSD/isomerase), a rate-limiting enzyme complex found in most steroid-metabolizing tissues, has not been purified from human placenta where it is equally distributed between the mitochondria and microsomes. The enzyme activities were co-solubilized from mitochondria using sodium deoxycholate (Edwards et al., 1976). Preliminary data is presented in this proposal that Brij 58 treatment of placental microsomes co-solubilizes 3-HSD and isomerase. The applicant proposes to purify solubilized 3-HSD/isomerase from both organelles by ammonium sulfate precipitation, affinity chromatography, and gel filtration chromatography. Should the 3-HSD and isomerase activities separate, 3-HSD activity will be followed. Purified enzyme homogeneity will be demonstrated: single band on SDS and non-denaturing polyacrylamide electrophoresis; single NH2-terminus; non-specific antibody formation by immunization of rabbits. 3-HSD/isomerase purified from microsomes and mitochondria will be separately characterized: molecular weight; subunit composition; amino acid composition; NH2-terminus sequence; stability in solution; pH and temperature optimums; substrate and cofactor kinetic profile; and product inhibition kinetic studies. These observations will demonstrate the degree of similarity between the mitochondrial and microsomal enzymes. To address whether pregnene and androstene substrates are oxidized at the same active site and whether the 3-HSD and isomerase activities reside at the same or separate centers on the purified protein, affinity alkylation and radioalkylation studies are proposed. Affinity alkylating analogs of substrate (pregnenolone, dehydroepiandrosterone), product (progesterone, androstenedione), and inhibitor (estrone, equilenin) steroids; an enzyme-generated affinity alkylator (estryne); and alkylating cofactor analogs (eg: fluorosulfonylbenzoyladenosine) will be used. The profiles from enzyme inactivation, substrate and cofactor protection from inactivation, and amino acids radioalkylated should answer these questions regarding the multiple activities of 3-HSD/isomerase. If dehydroepiandrosterone of fetal origin and maternal pregnenolone are oxidized at the same site, the fetus can induce """"""""progesterone withdrawal"""""""" via 3-HSD allowing prostaglandin-mediated myometrial contractions. Purification and study of 3-HSD/isomerase will clarify both the importance of this enzyme in the cascade of events which initiate labor and steroid metabolism at a macromolecular binding site.
Showing the most recent 10 out of 24 publications
