Study of human placental estradiol-17 beta dehydrogenase/20 alpha-hydroxysteroid dehydrogenase by preparative disc-gel electrophoresis.
The soluble enzyme, estradiol-17 beta dehydrogenase from human term placenta, appears to co-purify with a second soluble enzyme, 20 alpha-hydroxysteroid dehydrogenase. The enzyme, which had been partially purified by affinity chromatography, fractionated on a preparative electrophoresis gel to a homogeneous preparation containing both estradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities in a ratio of approximately 100:1. Analytical polyacrylamide disc-gels resolved this homogeneous preparation as a single band by both protein and activity staining techniques. Homogeneous enzyme inactivated and affinity-radioalkylated by 16 alpha-[2'-14C]bromoacetoxyprogesterone or 16 alpha-[2'-14C] bromoacetoxyestradiol 3-methyl ether, and when analyzed by SDS disc-gel electrophoresis, gave a single protein band which corresponded identically to the radioactivity peaks. These observations support the hypothesis that estradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase represent dual oxidoreductase activity in one enzyme. Preparative disc-gel electrophoresis, a technique which has not been previously adapted to purification of these human placental enzyme activities, was useful to rapidly (3 days) effect a 15-fold enrichment of the estradiol-17 beta dehydrogenase specific activity from "heat-treated cytosol". Thus, laboratory-scale preparative disc-gel electrophoresis is useful for rapid, small-scale enrichment of this soluble enzyme.
D015067:17-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D015089:20-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D004092:20-alpha-Dihydroprogesterone / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004589:Electrophoresis, Disc; D004958:Estradiol / Q000378:metabolism; D004960:Estradiol Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D006358:Hot Temperature; D006801:Humans; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D004092:20-alpha-Dihydroprogesterone; D004958:Estradiol; D015067:17-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases