PARTIAL PURIFICATION OF A NADPH-DEPENDENT 21-HYDROXYSTEROID DEHYDROGENASE FROM HUMAN PLACENTA

A NADPH-dependent 21-hydroxysteroid dehydrogenase was purified 70- to 117-fold from term human placenta. Properties of the enzyme were studied using 21-dehydrocortisol synthesized by an improved procedure. Final specific activity ranged from 0.026 to 0.048 μmoles NADPH oxidized per min per mg of protein. Optimum pH was at 6.7 in sodium phosphate and 6.3 in imidazole buffers. Km values for several 21-dehydrosteroids were approx. 1·10-3 M. In addition, methylglyoxal and phenylglyoxal, but not glyoxal, were reduced. Km value for NADPH was 1.2·10-5 M. Excess NADPH inhibited 21-dehydrocortisol reduction. The inhibition constant, KiB, was 1.3·10-4 M. NAD+ and NADP+ also inhibited the enzyme. Androgens, estrogens and progestagens were inactive as substrates, and did not inhibit reduction of 21-dehydrocortisol. The 21-hydroxysteroids were neither reduced nor oxidized by the enzyme system, and were weak competitive inhibitors of the reduction of 21-dehydrocortisol. Sulfhydryl groups on the enzyme appeared to be necessary for activity. Molecular weight was estimated at 33 000 to 35 000 by gel filtration. Oxidation of 21-hydroxysteroids by the dehydrogenase could not be demonstrated. It is suggested that 21-hydroxysteroid dehydrogenase functions to control the level of 21-dehydrosteroids in the placenta formed by other pathways. © 1969.