Purification and characterization of pig adrenal 20 alpha-hydroxysteroid dehydrogenase.

Two distinct molecules of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) from pig adrenal cytosol have been purified to homogeneity and characterized. Purification was achieved by ammonium sulphate precipitation followed by DEAE-cellulose column chromatography. 20 alpha-HSD activity was separated into two fractions including 20 alpha-HSD-I and 20 alpha-HSD-II, and these were further purified by affinity chromatography on 2',5'ADP-Sepharose, reactive red 120-agarose and some conventional column chromatography. Both enzymes catalyzed the reduction of 17 alpha-hydroxyprogesterone to 17 alpha, 20 alpha-dihydroxy-4-pregnen-3-one in the presence of NADPH as the preferred cofactor. The apparent Km and Vmax values against 17 alpha-hydroxyprogesterone were 26.2 microM and 1.3 nmol/min/mg for 20 alpha-HSD-I, and 118 microM and 19.4 nmol/min/mg for 20 alpha-HSD-II, respectively. Furthermore, remarkable differences between 20 alpha-HSD-I and 20 alpha-HSD-II were demonstrated under the influence of ionic strength, heat treatment and divalent cations. The molecular weight was estimated to be 39 kDa for 20 alpha-HSD-I and 40 kDa for 20 alpha-HSD-II by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and 30 kDa for both enzymes by gel filtration on Sephadex G-100. These were glycoproteins. There was a large difference in their isoelectric points (8.5 for 20 alpha-HSD-I and 5.5 for 20 alpha-HSD-II). The peptide mapping of the two distinct enzymes was greatly different although there was a slight difference in the amino acid composition.