Purification and characterization of 3 beta-hydroxysteroid-dehydrogenase/isomerase from bovine adrenal cortex.

The formation of 4-ene-3-ketosteroids from 3 beta-hydroxy-5-ene precursors is an obligatory step in the biosynthesis of hormonal steroids such as glucocorticoids, mineralocorticoids, estrogens and androgens. In the adrenal cortex, pregnenolone, 17 alpha-hydroxy-pregnenolone and dehydroisoandrosterone are converted to progesterone, 17 alpha-hydroxy-progesterone and androstenedione, respectively, by the enzymatic system 3 beta-hydroxy-5-ene steroid dehydrogenase and 3-keto-5-ene steroid isomerase (3 beta-HSD/I). The present work reports a two step purification procedure which yields an homogenous preparation of 3 beta-HSD/I from bovine adrenal cortex. It uses solubilization of the microsomal proteins followed by two chromatographic steps, i.e. DEAE-cellulose and heparine-sepharose columns. The enzyme was obtained as an homogeneous protein exhibiting an apparent molecular size of 45 kDa upon SDS-gel electrophoresis and of 81 kDa upon gel filtration. The purified enzyme exhibits both the 5-ene-3 beta-ol steroid dehydrogenase and isomerase activities in contrast to previous work using a more complex procedure which yielded a final preparation having lost its isomerase activity [Hiwatashi et al., Biochem. J. 98 (1985) 1519-1525]. N-terminal aminoacid (29 residues) sequence of the purified protein was determined and was found identical to that predicted from the nucleic acid sequence of the recently identified enzyme cDNA [Zhas et al. FEBS Lett. 259 (1989) 153-157].