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].
Cherradi
N
N
Unité INSERM 244, Département de Biologie Moléculaire et Structurale, Grenoble, France.
Guidicelli
C
C
Defaye
G
G
Chambaz
E M
EM
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism*; D000302:Adrenal Cortex / Q000201:enzymology*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013379:Substrate Specificity
D015096:3-Hydroxysteroid Dehydrogenases