Purification of the NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase from female rat pituitary cytosol.
The NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone; 5 alpha-DHP) to 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha-,5 alpha-tetrahydroprogesterone; 3 alpha,5 alpha-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification procedure. Specific activity of purified 3 alpha-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3 alpha-HSOR appears to be a monomer with an approximate molecular weight of 36 kDa and an isoelectric point of about 5.75. The purified enzyme appears as a single protein staining band (36 kDa) when examined by polyacrylamide gel electrophoresis and with both silver or Coomassie blue staining. Under non-dissociating electrophoretic conditions, all of the 3 alpha-HSOR activity co-migrated with the 36 kDa protein staining band. The purified enzyme in the presence of the preferred cofactor, NADPH, has an apparent Km for 5 alpha-DHP of 82 nM and a Vmax of 1.2 mumol of 3 alpha,5 alpha-THP formed per mg protein/30 min. The Km for NADPH was 0.71 microM. In the oxidative direction, the enzyme in the presence of NADP+ has a Km for 3 alpha,5 alpha-THP of 1.4 microM and a Vmax of 9.7 mumol of 5 alpha-DHP formed per mg protein/30 min. The Km for NADP+ was 1.6 microM.
Campbell
J S
JS
Department of Physiological Chemistry, University of Wisconsin-Madison 53706.
Karavolas
H J
HJ
D015096:3-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity
D015096:3-Hydroxysteroid Dehydrogenases; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)