[Purification and some properties of 3 alpha-hydroxysteroid dehydrogenase from pig adrenal cytosol].
A 3 alpha-reducing activity of 5 alpha-dihydrotestosterone (5 alpha-DHT) was found in pig adrenal cytosol. The enzyme (3 alpha-hydroxysteroid dehydrogenase: 3 alpha-HSD) has been purified to homogeneity from pig adrenal cytosol by ammonium sulfate precipitation followed by DEAE-cellulose, 2', 5'-adenosine diphosphate-Sepharose and Sephadex G-100 column chromatographies. The molecular weight was estimated to be 33,000 and 39,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric point was estimated to be 8.5 by isoelectric focusing. The Km and Vmax values for 5 alpha-DHT in the reduction were 10.2 microM and 10.6 nmol/min/mg. The enzyme utilized reduced nicotinamide adenine dinucleotide phosphate (NADPH) or reduced nicotine amide adenine dinucleotide (NADH) in the reduction as a cofactor, but it preferentially required NADPH rather than NADH. Furthermore, the purified enzyme catalyzed not only 3 alpha-reduction of 5 alpha-DHT (9.65 nmol/min/mg), but also catalyzed 20 alpha-reduction of 17 alpha-hydroxyprogesterone (0.58 nmol/min/mg). The enzyme activity of 3 alpha-HSD was strongly inhibited by Hg2+, but it was not inhibited by medroxyprogesterone acetate and some anti-inflammatory agents. No remarkable differences was demonstrated between 3 alpha-HSD and 20 alpha-HSD activity under the influence of heat treatment, divalent cation, anti-inflammatory agents and some inhibitory steroids. These results strongly suggest that 3 alpha-HSD purified from pig adrenal cytosol is a bi-functional enzyme catalyzing 3 alpha- and 20 alpha-HSD activities.
Nakajin
S
S
Faculty of Pharmaceutical Sciences, Hoshi University, Tokyo, Japan.
Fujii
S
S
Ohno
S
S
Shinoda
M
M
D015089:20-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D015096:3-Hydroxysteroid Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D000311:Adrenal Glands / Q000201:enzymology*; D000818:Animals; D002384:Catalysis; D002413:Cations, Divalent; D003600:Cytosol / Q000201:enzymology; D013196:Dihydrotestosterone / Q000378:metabolism; D004355:Drug Stability; D006358:Hot Temperature; D008970:Molecular Weight; D009249:NADP; D013552:Swine
D002413:Cations, Divalent; D013196:Dihydrotestosterone; D009249:NADP; D015096:3-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)