Purification and properties of human hepatic 3 alpha-hydroxysteroid dehydrogenase.
3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5 beta-[3H]dihydrocortisol (5 beta-DHF) as substrate. Electrophoretically homogeneous enzyme was obtained using a procedure that involved ammonium sulfate precipitation and three successive column chromatography steps: DEAE-cellulose, hydroxylapatite and Blue-Sepharose. The enzyme is a monomer since the native molecular weight was found to be 37,000, using a calibrated Sephadex G-75 column, and the denatured subunit molecular weight was determined to be 38,500, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The enzyme had a pI of 5.6-5.9. The 3-ketosteroids: cortisol, testosterone, progesterone and androstenedione, were not substrates for 3 alpha-HSD indicating that a saturated 4,5 double bond was required for substrate activity. The conformation at the 5 position, however, did not influence substrate activity since 5 alpha- and 5 beta-DHF and 5 alpha-dihydrotestosterone were all reduced at similar rates. The purified enzyme preferred NADPH to NADH as a cofactor and showed a broad peak of activity in the pH range of 6.8-7.4. The apparent Km for 5 beta-DHF was 18 microM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4 degrees C. Freezing and thawing of the enzyme resulted in a large loss of activity during early stages of the purification. This is the first report of the purification to homogeneity of 3 alpha-HSD from human tissue.
Iyer
R B
RB
Department of Medicine, New York Medical College, Valhalla.
Binstock
J M
JM
Schwartz
I S
IS
Gordon
G G
GG
Weinstein
B I
BI
Southren
A L
AL
D015096:3-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000911:Antibodies, Monoclonal; D003600:Cytosol / Q000201:enzymology; D005123:Eye / Q000201:enzymology; D006801:Humans; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D015096:3-Hydroxysteroid Dehydrogenases