Purification and properties of human hepatic 3α-hydroxysteroid dehydrogenase

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Abstract

3α-Hydroxysteroid dehydrogenase (3α-HSD) was purified greater than 500-fold from human liver cytosol. The purification was monitored using 5β-[3H]dihydrocortisol (5β-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 3α-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 5α- and 5β-DHF and 5α-dihydrotesterone 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 5β-DHF was 18 μM. The enzyme was markedly stabilized by 50 mM phosphate buffer containing 10 to 20% glycerol at 4°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 3α-HSD from human tissue.

References (35)

  • C. Kubli-Garfias et al.

    Induction of lordosis behavior in female rats by intravenous administration of progestins

    Horm. Behav.

    (1977)
  • C. Kubli-Garfias et al.

    In vitro inhibition of rat uterine contractility induced by 5α- and 5β-progestins

    Steroids

    (1979)
  • J.A. Gustaffson et al.

    Sex steroid induced changes in hepatic enzymes

    A. Rev. Physiol.

    (1983)
  • T.M. Penning et al.

    Prostaglandin dehydrogenase activity of purified rat liver 3α-hydroxysteroid dehydrogenase

    Biochem. Biophys. Res. Commun.

    (1987)
  • T.M. Penning et al.

    Inhibition of a major NAD(P)-linked oxidoreductase from rat liver cytosol by and by prostaglandins

  • A.L. Southren et al.

    5β-Dihydrocortisol: possible mediator of the ocular hypertension in glaucoma

    Invest. Ophthalmol. Vis. Sci.

    (1985)
  • A.L. Southren et al.

    Intraocular hypotensive effect of a topically applied cortisol metabolite: 3α,5β-tetrahydrocortisol

    Invest. Ophthalmol. Vis. Sci.

    (1987)
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