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Human Saturated Steroid 6-Hydroxylase¹

Cecil H. and Ida Green Center for Reproductive Biology Sciences and the Departments of Obstetrics-Gynecology and Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas 75235

Address all correspondence and requests for reprints to: Paul C. MacDonald, M.D., Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235-9051. E-mail: macdonald{at}grnctr.swmed.edu

This study was conducted to evaluate further the reaction catalyzed by the saturated steroid 6-hydroxylase of extrahepatic human tissues. Progesterone and 5-dihydroprogesterone (5-DHP) are plasma-borne precursors of 5-pregnan-3-ol-20-one, an anxiolytic/anesthetic steroid, and 5-pregnan-3ß-ol-20-one in extrahepatic human tissues. These two steroids are metabolized further by a saturated steroid 6-hydroxylase enzyme(s) that is distinct from the cytochrome P450 6-hydroxylase that catalyzes the 6-hydroxylation of ⁴-3-ketosteroids such as progesterone, cortisol, and testosterone. Products of this saturated steroid 6-hydroxylase, viz. 3ß/,6-dihydroxy-5-pregnan-20-ones, are major radiolabeled urinary metabolites (excreted as glucuronosides) of iv administered tritium-labeled 5-DHP in women and men. T47-D human breast cancer cells, which are rich in saturated steroid 6-hydroxylase activity, were used as the enzyme source in this study. The greatest total and the highest specific activity of saturated steroid 6-hydroxylase were localized in microsome-enriched preparations; enzyme activity was linear with incubation time up to 30 min and with microsome-enriched tissue protein concentrations between 0.05–0.5 mg/mL incubation mixture. The velocity of the reaction was similar in incubations in which the pH was varied from 6.0–8.0, and NADH and NADPH were equally effective in supporting the 6-hydroxylation of 5-pregnan-3ß-ol-20-one and 5-pregnan-3-ol-20-one. The more efficient substrates for this enzyme were 5-pregnan-3ß-ol-20-one and 5-pregnan-3-ol-20-one, and the apparent K_m (3.5 µmol/L) and maximum velocity (150 pmol/min·mg microsome-enriched protein) for these two substrates were indistinguishable. 5-Androstane-3ß,17ß-diol was less efficiently 6-hydroxylated, and 5-androstane-3,17ß-diol was an inefficient substrate. The addition of a variety of inhibitors of cytochrome P450 monooxygenases to the incubation mixtures did not diminish significantly the 6-hydroxylation of 5-pregnan-3ß-ol-20-one, findings consistent with those of other investigators who suggested that human saturated steroid 6-hydroxylase (of human prostate) is not a cytochrome P450.

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