Kinetic Data on the Formation of 17β–Hydroxy–5α–androstan–3–one in Renal Nuclei from Male and Female Rats

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Abstract
The 5α–reductase that promotes conversion of testosterone to 17β–hydroxy–5α–androstan– 3–one (dihydrotestosterone) has been further investigated in the rat kidney. Enzyme assays were performed by the incubation of 3H—labeled substrate in the presence of an NADPH—generating system. Radiometabolites were separated by paper— and thin layer chromatography, and quantitated by liquid scintillation counting. Comparison of the enzyme activities in nuclear preparations with an increasing degree of purity confirms that appreciable part of the total 5α—reductase activity in male and female kidneys is closely associated with the nuclear fraction. Part of the 3α–hydroxysteroid dehydrogenase activity cannot be dissociated from the purified nuclei in male rats only. The kinetics of the 5α–reductase were studied by the in vitro incubation of renal nuclei with l,2–3H–testosterone. It was demonstrated that the enzyme exhibits an absolute requirement for NADPH. The reaction proceeds optimally at a pH about neutrality and the apparent Km for testosterone is O.8μM. The enzyme is stable at — 20 C for at least 3 days, and the activity is markedly enhanced by raising the temperature of the incubation mixture from 10 C up to 37 C. In the absence of substrate and cofactors, however, enzyme denaturation occurs very rapidly at the latter temperature. Various steroids with a Δ4–3– keto structure and without an oxygen function at C–ll inhibit the 5α–reduction of testosterone. The potent inhibitors which were tested underwent 5α–reduction when incubated with renal nuclei. The fact that mutual inhibitory effects can be demonstrated in mixed substrate incubations suggests that the mechanism of inhibition is a competition for the active site of the 5α–reductase. Physiological androgen antagonists such as the estrogens and pharmacologically active antiandrogens such as cyproterone do not interfere with the formation of dihydrotestosterone. (Endocrinology91: 54, 1972)

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