20β-Hydroxysteroid Dehydrogenase of Neonatal Pig Testis: Purification and Some Properties1

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Abstract
20β-Hydroxysteroid dehydrogenase was purified from a cytosol fraction of neonatal pig testes to homogeneity as demonstrated by polyacrylamide gel electrophoresis (PAGE) and by isoelectric focusing. The molecular weight was estimated to be 30,500 using PAGE with sodium dodecyl sulfate and the gel filtration method. Molecular estimations showed that the purified enzyme consisted of a single polypeptide chain. It catalyzed the reduction of 17α-hydroxyprogesterone to 17α 20β-dihydroxypregn-4-en-3-one with NADPH. Furthermore, the C21-steroids, such as progesterone, pregnenolone, 17α-hydroxypregnenolone, deoxycorticosterone, and deoxycortisol were also reduced by the purified enzyme. Apparent Km values for 17α-hydroxyprogesterone, progesterone, pregnenolone, and deoxycorticosterone were 9.4, 1.5, 4.0, and 8.6 μM, respectively. The enzyme did not show 20α-hydroxysteroid dehydrogenase activity. The maximum rate of enzyme activity was observed at 45°C and optimum pH was at pH 5.5. The enzyme activity was strongly inhibited by heavy metal ions such as Hg2+ and Cu2+.