Purification and properties of NADP-dependent 17 beta-hydroxysteroid dehydrogenase solubilized from porcine-testicular microsomal fraction.

Abstract

17β‐Hydroxysteroid dehydrogenase, which catalyzes oxidoreduction between androstenedione and testosterone, has been solubilized by sonication from porcine testicular microsomal membrane for the first time. From the solubilized enzyme preparation, the dehydrogenase activity was precipitated by between 40 and 75% saturation of ammonium sulfate. After the precipitate was dissolved, the enzyme solution was applied to a Sephadex G‐100 column and the dehydrogenase activity emerged at 1.80 void volumes. For further purification, it was subjected to DEAE‐cellulose column chromatography; by eluting with a linear gradient of increasing phosphate concentration, the enzyme activity between 15 and 20 mM phosphate. Finally, when the partially purified enzyme preparation was applied to a Bio‐Gel P‐100 column, the dehydrogenase activity emerged at 1.46 void volumes. The final enzyme preparation was purified 1880‐fold in specific activity compared to the microsomal fraction.The purified enzyme preparation approached apparent homogeneity, checked by discontinuous polyacrylamide gel electrophoresis at pH 8.3 and by thin‐layer gel chromatography. The final preparation was able to produce 67.7 nmol testosterone from androstenedione per min per mg protein at 37°C in the presence of NADPH, and could be stored at–20°C for 6 months with no significant loss of the enzyme activity. The molecular weight and molecular radius of the 17β‐hydroxysteroid dehydrogenase was estimated to be about 35500 and 2.37 nm, respectively, from the results of the gel filtration chromatography. In the phosphate buffer pH 7.4 maximal rate of reduction of androstenedione by the enzyme was observed at 50°C, and Q10 for the enzyme was 3.7, while the activation energy was 19500 cal. The rate of reduction of androstenedione at 37.5°C was about a half of the maximal one at 50°C in the phosphate buffer, whereas the reduction rate at 37°C was higher than the one at 50°C in the citric acid‐Na2HPO4 buffer pH 7.3. The enzyme for androstenedione showed a pH optimum between 6.5 and 7.5 in the presence of NADPH at 37°C but the optimal pH of the enzyme was shifted to 8.5 by raising the incubation temperature to 50°C. Estrone was the acceptor of the hydrogen released from NADPH by the 17β‐hydroxysteroid dehydrogenase and dehydroepiandrosterone served a similar purpose for androstenedione. The oxidation of testosterone by the enzyme in the presence of NADP+ proceeded at the same rate as the reduction of androstenedione. The purified enzyme preparation was devoid of any alcohol or 3‐hydroxyhexobarbital dehydrogenase activities or other enzymes related to testicular steroidogenesis.