Porcine testicular 17β-hydroxysteroid dehydrogenase: Affinity chromatography with dye-ligand agarose and demonstration of multiple forms of the enzyme

Abstract

17β-Hydroxysteroid dehydrogenase of porcine testes was purified by an affinity chromatography on agarose coupled with Procion Red HE3B. The purified enzyme preparation approached apparent homogeneity checked by SDS-polyacrylamide gel electrophoresis. By zymographic analysis of the dehydrogenase preparation on polyacrylamide gel disc electrophoresis, this preparation was separable into at least three protein bands which had the dehydrogenase activities. Furthermore, by isoelectric focusing in polyacrylamide gel, four enzyme fractions were distinctly separated, and their isoelectric points were determined as pH 4.9, 5.0, 5.5, and 5.6. The heterogeneity of the dehydrogenase was mainly due to the presence of a different net charge on the molecule, but not difference in molecular size. The molecular weight of these distinct enzymes was estimated as 34,000 by the disc gel electrophoresis. According to our zymographic analysis, this enzyme catalyzed oxidation of testosterone to androstenedione in the presence of NADP + but did not significantly convert androst-5-ene-3β,17β-diol to dehydroepiandrosterone.