DPN- AND TPN-17β-HYDROXY-C19-STEROID DEHYDROGENASES; INTRACELLULAR LOCALIZATION IN DOG PROSTATE

Abstract

Both the DPN- and TPN-linked 17β-hydroxy-C19-steroid dehydrogenase activities of the dog prostate were localized in the microsomal fraction. The usual procedure of tissue fractionation resulted in a loss of DNA from the nuclei and an unsatisfactory separation of the subcellular components. Initial homogenization in 2.2 m sucrose, however, not only yielded clean nuclei but also permitted an effective separation of the mitochondrial, microsomal, and soluble fractions. The completeness of the separations was characterized by the determination of protein, DNA, RNA, succinic dehydrogenase and esterase activities of each fraction. The microsomal preparation retained its dehydrogenase activity on storage at –20° C. It did not metabolize the 3α-hydroxy- or the Δ5-3β-hydroxy groups. The pH of maximum activity was slightly different in the presence of DPN (10.6) than in TPN (10.2). The two coenzymes competed for the active site on the dehydrogenase. The Km value for the substrate (testosterone) was the same in the presence of each coenzyme, but the maximum activity was approximately three times greater with DPN than TPN.