Relationship between Steroids and Pyridine Nucleotides in the Oxido-Reduction Catalyzed by the 17 beta-Hydroxysteroid Dehydrogenase Purified from the Porcine Testicular Microsomal Fraction

Abstract

The 17β-hydroxysteroid dehydrogenase which was purified from porified from porcine testicular microsomal fraction [Inano, H. and Tamaoki, B. (1974) Eur. J. Biochem. 44, 13–23] catalyzed the reduction of androstenedione to testosterone with the accompanying oxidation of equimolar NADPH. For the oxido-reduction of the steroids, the 17β-hydroxysteroid dehydrogenase preferred NADP(H) to NAD(H). Transhydrogenation from NADPH to NAD⁺ or NADH to NADP⁺ through the cyclic oxido-reduction of the steroids by the purified 17β-hydroxysteroid dehydrogenase preparation was not spectrophotometrically detectable, because of selective preference of the testicular 17 β-hydroxysteroid dehydrogenase against NADP(H). To examine stereospecific transfer of the hydrogen from NADPH to androstenedione by the purified 17 β-hydroxysteroid dehydrogenase, the following tritiated cofactors were synthesized: [4-³H]NADP⁺ was prepared by catalytic replacement from non-radioactive NADP⁺ and ³H₂O in the presence of potassium cyanide. Then, [4-pro-R-³-H]NADPH was enzymatically synthesized from the [4-³H]NADP⁺ by glucose 6-phosphate and its dehyudrogenase. On the other hand, [4-pro-S-³H]NADPH was prepared from the [4-³H]NADP⁺ by isocitrate and isocitrate dehydrogenase. When androstenedione was incubated with the 17 β-hydroxysteroid dehydrogenase in the presence of these sterospecifically ³H-labeled cofactors, only the tritium located at 4-pro-S position of the nicotinamide moiety of NADPH was transferred to testosterone. The location of the tritium in the testosterone molecule produced, 17 α-position of the steroid, was assigned by the fact that the tritium of the testosterone remained in its molecule after acetylation, but was completely lost by oxidation.