Relationship between steroids and pyridine nucleotides in the oxido-reduction catalyzed by the 17 beta-hydroxysteroid dehydrogenase purified from the porcine testicular microsomal fraction.
The 17 beta-hydroxysteroid dehydrogenase which was purified 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 17 beta-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 17 beta hydroxysteroid dehydrogenase preparation was not spectrophotometrically detectable, because of selective preference of the testicular 17 beta-hydroxysteroid dehydrogenase against NADP(H). To examine stereospecific transfer of the hydrogen from NADPH to androstenedione by the purified 17 beta-hydroxysteroid dehydrogenase, the following tritiated cofactors were synthesized: [4-3-H]NADP+ was prepared by catalytic replacement from non-radioactive NADP+ and 3H2O in the presence of potassium cyanide. Then, [4-pro-R3H]NADPH was enzymatically synthesized from the [4-3H]NADP+ by glucose 6-phosphate and its dehydrogenase. 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 beta-hydroxysteroid dehydrogenase in the presence of these stereospecifically 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, 17alpha-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.
D000735:Androstenedione; D000818:Animals; D003687:Dehydroepiandrosterone; D004958:Estradiol; D004970:Estrone; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D009243:NAD; D009249:NADP; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship; D013552:Swine; D013737:Testis / Q000201:enzymology*; D013739:Testosterone; D013997:Time Factors
D009243:NAD; D004970:Estrone; D013739:Testosterone; D000735:Androstenedione; D003687:Dehydroepiandrosterone; D004958:Estradiol; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases