Purification and properties of a new testosterone 17beta-dehydrogenase (NADP+) from guinea-pig liver.
As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.
D000731:Androstanes; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006913:Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009249:NADP; D010710:Phosphates / Q000494:pharmacology; D013696:Temperature; D013739:Testosterone
D000731:Androstanes; D010710:Phosphates; D013739:Testosterone; D009249:NADP; D006913:Hydroxysteroid Dehydrogenases