(NADP+) from Guinea-Pig Liver

As a result of studies of guinea-pig liver testosterone 17/J-dehydrogenase (NADP+) (EC 1.1.1.64), anew testosterone 17f8-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105000g-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 17fl-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pHoptima 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. 5a-Androstanes, such as 5a-androstane-3a, 1 7fi-diol, 5a-androstane3fi,17fi-diol and 17f8-hydroxy-5a-androstan-3-one, and 5fi-congeners, such as 51J-androstane-3a,17fi-diol, 5fl-androstane-3f6,17fi-diol and 17f6-hydroxy-5fl-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.