ABSTRACT
A 3 beta-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5 alpha-pregnane-3,20-dione to 5 alpha-pregnan-3 beta-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 8.0. The reaction had an optimum incubation temperature of 25 degrees C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5 alpha-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 microM for 5 alpha-pregnane-3,20-dione and 50-120 microM for the co-substrate NADPH2. In order to localize 3 beta-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3 beta-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity.
