3-ALPHA-HYDROXYSTEROID-5-BETA-OXIDOREDUCTASE IN TISSUE-CULTURES OF DIGITALIS-LANATA

Putative intermediates of cardenolide biosynthesis, namely progesteron e, pregnenolone, 5 beta-pregnane-3,20-dione or 5 beta-pregnan-3 beta-o l-20-one, were administered to light- or dark-grown shoot cultures of Digitalis lanata. The unsaturated compounds were reduced to their resp ective 5 alpha-pregnanes, 5 beta-pregnane-3,20-dione was reduced to 5 beta-pregnan-3 alpha-ol-20-one and 5 beta-pregnan-3 beta-ol-20-one was isomerized to the respective 3 alpha-pregnane. Suspension cultures of Digitalis lanata, on the other hand, accumulated both the 3 alpha- an d the 3 beta-isomer of 5 beta pregnan-3-ol-20-one when incubated in th e presence of 5 beta-pregnane-3,20-dione. When 5 beta-pregnan-3 alpha- ol-20-one was administered the cultured cells accumulated large amount s of the 3 beta-isomer together with small amounts of 5 beta-pregnane- 3,20-dione, which may be regarded as an intermediate during the isomer ization reaction. Cell-free, buffered extracts from light-grown shoots were shown to reduce 5 beta-pregnane-3,20-dione almost exclusively to 5 beta-pregnan-3 alpha-ol-20-one when 0.05 M MgCl2 were present in th e incubation mixture. Under these conditions the formation of 5 beta-p regnan-3 beta-ol-20-one was inhibited. The enzyme activity could be re covered from membrane-free supernatants. Optimum enzyme activity occur red at pH 7.0 and 42 degrees C. The energy of activation was 56.2 kJ/m ol and the enzyme reaction was found to be NADPH-dependent. SH reagent s were essential for enzyme activity. The enzyme seems to be specific for 5 beta-pregnan-3-ones since neither 5 alpha-pregnane-3-ones nor De lta(4)/Delta(5)-pregnenes were reduced. The NADPH:5 beta-pregnane 3 al pha-hydroxysteroid-5 beta-oxidoreductase described here may play a rol e in the regulation of cardenolide biosynthesis by removing precursors , such as 5 beta-pregnane-3,20-dione, from the pathway.