3 alpha-Hydroxysteroid-5 beta-oxidoreductase in tissue cultures of Digitalis lanata

Putative intermediates of cardenolide biosynthesis, namely progesterone, pregnenolone, 5 beta-pregnane-3,20-dione or 5 beta-pregnan-3 beta-ol-20-one, were administered to light- or dark-grown shoot cultures of Digitalis lanata. The unsaturated compounds were reduced to their respective 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- and the 3 beta-isomer of 5 beta-pregnan-3-ol-20-one when incubated in the presence of 5 beta-pregnane-3,20-dione. When 5 beta-pregnan-3 alpha-ol-20-one was administered the cultured cells accumulated large amounts 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 isomerization 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 the incubation mixture. Under these conditions the formation of 5 beta-pregnan-3 beta-ol-20-one was inhibited. The enzyme activity could be recovered from membrane-free supernatants. Optimum enzyme activity occurred at pH 7.0 and 42 degrees C. The energy of activation was 56.2 kJ/mol and the enzyme reaction was found to be NADPH-dependent. SH reagents were essential for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)