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The in vitro transformation of pregnenolone into progesterone in Digitalis lanata tissues was shown to be catalyzed by a 3β-hydroxysteroid dehydrogenase/ketosteroid isomerase (3β-HSD). Product formation was monitored by HPLC. The enzyme could be partially characterized and 3β-HSD activities were measured in various Digitalis lanata tissues and in cell cultures of other plant species. Since no correlation was observed between biosynthetic competence of the tissue and 3β-HSD activity, it was concluded that this enzyme does not play a major role in regulating cardenolide biosynthesis.

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The conversion of pregnenolone to progesterone is an intermediate step in cardiac glycoside biosynthesis (2, 3) and has been observed in vivo in plants (4), in tissue culture (5), and in leaf homogenates (6) of Digitalis species.During our attempts to study the biosynthetic pathway on an enzymatic level, we established a method for assaying 313-HSD in vitro. Incubations were performed using acetone powders prepared from an undifferentiated embryogenic Digitalis lanata Ehrh. (Scrophulariaceae) suspension culture together with pregnenolone and NAD. The reaction was stopped by ether extraction of progesterone. Testosterone was used as an internal standard and product formation was monitored by HPLC.The enzyme was characterized with respect to its cofactor requirement, pH optimum, temperature optimum, and protein and time dependence. The influences of SH-reagents, ascorbic acid and pregnenolone solubilizing solvents, were investigated.The KM values for pregnenolone and NAD were determined. Progesterone formation by an oxidase, which did not require NAD, was observed but could be inhibited by appropriate incubation temperatures. Using the optimized standard assay, 3f-HSD activity was monitored in embryogenic Digitalis lanata cultures of various differentiation stages, as well as in cell cultures of other plant species. No correlation between the state of differentiation, cardenolide production, and 313-HSD activity could be observed. No 33-HSD activity was detected in alkaloid-producing cell cultures of Ruta graveolens and Berberis wilsoniae. However, significant progesterone formation was found in Ginseng, Strophanthus, and Tobacco cultures, indicating the role of the 313-HSD for steroid metabolism. AcknowledgementsThe authors wish to thank Ms. P. Baur for her expert technical assistance.References 1.Tissue callus cultures originating from Nerium oleander L. were established on a modified medium according to Abou-Mandour (1). It was found that various concentrations of PVP, 2,4-D, and coconut water have only a minimal, often not statistical significant, influence on the growth rate of the calli. Silica gel column separation of a methanolic extract of the calh revealed the presence of ursolic acid, sitosterolglucoside, sterols (cholesterol, sitosterol), and flavonoids (rutin, kaempferol-rhamnosido-glucoside). Pregnanes, eardenolides with 2,6-dideoxysugars which are typical components of the Nerium oleander plant, were not found in this extract.The biotransformation of the cardenolide aglycone digitoxigenine in a suspension culture of Nerium oleander (leaf) was examined. It was shown that, in contrast to results obtained with leaves of Nerium oleander (2), the digitoxigenine was not only glucosylated, but also transformed to gitoxigenine, digitoxigenone, and epi-digitoxigenine. These metabolites were identified by comparison with authentic substances by TLC and HPLC. In one HPLC-system it was possible to separate the structural isomers uzarigenine, digitoxigenine, and epi-digitoxigenine.Acknowledgements