CHARACTERIZATION AND LOCALIZATION OF PROGESTERONE 5-ALPHA-REDUCTASE FROM CELL-CULTURES OF FOXGLOVE (DIGITALIS-LANATA EHRH)

Progesterone 5α-reductase, which catalyses the reduction of progesterone to 5α-pregnane-3,20-dione, was isolated and characterized from cell cultures of Digitalis lanata (foxglove). Optimum enzyme activity was observed at pH 7.0, and the enzyme had an apparent K(m) value of 30 μM for its substrate progesterone. THe enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent K(m) value is 130 μM. The optimum temperature was 40°C; at temperatures below 45°C, the product 5α-pregnane-3,20-dione was reduced by a second reaction to 5α-pregnan-3β-ol-20-one. Progesterone 5α-reductase activity was not dependent on bivalent cations. In the presence of EDTA, 0.1 mM-Mn2+ had no influence on enzyme activity, whereas 0.1 mM-Ca2+, -Co2+ and -Zn2+ decreased progesterone 5α-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5α-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5α-reductase was found to be located in the endoplasmic reticulum