Characterization and localization of progesterone 5α-reductase from cell cultures of foxglove (Digitalis lanata EHRH)

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Abstract
Progesterone 5.alpha.-reductase, which catalyses the reduction of progesterone to 5.alpha.-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 Km value of 30 .mu.M for its substrate progesterone. The enzyme needs NADPH as reductant, which could not be replaced by NADH. For NADPH, the apparent Km value is 130 .mu.M. The optimum temperature was 40.degree. C; at temperatures below 45.degree. C, the product 5.alpha.-pregnane-3,20-dione was reduced by a second reaction to 5.alpha.-pregnan-3.beta.-ol-20-one. Progesterone 5.alpha.-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 progesteone 5.alpha.-reductase activity. Only 0.1 mM-Mg2+ was slightly stimulatory. EDTA and thiol reagents such as dithiothreitol stimulate progesterone 5.alpha.-reductase activity. By means of linear sucrose gradient fractionation of the cellular membranes, progesterone 5.alpha.-reductase was found to be located in the endoplasmic reticulum.

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