Characterization and localization of progesterone 5 alpha-reductase from cell cultures of foxglove (Digitalis lanata EHRH).
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 microM 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 microM. The optimum temperature was 40 degrees C; at temperatures below 45 degrees 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 progesterone 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.
Wendroth
S
S
Institut für Biologie I, Universität Tübingen, Federal Republic of Germany.
Seitz
H U
HU
D002412:Cations; D002478:Cells, Cultured; D004070:Digitalis / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011374:Progesterone / Q000378:metabolism; D013696:Temperature
D002412:Cations; D008670:Metals; D011374:Progesterone; D009249:NADP; D008623:Mercaptoethanol; D004492:Edetic Acid; D010088:Oxidoreductases; C022447:3-oxo-5-alpha-steroid 4-dehydrogenase (NADP(+)); D004229:Dithiothreitol