Abstract
Different subcellular fractions (purity checked by electron microscopy and respective marker enzymes) were incubated with 0.1 muCi 14C-progesterone (10 muM) in 0.15 M phosphate buffer at pH 7.4 and 37 C under air for varying periods of time in the presence of NAD(P)H (500 muM). By the preparation of chromic acid oxidation products and acetates, thin-layer chromatography, and crystallisation to constant specific activity, the following metabolites were identified: 20alpha-hydroxypregn-4-en-3-one, 20alpha-hydroxy-5alpha-pregnan-3-one, 20alpha-hydroxy-5beta-pregnan-3-one, 5alpha-pregnane-3,20-dione, and 5beta-pregnane-3,20-dione, indicating the presence of a 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and 5alpha- and 5beta-reductases. Most of the 20alpha-HSD activity was located in mitochondria (associated mainly with outer membranes) and microsomes. Purified nuclei and cytosol contained 1/6 to 1/18 of the activity of mitochondria and microsomes, respectively. SUBFRACTIONS OF ENDOMETRIAL CELLS ONLY CONTAINED EITHER 5ALPHA- OR 5BETA-REDUCTASE ACTIVITY. 5alpha-reductase activity was mainly associated with microsomes, 5beta-reductase activity was found only in the cytosol. While in normal endometrium specific enzyme activities in subcellular fractions depended on the phase of the cycle, in endometrial carcinoma it depended on the degree of tumour differentiation. The highest values of 5alpha-reductase activity were found in the early proliferative phase. 20alpha-HSD activity was highest in the middle of the secretory phase. The specific activity of the 5alpha-reductase increased with decreasing differentiation of the tumour while the specific activity of the 20alpha-HSD decreased. Kinetic parameters (Km-values, coenzyme requirements and maximum velocities) were determined. The Km-value for progesterone of the 20alpha-HSD in proliferative endometrium was significantly higher than in secretory endometrium, while the Km-values of the 5alpha- and 5beta-reductases were considerably lower during the proliferative than secretory phase.
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