On the mitochondrial 17β-hydroxysteroid dehydrogenase from human endometrium and endometrial carcinoma: Characterization and intramitochondrial distribution

Abstract

To determine intramitochondrial location of an endometrial 17β-hydroxysteroid dehydrogenase (17β-HSD), purified mitochondria from secretory endometrium were separated into inner and outer membranes, matrix and an intermembrane fraction. The purity of each fraction was monitored by marker enzymes. A radiochemical assay was used for the determination of 17β-HSD activities. It was found that the 17β-HSD was mainly located in the outer membranes of the mitochondria. Kinetic parameters and substrate specificity of the enzyme were determined. The conversion of estradiol (E 2) to estrone (E 1) by purified mitochondria was linear with time and protein concentration. The optimum temperature was approximately 40°C and the optimum pH 9·5. For the reduction of E 1 the optimum pH was 6·0. With NAD E 2 was oxidized approximately 15 times more rapidly than with NADP. The apparent K m -values for E 2 (in the presence of NAD) were 13 × 10 −6 M and 4 × 10 −6 M in proliferative and secretory endometrium, respectively. The maximal velocity was highest in secretory endometrium. Testosterone and androstenedione could also serve as substrates. In normal endometrium they were interconverted at approximately 50% of the rate of E 2 and E 1. In endometrial carcinoma androstenedione was reduced twice as rapidly as estrone. Sulfhydryl groups were shown to be essential for catalysis. The mitochondrial 17β-HSD was similar in character to the cytoplasmic, microsomal and nuclear enzymes described previously.