Further characterization of 17 beta-hydroxysteroid dehydrogenase activity in cultured myometrial cells: cofactor...

To further characterize 17 beta-hydroxysteroid dehydrogenase (17 beta-SDH) from cultured ovine myometrial cells, an assay was established in whole cell homogenates and cell subfractions. Tritiated estradiol (E2) was incubated in the presence of an excess of cofactor and estrone (E1) formed purified by thin-layer chromatography. The enzyme activity was linear with time up to 2 hours and with protein concentration up to 0.7 mg/ml at the substrate concentration used (5 X 10(-9) M). The routine assay was for 30 min in the presence of 0.5 mg/ml of protein. Both NAD+ or NADP+ could sustain enzyme activity but NAD+ was twice as much efficient. Most of the enzyme activity was associated with the microsome and mitochondrial membranes. The addition of an excess (1000 microM) of NAD+ to the incubation medium prevented the progressive decline observed with time in a given subculture in the intact cell monolayer assay, supporting our previous hypothesis that this decline was due to cofactor depletion. In contrast, the slow and irreversible decline of enzyme activity observed in successive subcultures was not prevented by the addition of cofactor to the homogenate and thus reflects another phenomenon, probably a change in metabolism with age.