Determination of delta 5 3 beta-hydroxysteroid dehydrogenase activity in intact isolated rat Leydig cells

A method for the determination of delta 5 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) activity in intact isolated Leydig cells was established. This method utilizes the conversion of [7-3H]dehydroepiandrosterone (1.04 mumole) to androstenedione and expresses the activity of the enzyme as mumoles of androstenedione produced/microgram DNA/h. The reaction is limited to 0.5 - 4 micrograms DNA of Leydig cells/ml (equivalent to 0.1-0.8 million of Leydig cells/ml) and to 1 h of incubation at 34 degree C. The 3 beta-HSD activity of 44 suspensions of Leydig cells isolated from adult rats was found to be 1.13 +/- 0.03 (SE) mumoles/microgram DNA/h. This new method for direct measurement of 3 beta-HSD activity in intact Leydig cells was found to be rapid, easy to perform and highly reproducible.