Determination of Δ53β-hydroxysteroid dehydrogenase activity in intact isolated rat Leydig cells

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Abstract
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 .mu.mol) to androstenedione and expresses the activity of the enzyme as .mu.mol of androstenedione produced/.mu.g DNA per h. The reaction is limited to 0.5-4 .mu.g 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 1.13 .+-. 0.03 (SE) .mu.mol/.mu.g DNA per h. This new method for direct measurement of 3.beta.-HSD activity in intact Leydig cells was rapid, easy to perform and highly reproducible.