Development of a quantitative assay method for 3 beta-hydroxy-delta 5-steroid dehydrogenase in the rat testis

We investigated the first step of the sex steroid hormone biosynthesis pathway by assaying the activities of 3 beta-hydroxy-delta 5-steroid dehydrogenase, the rate-limiting enzyme in this pathway. We have developed a simple and rapid colorimetric assay for 3 beta-hydroxy-delta 5-steroid dehydrogenase in rat testis. The supernatant from rat testis tissue homogenates were used for the enzyme assay. The enzyme activity was determined by measuring the absorbance at 570nm which indicates the rate of conversion of pregnenolone into progesterone in the presence of NAD, using phenazine methosulfate and nitro blue tetrazolium as the color reagent. The activity of this enzyme ranged from 4.57+/-1.34 to 10.56+/-2.13 nmol/mg protein/min with a mean activity of 8.96+/-1.27 nmol/mg protein/min. The K(m) of the enzyme at an optimum pH of 7.25 was about 4.7+/-0.12 nM.