Enzyme assay for 5 alpha-reductase Type 2 activity in the presence of 5 alpha-reductase Type I activity in rat testis

The relative abundance and physiological role of 5 alpha -reductase (5 alph aR) isoforms in rat testis, in particular 5 alpha -reductase Type 2 (5 alph a R2) are poorly understood. Investigation of 5 alpha R2 activity using enz yme kinetic studies was hampered by the high concentrations of 5 alpha -red uctase Type 1 (5 alpha R1) in rat testis. Therefore. an assay was developed which exploited the differences in pH optima of the two isoforms. The 5 al phaR assays measured the conversion of (3)[H]-testosterone to 5 alpha -redu ced metabolites (dihydrotestosterone + 3 alpha -Androstanediol) at pH 5.0 a nd 7.0. To compensate for the overlap of 5 alpha R1 activity at pH 5.0, the amount of 5 alpha R1 activity at pH 5.0 was determined by measuring recomb inant rat 5 alpha R1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5 alpha R1 was determined to be 12.4 +/- 1.4% (mean +/- S.D., n = 14). The 5 alpha R2 assay was validate d by determining recombinant rat 5 alpha R2 activity in the presence of rec ombinant rat 5 alpha R1 activity in COS cells. A 99.3 +/- 14.7% recovery of 5 alpha R2 activity was obtained when comparing 5 alpha R2 activity recove red versus activity added. 5 alpha R1 and 5 alpha R2 activities were then a ssayed in rat testis extracts from 30, 75 and 147 days. Both isoforms marke dly declined (50-100-fold) over this age range, with 5aR1 as the predominan t isoform. In conclusion, an enzymatic assay that detects 5 alpha R2 activi ty in the presence of high concentrations of 5 alpha R1 was developed and i s applicable in the measurement of 5 alpha R2 activity in rat testis. (C) 2 001 Elsevier Science Ltd. All rights reserved.