Enzyme assay for 5alpha-reductase type 2 activity in the presence of 5alpha-reductase type 1 activity in rat testis.
The relative abundance and physiological role of 5alpha-reductase (5alphaR) isoforms in rat testis, in particular 5alpha-reductase Type 2 (5alphaR2) are poorly understood. Investigation of 5alphaR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5alpha-reductase Type 1 (5alphaR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5alphaR assays measured the conversion of 3[H]-testosterone to 5alpha-reduced metabolites (dihydrotestosterone+3alpha-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5alphaR1 activity at pH 5.0, the amount of 5alphaR1 activity at pH 5.0 was determined by measuring recombinant rat 5alphaR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5alphaR1 was determined to be 12.4+/-1.4% (mean+/-S.D., n=14). The 5alphaR2 assay was validated by determining recombinant rat 5alphaR2 activity in the presence of recombinant rat 5alphaR1 activity in COS cells. A 99.3+/-14.7% recovery of 5alphaR2 activity was obtained when comparing 5alphaR2 activity recovered versus activity added. 5alphaR1 and 5alphaR2 activities were then assayed in rat testis extracts from 30, 75 and 147 days. Both isoforms markedly declined (50-100-fold) over this age range, with 5alphaR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5alphaR2 activity in the presence of high concentrations of 5alphaR1 was developed and is applicable in the measurement of 5alphaR2 activity in rat testis.
Pratis
K
K
Prince Henry's Institute of Medical Research, P.O. Box 5152, Vic. 3168, Clayton, Australia.
O'Donnell
L
L
Ooi
G T
GT
McLachlan
R I
RI
Robertson
D M
DM
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis* / Q000145:classification* / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002457:Cell Extracts; D004822:Epididymis / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D015203:Reproducibility of Results; D013737:Testis / Q000201:enzymology*
D002457:Cell Extracts; D011994:Recombinant Proteins; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase