TY - JOUR
T1 - Enzyme assay for 5α-reductase Type 2 activity in the presence of 5α-reductase Type 1 activity in rat testis
AU - Pratis, Kyriakos
AU - O'Donnell, Liza
AU - Ooi, Guck T.
AU - McLachlan, Robert I.
AU - Robertson, David M.
PY - 2000/12/1
Y1 - 2000/12/1
N2 - The relative abundance and physiological role of 5α-reductase (5αR) isoforms in rat testis, in particular 5α-reductase Type 2 (5αR2) are poorly understood. Investigation of 5αR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5α-reductase Type 1 (5αR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5αR assays measured the conversion of 3[H]-testosterone to 5α-reduced metabolites (dihydrotestosterone + 3α-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5αR1 activity at pH 5.0, the amount of 5αR1 activity at pH 5.0 was determined by measuring recombinant rat 5αR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5αR1 was determined to be 12.4 ± 1.4% (mean ± S.D., n = 14). The 5αR2 assay was validated by determining recombinant rat 5αR2 activity in the presence of recombinant rat 5αR1 activity in COS cells. A 99.3 ± 14.7% recovery of 5αR2 activity was obtained when comparing 5αR2 activity recovered versus activity added. 5αR1 and 5αR2 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 5αR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5αR2 activity in the presence of high concentrations of 5αR1 was developed and is applicable in the measurement of 5αR2 activity in rat testis.
AB - The relative abundance and physiological role of 5α-reductase (5αR) isoforms in rat testis, in particular 5α-reductase Type 2 (5αR2) are poorly understood. Investigation of 5αR2 activity using enzyme kinetic studies was hampered by the high concentrations of 5α-reductase Type 1 (5αR1) in rat testis. Therefore, an assay was developed which exploited the differences in pH optima of the two isoforms. The 5αR assays measured the conversion of 3[H]-testosterone to 5α-reduced metabolites (dihydrotestosterone + 3α-Androstanediol) at pH 5.0 and 7.0. To compensate for the overlap of 5αR1 activity at pH 5.0, the amount of 5αR1 activity at pH 5.0 was determined by measuring recombinant rat 5αR1 expressed in COS-7 cells at pH 5.0 and 7.0. The amount of activity at pH 5.0 that was attributed to 5αR1 was determined to be 12.4 ± 1.4% (mean ± S.D., n = 14). The 5αR2 assay was validated by determining recombinant rat 5αR2 activity in the presence of recombinant rat 5αR1 activity in COS cells. A 99.3 ± 14.7% recovery of 5αR2 activity was obtained when comparing 5αR2 activity recovered versus activity added. 5αR1 and 5αR2 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 5αR1 as the predominant isoform. In conclusion, an enzymatic assay that detects 5αR2 activity in the presence of high concentrations of 5αR1 was developed and is applicable in the measurement of 5αR2 activity in rat testis.
KW - 5α-Reductase
KW - Androgen
KW - Spermatogenesis
KW - Testis
UR - http://www.scopus.com/inward/record.url?scp=0034351821&partnerID=8YFLogxK
U2 - 10.1016/S0960-0760(00)00139-4
DO - 10.1016/S0960-0760(00)00139-4
M3 - Article
C2 - 11179911
AN - SCOPUS:0034351821
SN - 0960-0760
VL - 75
SP - 75
EP - 82
JO - The Journal of Steroid Biochemistry and Molecular Biology
JF - The Journal of Steroid Biochemistry and Molecular Biology
IS - 1
ER -