Human Prostate Androgen Receptor Quantitation: Effects of Temperature on Assay Parameters1

When cytoplasmic extracts of human prostatic tissues were split to permit quantitation of total androgen receptor (RCT) content by saturation analysis at 15°and 2°,we observed that 30% (10 of 32) of the specimens yielded statistically increased values for RCT following incubation at 15°as compared to 2°. Considering only those specimens (13 of 32) showing statisti cally differentiated RCT yield, 77% (10 of 13) yielded greater RCT content following incubation at 15°.The families of as sociation constants (Ka) obtained for RCT determinations at 2° and 15° were not statistically differentiated. The increased yield of RCT content determined at 15°was 95% (mean) and 20 to 350% (range). Nuclear androgen receptor content deter mined at 15° was greater for 25% (2 of 8) of the patient specimens when compared to split determinations performed at 2°. Incubation of nuclear extracts at 15° resulted in a significant 3-fold reduction in receptor Ka for methyltrienolone (R1881). This did not appear to affect assay precision. These studies showed that incubation at 15°is preferable to incuba tion at 2°for quantitation of RCT and nuclear androgen recep tor content by saturation analysis. Single saturating dose determinations of RCT consistently yielded underestimates. The extent of underestimate was vari able from specimen to specimen and was both ligand concen tration and assay temperature dependent. Our data suggest that results of single saturating dose determinations of RCT require cautious interpretation.