Exchange assay for androgen receptors in the presence of molybdate

Several reports have shown that sodium molybdate stabilizes steroid hormone receptors. We have utilized these observations to develop an exchange assay for the androgen receptor at elevated temperatures. Exchange was found to be complete after 30 min at 30 degrees C. Receptor degradation was negligible during this treatment. Scatchard analysis indicated that the dissociation constant of the androgen receptor was similar both in the absence (Kd = 3.9 nM) and presence (Kd = 2.9 nM) of molybdate. Steroid specificity of the androgen receptor was unaltered by this treatment. The exchange procedure was reproducible, with an interassay variation of 2.45% and intraassay variation less than 10.0%. Using this assay, highest concentrations of androgen binding were measured in androgen target tissues of the rat (Dunning R3327 tumor, prostate and seminal vesicle; 23.37, 20.20 and 19.84 fmol/mg protein respectively). Lower concentrations were observed in other tissues (lung, brain, heart, spleen, liver and kidney; 9.06, 5.63, 3.50, 2.42, 2.33 and 1.36 fmol/mg protein respectively). These results demonstrate that molybdate stabilization of the androgen receptor allows efficient steroid exchange without significant alteration of the receptor's steroid binding properties. Furthermore, this exchange assay can be used to obtain a reasonable measurement of receptor concentrations in different androgen target tissues.