A new procedure for the quantitation of nuclear and cytoplasmic androgen receptors.

Abstract

Experimental conditions for the measurement of prostatic androgen receptors occupied with unlabeled hormone are described. The assay allows quantitation of cytoplasmic and nuclear receptors occupied with unlabeled ligand by exchange with 17 beta-hydroxy-17 alpha-methyl [3H]estra-4,9,11-trien-3-one at 0 degrees C. The mercurial reagent mersalyl acid (0.2 mM) is used to promote dissociation of more than 90% receptor-bound steroid. The half-time of this reaction is 10-15 min. The reaction is reversible by addition of dithiothreitol or monothioglycerol. Treatment of the receptor with mersalyl and displacement of this reagent by thiol reagents does not alter the steroid binding affinity of the receptor or the sedimentation characteristics. Binding is measured by the hydroxylapatite procedure and all buffers contain 10 mM sodium molybdate. This assay circumvents many of the difficulties associated with conventional exchange assays performed either at elevated temperature or at 16 degrees C for 20-24 h, such as receptor degradation or incomplete exchange.