Studies on the characterization of rat prostate androgen receptors

Abstract

In the presence of sodium molybdate and protease inhibitors, two forms of androgen-receptor complexes were observed which sedimented in the areas of 8–9S and 5–7S by SDG centrifugation. The intermediary 5–7S form was better seen when complexes were incubated at low KCI concentrations. The sedimentation coefficient of this form fluctuated between 5 and 7S depending on the KCI concentration. At high ionic strength (0.6M KCl) in all media, one form only was observed having a sedimentation coefficient value of 4.3S. By gel exclusion chromatography, we also observed two specific entities at 75A and 68A; in the presence of 0.6M KCl, however, two entities were found at 68Å and 43Å. The constant presence of protease inhibitors in all buffers was necessary to separate the intermediary 68A form. We calculated molecular weights of about 270 kDa, 190 kDa, and 80 kDa respectively for these three forms. [³H]R1881-receptor complexes bound to DEAE-cellulose and were eluted in the absence of glycerol at O.1M and 0.2M KCl. Material found at 0.1M KCl sedimented in the areas of 5–7S and 8–9S in nearly equal proportion, and that found at 0.2M KCI sedimented in the 8-9S area only. When the cytosol was chromatographed at a fast flow rate (4ml/min), untransformed 8–9S receptors did not bind to phosphocellulose, but transformed complexes were retained, could be eluted with 0.4M KCl and sedimented in the 4S area on KCI free SDG centrifugation. When the excluded untransformed 8–9S complexes were re-chromatographed at a slow flow rate (1ml/min), they were retained on phosphocellulose, and could be eluted with 0.3M KCI. Complexes of this latter fraction still sedimented in the 8–9S area on KCl free SDG centrifugation. The first fast flow rate chromatography was thus necessary to eliminate destabilizing components, presumably proteases. In conclusion, we were able to demonstrate and to characterize an intermediary 5-7S (6.6S after desalting) form of androgen receptors in rat ventral prostate. Also, using specific experimental conditions, we were able to conserve the 8–9S form during a series of manipulations including phosphocellulose chromatography. The presence of a putative ‘ζ’ subunit which may participate in the formation of the 8-9S form is postulated.