We have investigated the stabilizing effects of sodium molybdate on the androgen receptor in mouse kidney cytosol. In these studies, this receptor was measured with [3H]-methyltrienolone binding using ion exchange filters to separate bound and free steroids. Maximal in vitro stabilization of the androgen receptor occurs in the presence of 5–20 mM Na2MoO4 and is highly dependent on the final pH of the buffer. There is a narrow (pH) optimum for stabilization of the steroid-unoccupied receptor between pH 6.6–7.1. The pH optimum of the steroid-occupied receptor is only slightly broader (pH 6.4–7.5). Since 24 h are required for low concentrations of [3H]methyltrienolone to come to equilibrium with the androgen receptor, stabilization by sodium molybdate results in an increase in the number of binding sites detected by saturation analysis without a consistent effect on the equilibrium binding constant (Kd). The rates of association and dissociation of the steroid-receptor complex in the presence and absence of molybdate were: k1 equals 2.9 × 108 M-1 h-1 and k-1 equals 0.044 h-1 (mean of two determinations) with molybdate; and k1 equals 3.1 ×times; 108 M-1 h-1 and k-1 = 0.023 h-1 (mean of two determinations) without molybdate. Finally, the influence of molybdate on the thermolability of the unoccupied androgen receptor was evaluated. Molybdate significantly increased the t1/2 of inactivation of the receptor at all temperatures examined ranging from 4–41.5 C. Arrhenius analysis of the rates of degradation showed that the receptor has a 21% higher apparent enthalpy (∆H) of inactivation in the presence of molybdate than in its absence (∆Hmolyhdate Equals 31.9 kCal/mol; ∆Hwithout molyhdate equals 26.4 kCal/mol). We conclude that 1) sodium molybdate is a useful reagent for the assay and isolation of androgen receptors, and 2) the observations are consistent with the suggestion that molybdate stabilizes the androgen-binding site at least partially through a direct effect on the receptor.
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