Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate.

Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of 3H-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for 3H dihydrotestosterone being somewhat higher than for 3H testosterone. Equilibrium dissociation constants for 3H dihydrotestosterone and 3H testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for 3H dihydrotestosterone and 2 X 10(8) M-1 h-1 for 3H testosterone) and dissociation (t 1/2 congruent to 40 hr for 3H dihydrotestosterone and 15 h 3H testosterone). Evaluation of the effect of temperature on androgen receptor binding of 3Htestosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of 3H testosterone or 3H dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S. Differences in sedimentation coefficients were observed following (NH4)2SO4 precipitation which did not influence the binding properties of the receptors. These results, together with measurements of3alpha/beta-hydroxysteroid oxidoreductase activity in vitro, suggest that organ differences in receptor binding of 3H dihydrotestosterone and 3H testosterone in vivo result from relative differences in intracellular concentrations of these androgens rather than from differences in receptor affinities.