Identification of Limited Capacity Androgen Binding Components in Nuclear and Cytoplasmic Fractions of Canine Prostate

Abstract

Nuclear and cytoplasmic androgen receptors were identified in canine prostate preparations. Saturation analysis employing 5α-dihydrotestosterone and tissue from intact dogs demonstrated that crude nuclear pellets contained 85 femtomoles of KCI-extractable and 106 femtomoles of ethanol-extractable receptor sites per 100 μg DNA. The apparent 5α-dihydrotestosterone association constant, 3-3.5 × 10⁸ M⁻¹, and steroid binding specificity of both receptor fractions were identical. 5α-Dihydrotestosterone, 19-nortestosterone, 17β-testosterone, and 5α-androstane-3λ,17β-diol were effective inhibitors of the nuclear binding of radiolabeled 5α-dihydrotestosterone, whereas 5α-androstane-3α,17α-diol, δ⁴-androstene-3,17-dione, progesterone, estradiol, and cortisol were not. The sedimentation constant of the KCl-extractable nuclear receptor on linear sucrose gradients was 4-5S. Cytoplasmic saturation analysis, employing tissue obtained from dogs 72 hr post-orchiectomy, demonstrated that 5α-dihydrotestosterone and the synthetic androgen R 1881 (17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one) were bound to an identical number of receptor sites, 34.3 ± 5.9 femtomoles per mg cytosol protein, with apparent association constants of 0.73 ±0.06 × 10⁹ M⁻¹ and 1.9 ± 0.2 × 10⁹ M⁻¹ respectively. Receptor binding of radiolabeled R 1881, measured at 2°, was readily inhibited by 5α-dihydrotestosterone and 19-nortestosterone; was modestly inhibited by 5α-androstane-3α,17β-diol, estradiol, and progesterone, and was not affected by δ⁴-androstene-3,17-dione, 5α-androstane-3α,17α-diol or cortisol. Estradiol and progesterone inhibition of R 1881 binding was markedly diminished at 15°, whereas androgen inhibition of R 1881 binding was temperature invariant. The sedimentation constant of the cytoplasmic R 1881- or 5α-dihydrotestosterone-receptor complex was 9-10S on linear sucrose gradients. The canine prostate nuclear and cytoplasmic androgen receptors had properties similar to those of the respective rat ventral prostate androgen receptors.