Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate

Abstract

Saturation protocols were developed for measurement of unoccupied (R) and steroid-occupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2°C) for the synthetic androgen R1881 (17β-hydroxy-17α-methyl-estra-4,9,11-trien-3-one) and the synthetic progestin R5020 (17α,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) respectively was 10.7 ± 1.4 and 14.3 ± 3.2 fmoles per mg cytosol protein and the apparent steroid affinity respectively was 9.6 ± 0.8 and 1.6 ± 0.4 × 10 8 M −1. Steroid specificity of the unoccupied cytoplasmic R1881 and R5020 binding sites was similar. When R1881 and R5020 were employed as probes of total, R plus RA, cytoplasmic binding components (20–24 hr incubation at 15°C) saturable binding of R5020 was not detectable. Total cytoplasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 ± 3.3 fmoles per mg cytosol protein and 2.7 ± 0.6 × 10 7 M −1. R5020 was a poor inhibitor of R1881 binding to total cytoplasmic R1881 binding components. Incubation (4 hr at 37°C) of human prostate mince with 5α-DHT (17β-hydroxy-5α-androstan-3-one) demonstrated a single class of KCI extractable nuclear binding component(s) which sedimented at 4–5S on linear sucrose gradients containing 0.6 M KCI. Binding site concentration and apparent affinity for 5α-DHT were 15 fmoles per 100 μg DNA and 1.0 × 10 8 M −1. The crude nuclear pellet obtained from human prostate homogenates contained binding components which were detectable by incubation of the clarified nuclear KCI extract with R1881 at 15°C for 20–24 hr. Binding site concentration and apparent affinity for R1881 were 29 ± 8 fmoles per 100 μg DNA and 3.1 ± 0.4 × 10 8 M −1. Steroid specificity of the nuclear 5α-DHT and R1881 binding sites was comparable. Unoccupied KCI-extractable R1881 binding sites were not detected subsequent to 2 hr incubation at 2°C. The data were consistent with identification of the nuclear binding components and the R1881 cytoplasmic binding component measured subsequent to incubation at 15°C as human prostate androgen receptors.