Characterization of a [ 3H]methyltrienolone (R1881) binding protein in rat liver cytosol

Abstract

The binding of radiolabelled methyltrienolone 17β-hydroxy-17α-methyl-estra-4, 9, 11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [ 3H] R 1881 macromolecular complex which sediments in the 8–9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [ 3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [ 3H] R1881 was recovered by thin layer chromatography. Characteristics of this [ 3H] R1881-8S binding protein include high affinity ( K d = 2.3 ± 41 nM) and low binding capacity (18.8 ± 3.3 fmol/mg cytosol protein), precipitability in 0–33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [ 3h] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a K d = 2.8 nM for [ 3H] R1881, and is androgen specific (testosterone > 5α-dihydrotestosterone > estradiol > progesterone > cyproterone acetate > di-ethylstilbestrol > dexamethasone > triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.