Identification of a specific binding site for the anabolic steroid stanozolol in male rat liver microsomes

Abstract

Male rat liver microsomes contain a [H-3]dexamethasone binding site, capable of binding glucocorticoids and progesterone. We have shown previously that the 17 alpha-alkylated androgen, stanozolol, can inhibit the [H-3]dexamethasone binding to microsomes through a negative allosteric mechanism, which gives rise to the possibility of its interaction with a different binding site. In this study, the existence of a single-saturating binding site, capable of binding the radioactive steroid with a maximum number of the specific binding site of 49 +/- 2 pmol/mg of protein and a K-d of 37 +/- 1.3 nM was demonstrated by using [H-3]stanozolol. In competition experiments, only stanozolol and danazol were able to compete with [H-3]stanozolol for its binding to microsomes, among more than 60 steroids and other compounds tested. The binding of [H-3]stanozolol was depressed after protease treatment of the microsomes, or after the administration of cycloheximide to adult male rats for 24 hr, which suggest its proteic nature. The [H-3]stanozolol binding site was detected in many tissues of the rat, with the highest concentrations being found in the liver. It was detected from birth,increasing afterward in concentration and reaching a peak at 2 to 3 months of age. This is the first experimental verification of the existence in liver microsomes of a specific binding site for some 17 alpha-alkylated androgens, such as stanozolol and danazol, different from the androgen receptor or the [H-3]dexamethasone binding site.