Antiestrogen binding sites in rat liver nuclei

Abstract

Isolated, intact rat liver nuclei have high-affiity ( K d=10 −9 M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [ 3H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [ 3H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [ 3H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity ( K d=10 −9 M) [ 3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000× g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity ( K d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000× g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity ( K d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.