Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea (+/- 0.4 M KCl) buffers and control buffers (+/- 0.4 M KCl) by chromatography through small columns of Sephadex G-25 or by dialysis at 0-6 degrees C. Equilibrium dissociation constants (Kd) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17 beta-[3H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol (Kd = 0.36 +/- 0.09 nM, n = 14) which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity (Kd = 3.45 +/- 0.86 nM, n = 6) to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. However, KCl did help prevent the reduction in binding capacity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification.