Reversible, positive cooperative interaction of 11β-chloromethyl-[ 3H]estradiol-17β with the calf uterine estrogen receptor

Abstract

The binding of 11β-chloromethyl-[ 3H]estradiol-17β [ 3H]CME 2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4–1.5. This positive cooperative interaction of [ 3H]CME 2 was indistinguishable from the typical cooperative interaction of [ 3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME 2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics [ 3H]CME 2 from the receptor was biphasic, composed of a fast dissociating component (15%, t 1 2 = 4 min at 0°C; 9%, t 1 2 = 4 at 28°C) and a slow dissociating component (85%, t 1 2 > 50 h at 0°C; 91 %, t 1 2 > 50 h at 28°C). The dissociation kinetics of [ 3H]estradiol was also biphasic: the t 1 2 of the fast dissociating component was 4 min at 0 and 28°C and ∼ 200 min for the slow dissociating component at both temperatures. The fraction of the slow [ 3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [ 3H]CME 2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME; are discussed.