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

The binding of 11β-chloromethyl-[³H]estradiol-17β [³H]CME₂) 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]CME₂ was indistinguishable from the typical cooperative interaction of [³H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME₂ for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics [³H]CME₂ 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]CME₂ is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME; are discussed.