Search

Immobilization of the estrogen receptor to the N-hydroxysuccinimide ester of succinylethylenediaminocarboxymethyl agarose (Reagent B) is described and compared with that to the charged N-hydroxysuccinimide ester derivative (Reagent A), previously described. The time course for immobilization was examined. Thirty-six percent of the input receptor was immobilized within 1 h. The optimum pH in immobilization is 7.0–7.4. The dissociation rate of [ 3 H]estradiol(3,17β−1,3,5(10)-estratriene) from the [ 3 H]estradiol-receptor complex immobilized to Reagent B was similar to that in Reagent A. The receptor immobilized to Reagent B was saturated with estradiol at 5 h. The [ 3 H]estradiol concentration necessary for saturation was 10 nM. The dissociation constant (K D ) for the receptor immobilized to Reagent B was 0.95 × 10 −9 M.

Covalent immobilization of the soluble estrogen receptor from a rabbit uterus to N-hydroxysuccinimide ester derivative of agarose is shown. At first, the condition for the immobilization reaction was examined. The non-immobilized receptor was extracted with 0.4 M NaCl-containing medium. Sixty seven to 80% of the input receptor were immobilized within 30 min at 0°C in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4). The immobilized [ 3 H]estradiol (3,17β-dihydroxy-1,3,5(10)-estratriene) -receptor complex was stable for at least 24 h. The optimum pH for immobilization was 7.4. Ca 2+ or Na + ions in the reaction media decreased the yields in immobilization of the receptor to the reagent with an electrostatically positive spacer arm. Next, influences of immobilization on the receptor were examined. The dissociation rate of [ 3 H]estradiol from the immobilized receptor was a little slower than that from the native receptor. The estrogen-free immobilized receptor was saturated by incubating with 10 nM [ 3 H]estradiol for 10 h at 0°C in 0.1 M HEPES (pH 7.4). From Scatchard plot analysis, it was found that the hormone binding affinity in the immobilized receptor decreased to approximately one-fourth of that in the native receptor.

The extraction of [3H]estradiol- and [3H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [3H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [3H]tamoxifen-receptor complex extracted with 0.4 M KC1. The nuclear [3H]estradiol-receptor complex extracted with 0.4 M KC1 sediments at 4 S. The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.