Covalent immobilization of the estrogen receptor to an electrostatically neutral N-hydroxysuccinimide ester derivative of agarose.
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 [3H]estradiol(3,17 beta-1,3,5(10)-estratriene) from the [3H]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 [3H]estradiol concentration necessary for saturation was 10 nM. The dissociation constant (KD) for the receptor immobilized to Reagent B was 0.95 X 10(-9) M.
Ikeda
M
M
Department of Biochemistry, Kawasaki Medical School, Okayama, Japan.
Kusaka
T
T
D001667:Binding, Competitive; D004054:Diethylstilbestrol / Q000378:metabolism; D004560:Electricity; D004958:Estradiol / Q000378:metabolism; D006531:HEPES; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008744:Methylamines; D011960:Receptors, Estrogen; D012685:Sepharose / Q000031:analogs & derivatives
D008744:Methylamines; D011960:Receptors, Estrogen; D004958:Estradiol; C032392:Affi-Gel 10; D004054:Diethylstilbestrol; D012685:Sepharose; C027451:methylamine; D006531:HEPES