Study on interactions of endocrine disruptors with estrogen receptor-beta using fluorescence polarization.
In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities.
Suzuki
Sachiko
S
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Ohno
Ken-ichi
K
Santa
Tomofumi
T
Imai
Kazuhiro
K
D001667:Binding, Competitive / Q000187:drug effects; D004785:Environmental Pollutants / Q000494:pharmacology; D047629:Estrogen Receptor beta; D016231:Fluorescence Polarization Immunoassay; D005456:Fluorescent Dyes; D006728:Hormones / Q000494:pharmacology; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D004364:Pharmaceutical Preparations; D048789:Phytoestrogens; D028321:Plant Preparations / Q000494:pharmacology; D011960:Receptors, Estrogen / Q000187:drug effects*; D011994:Recombinant Proteins; D013816:Thermodynamics
D004785:Environmental Pollutants; D047629:Estrogen Receptor beta; D005456:Fluorescent Dyes; D006728:Hormones; D007529:Isoflavones; D004364:Pharmaceutical Preparations; D048789:Phytoestrogens; D028321:Plant Preparations; D011960:Receptors, Estrogen; D011994:Recombinant Proteins