title: Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol.
abstract: An estrogen binding site of moderate affinity (Kd approximately 10 nM) and high capacity (approximately 25-70 pmol/g of tissue) was measured in DES-stimulated chick oviduct cytosol. Saturation analysis by [3H]estradiol exchange demonstrated that this binding site displayed sigmoidal binding characteristics suggesting a cooperative binding mechanism. Competition analysis with a number of compounds demonstrated that the bioflavonoid luteolin was a better competitor for binding to type II sites in chick than either estradiol or DES. Steroid specificity was demonstrated by the inability of 17 alpha-estradiol, progesterone, testosterone, corticosterone, and the triphenylethylene antiestrogen nafoxidine (U-1100A) to compete for [3H]-17 beta-estradiol binding to chick oviduct cytosol preparations. In addition, the binding site appeared to be sensitive to sulfhydryl reducing reagents as evidenced by a 75% reduction in binding activity in the presence of dithiothreitol. Both prelabeling and postlabeling procedures used in conjunction with Sephacryl S-300 chromatography resulted in a single major peak of type II binding activity representing a molecular weight in the 40,000 range. Type II binding activity was recoverable after precipitation with ammonium sulfate, and this material was subjected to a variety of column chromatography procedures in order to achieve further purification of the type II site. Significant purification of the site was achieved with a bioflavonoid-Sepharose (quercetin-Sepharose) affinity matrix. The purified type II sites eluted from quercetin-Sepharose displayed the same sigmoidal binding curves characteristic of native cytosol.
authors.affiliation: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: C L
authors.initials: CL
authors.lastname: Densmore
authors.forename: B M
authors.initials: BM
authors.lastname: Markaverich
authors.forename: B W
authors.initials: BW
authors.lastname: O'Malley
authors.forename: J H
authors.initials: JH
authors.lastname: Clark
mesh_terms: D000645:Ammonium Sulfate; D000818:Animals; D011232:Chemical Precipitation; D002645:Chickens; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism; D004958:Estradiol / Q000493:pharmacokinetics; D010057:Oviducts / Q000032:analysis*; D011960:Receptors, Estrogen / Q000032:analysis / Q000302:isolation & purification*
chemical_list: D011960:Receptors, Estrogen; D004958:Estradiol; D004247:DNA; D000645:Ammonium Sulfate