Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol

• Published in: Biochemistry (Easton) 1989-09, Vol.28 (19), p.7788-7796
• Main Authors: Densmore, Charles L, Markaverich, Barry M, O'Malley, Bert W, Clark, James H
• Format: Article
• Language: English
• Subjects: aislamiento tecnica; Ammonium Sulfate; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; characterization; Chemical Precipitation; Chickens; chicks; Chromatography, Gel; Chromatography, Ion Exchange; Cytosol - analysis; DNA - metabolism; estradiol; Estradiol - pharmacokinetics; estrogenos; estrogens; Fatty acids; Fundamental and applied biological sciences. Psychology; isolation; isolement; Lipids; oestrogene; oestrogens; Other biological molecules; oviduct; oviducte; oviductos; oviducts; Oviducts - analysis; pollito; poussin; purification; receptors; Receptors, Estrogen - analysis; Receptors, Estrogen - isolation & purification; types
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00445a039

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.