Characterization and partial purification of an estrogen type II binding site in chick oviduct cytosol.
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.
Densmore
C L
CL
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Markaverich
B M
BM
O'Malley
B W
BW
Clark
J H
JH
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*
D011960:Receptors, Estrogen; D004958:Estradiol; D004247:DNA; D000645:Ammonium Sulfate