TY - JOUR
T1 - Studies on the Uterine, Cytoplasmic Estrogen Binding Protein. Thermal Stability and Ligand Dissociation Rate. An Assay of Empty and Filled Sites by Exchange
AU - Katzenellenbogen, John A.
AU - Johnson, Howard J.
AU - Carlson, Kathryn E.
PY - 1973/10/1
Y1 - 1973/10/1
N2 - The thermal stability and the rate of estradiol dissociation from the cytoplasmic estrogen binding protein of rat uterus have been determined at 0, 25, 30, and 37°. Unfilled sites are very labile, but estrogen-filled sites do not undergo detectable degradation over a 24-hr period at temperatures up to 30°. At 37°, however, 50% of the sites are lost over an 8-hr period, while the remainder are stable for up to 24 hr. Dissociation of bound estradiol, as determined by exchange, is complete within 24 hr at 25° and within 8 hr at 30°. Comparison of freshly prepared and exchanged cytosol (24 hr, 25°) on high and low salt-sucrose density gradients shows that considerable aggregation of the binding activity occurs during the exchange period. A procedure for assaying soluble, cytoplasmic estrogen binding sites by exchange has been designed, using the optimum conditions for rapid exchange without degradation of binding capacity. The assay is convenient, quantitative, and linear up to 3 uterine equivalents/ml. Using this assay procedure, the concentration of estrogen binding sites can be determined, regardless of whether they are empty at the time of assay or filled with estradiol, a nonsteroidal estrogen (hexestrol), or an antiestrogen (dimethylstilbestrol, U 11,100A, or CI-628). By following the time course of exchange, the dissociation rate of an unlabeled ligand can be determined.
AB - The thermal stability and the rate of estradiol dissociation from the cytoplasmic estrogen binding protein of rat uterus have been determined at 0, 25, 30, and 37°. Unfilled sites are very labile, but estrogen-filled sites do not undergo detectable degradation over a 24-hr period at temperatures up to 30°. At 37°, however, 50% of the sites are lost over an 8-hr period, while the remainder are stable for up to 24 hr. Dissociation of bound estradiol, as determined by exchange, is complete within 24 hr at 25° and within 8 hr at 30°. Comparison of freshly prepared and exchanged cytosol (24 hr, 25°) on high and low salt-sucrose density gradients shows that considerable aggregation of the binding activity occurs during the exchange period. A procedure for assaying soluble, cytoplasmic estrogen binding sites by exchange has been designed, using the optimum conditions for rapid exchange without degradation of binding capacity. The assay is convenient, quantitative, and linear up to 3 uterine equivalents/ml. Using this assay procedure, the concentration of estrogen binding sites can be determined, regardless of whether they are empty at the time of assay or filled with estradiol, a nonsteroidal estrogen (hexestrol), or an antiestrogen (dimethylstilbestrol, U 11,100A, or CI-628). By following the time course of exchange, the dissociation rate of an unlabeled ligand can be determined.
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U2 - 10.1021/bi00745a011
DO - 10.1021/bi00745a011
M3 - Article
C2 - 4745661
AN - SCOPUS:0015867364
SN - 0006-2960
VL - 12
SP - 4092
EP - 4099
JO - Biochemistry
JF - Biochemistry
IS - 21
ER -