ESTROGEN BINDING-SITES IN THE NUCLEUS OF NORMAL AND MALIGNANT HUMAN-TISSUE - OPTIMIZATION OF AN EXCHANGE ASSAY FOR THE MEASUREMENT OF SPECIFIC BINDING

Abstract

An exchange assay was validated and used to measure the total concentration of estrogen receptors [necessary for a response to endocrine manipulation] in the nuclei of cells from human breast tumors and myometrium. Tissue homogenates were centrifuged through 1.2 M sucrose pads to separate crude chromatin from soluble and membranous fractions. The yield of DNA by this procedure was .apprx. 80%. Total binding sites were measured by incubating the sedimented pellets with [3H-] estradiol at 30.degree. for 60 min and then precipitating the receptor-steroid complexes with protamine sulfate. Saturation analysis by this procedure provides evidence for the presence of a specific, nuclear estrogen-binding site in adition to the established estrogen receptor in both these tissues.