HETEROGENEITY OF NUCLEAR ESTROGEN-BINDING SITES IN THE RAT UTERUS - A SIMPLE METHOD FOR THE QUANTITATION OF TYPE-I AND TYPE-II SITES BY [ESTRADIOL-H-3 EXCHANGE

Estrogen administration to mature-ovariectomized rats causes the activation or stimulation of secondary nuclear estrogen-binding sites (type II) in the uterus which can interfere with estrogen receptor (type I) measurement. Quantitation of type I sites in the presence of the type II site is very difficult and can only be achieved by graphic analysis of saturation curves which employ a wide range (0.4-40 nM) of [3H]estradiol concentrations in nuclear exchange assay. The studies presented describe simple methods which can be used to separately quantitate both nuclear estrogen-binding sites using a single concentration of [3H]estradiol. Since the nuclear type II site does not bind [3H]estradiol in the presence of reducing agent, type I sites can be easily quantitated by incubating nuclei (37.degree. C for 30 min) in Tris-EDTA buffer containing 0.1-1.00 mM dithiothreitol using a single saturating concentration of [3H]estradiol. Conversely, a single concentration of [3H]estradiol (40-80 nM) can be used to quantitate the nuclear type II site by incubating nuclei in Tris-EDTA buffer under conditions (4.degree. C for 60 min) which do not measure occupied nuclear estrogen receptor. By using the appropriate buffer system, type I and type II sites can be easily separated in mixed binding systems. In addition, nafoxidine does not bind to the nuclear type II site. Therefore it can be used as a competitive inhibitor of [3H]estradiol binding to type I sites and permit the measurement of type II sites without interference from type I sites. These techniques should be applicable to autoradiographic or fluorescence studies which cannot discriminate between steroid binding to these 2 classes of nuclear estrogen-binding sites.