The estrogen receptor (ER) of hen oviduct cytosol was purified by ammonium sulfate precipitation and heparin- agarose chromatography for use in cell-free nuclear binding assays. Under conditions of constant protein concentration, the ER displayed a high affinity, saturable binding to purified hen oviduct nuclei in a cell-free assay. The nuclear binding was shown not to be due to exchange with endogenous nuclear estrogen receptors. Low ionic strength and excess protein, which lowers the effective ionic strength, were found to result in nonsaturable binding, obscuring the saturable high affinity nuclear binding sites for ER. Proteolytic activity also caused an increase in binding due to destruction of proteins that mask many binding sites. The nuclear binding displayed tissue specificity, with maximal binding to oviduct nuclei, intermediate binding to liver and kidney nuclei, and little or no binding to spleen, lung, and erythrocyte nuclei. Scatchard analysis of the cell-free binding revealed a single class of high affinity (Kd = 1.8 × 10-10 M) and low capacity (3000-5000 sites/nucleus) nuclear binding sites. This capacity was shown to be equivalent to the saturable binding measured in vivo. The saturable binding of [3H]ER in vivo to this class of sites correlated with the stimulation of RNA polymerase I and II activities. The specificity of nuclear acceptor sites for estrogen in the avian oviduct was then investigated using competitive binding with the progesterone receptors. The in vivo nuclear binding of [3H]estradiol was reduced by coinjection of unlabeled estradiol but was not affected by coinjection of unlabeled progesterone. Reciprocally, the nuclear binding of [3H]progesterone was reduced by coinjection of unlabeled progesterone, but was not affected by coinjection of unlabeled estrogen. The cell-free nuclear binding assay supported these findings. An excess of nonradioactively labeled ER effectively competed with [3H]ER binding to nuclei, but had no effect on [3H]progesterone receptor nuclear binding. These data support the existence of distinct nuclear acceptor sites for these two steroid receptors in the avian oviduct and, therefore, suggest that the antagonism of these two steroids does not occur at the level of nuclear binding.
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