Characterization of Estrogen Receptor, Estrogen Receptor Binding Factors and a Protease in the Nuclei of Gilt Uterus

Publisher Website
Google Scholar
Abstract
An analysis of the constituents of the estrogen receptor (ER) system of the nuclei of the gilt uterus is described. The nuclei obtained from gilt uteri incubated in vitro with [3H]estradiol were extracted with 0.4 m KC1. The Stokes radius (44 A) and the sedimentation coefficient (4.5S) of the extracted ER measured in the presence of 0.4 m NaSCN were identical with those of vero-ER which had been previously shown to be the only native ER unit molecule of the cytoplasmic ER system. It was assumed that vero-ER in the cytosol translocates into the nucleus in the intact form. When the nuclei were extracted with NaSCN, the extracted ER had a sedimentation coefficient of 2.9S and a Stokes radius of 24 A (“2.9S” ER). A protease which hydrolyzed vero-ER into “2.9S” ER was present in the nuclear extract with NaSCN. In contrast to the cytoplasmic protease previously described, the nuclear protease was not inhibited by antipain or leupeptin. Analysis of the endogenous nuclear ER showed that the hydrolysis of vero-ER into “2.9S” ER by the nuclear protease also took place in vivo. To analyze the factors which bind specifically with vero-ER, the nuclei obtained from fresh uteri were extracted with 0.4 m NaSCN and then subjected to gel filtration in the presence of the chaotropic salt. Fractions near the void volume contained a factor (aggregating factor, AF) which bound with vero-ER to form ER migrating to the bottom in sucrose gradient cen-trifugation under hypotonic conditions. AF was degraded by DNase treatment, but was not affected by RNase or trypsin. Component A (Stokes radius, 37 A) and component B (Stokes radius, 18.5 A), which had been previously shown to be constituents of the cytoplasmic ER system as factors which bind specifically with vero-ER, were shown to be present also in the nuclei.