A controlled trypsinization procedure is reported which results in more efficient solubilization of the nuclear estrogen receptor from human endometrium and rat uterine tissue than does extraction with high salt (0.4 M KCl) alone. The trypsin-liberated nuclear receptors in both species sediment at 3-4S on both high and low salt gradients, whereas the salt-extracted receptors sediment more rapidly (5.6S, rat and 4.0S, human) on high salt gradients and aggregate under low ionic strength conditions. Rates of dissociation of trypsin and KCl solubilized nuclear estrogen receptor complexes of human endometrium and rat uterus are similar, with half-times of dissociation of 55-60 h at 0-4°C. KD, determinations of estrogen binding to trypsin solubilized nuclear receptors indicate that these nuclear receptors in both species have a very high affinity for estradiol. KD = 2.6 × 10-10M (rat receptor) and 9.0 × 10-10 M (human receptor). Isoelectric focusing in polyacrylamide gels gives an apparent isoelectric point of 6.3 for the human nuclear receptor, 6.6 for the rat nuclear receptor, and 6.4 for the trypsin-treated cytoplasmic estrogen receptors of human and rat uterus. Molecular weights and anhydrous molecular radii (R) estimated by electrophoretic mobility on different percent acrylamide gels (Ferguson plots) show the nuclear and cytoplasmic trypsin-treated receptor forms in both rat and human to be very similar, with calculated Rs of 2.35-2.43 nm (corresponding to molecular weights between 45,000-50,000). These studies document great similarity in physicochemical properties and binding characteristics of the nuclear forms of the estrogen receptor from human and rat uterine tissue, and considerable similarity between nuclear and cytosol receptor forms. In addition, the efficient solubilization of nuclear estrogen receptor by the mild trypsinization method described here should provide a technique useful in analyzing the molecular, properties of estrogen receptors from normal and neoplastic estrogen target tissues.
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