Isopycnic banding in metrizamide of the uterine cytosol and nuclear estradiol receptor.
We have characterized the cytosol and the nuclear estrogen receptors (RE) of immature lamb uterus and their complexes formed with DNA and chromatin by using metrizamide isopycnic gradients. In low salt, the cytosol RE had a density of 1.238 +/- 0.002 g/cm3. This density was increased by Ca2+-activated proteolysis (1.275 g/cm3) and heat transformation of the receptor (1.257 g/cm3) and lowered by trypsin treatment (1.20 g/cm3), DNA binding (1.20 g/cm3) or molybdate treatment. In high salt (0.5 M KCl) both the native and the heat-"transformed" cytosol RE banded at the same density of 1.26 g/cm3. The 8S RE had the same density when bound to E2 or to 4-hydroxy-tamoxifen. Endometrial nuclei purified after nuclear translocation of RE were digested by micrococcal nuclease to solubilize the nuclear RE under low salt conditions. The majority of the extracted nuclear RE had a density similar to that of the 8S cytosol RE (1.239 +/- 0.002) and thus was different from the proteolyzed and heat "transformed" forms. Conversely, after a slight digestion of the nuclei, RE banded with chromatin at 1.208 +/- 0.001 g/cm3. In low salt, both forms of the nuclear RE, but not the trypsin proteolyzed nuclear RE, were displaced by naked DNA added in vitro. We conclude that the cytosol RE and the free nuclear RE have, in low salt, the same density and DNA-binding ability and that metrizamide isopycnic analysis is a good method for quantifying the interactions of the receptor with DNA and chromatin.
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis; D002500:Centrifugation, Isopycnic; D002843:Chromatin / Q000378:metabolism; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism; D005260:Female; D008793:Metrizamide; D008836:Micrococcal Nuclease / Q000378:metabolism; D011959:Receptors, Estradiol; D011960:Receptors, Estrogen / Q000302:isolation & purification*; D012756:Sheep; D014599:Uterus / Q000032:analysis*
D002843:Chromatin; D011959:Receptors, Estradiol; D011960:Receptors, Estrogen; D004247:DNA; D008836:Micrococcal Nuclease; D008793:Metrizamide