Limited proteolysis of cytoplasmic and nuclear uterine estradiol receptors yields identical estradiol-binding fragments.
Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus has been demonstrated to yield in a high-salt buffer a stable estradiol-binding molecule with the following characteristics: sedimentation coefficient 4.0 +/- 0.1 S; Stokes radius 3.5 +/- 0.05 nm; molecular weight 60000 (for an assumed v value of 0.73 ml g-1) and frictional ratio 1.36. Nuclear KCl extracts, prepared from uteri preincubated at 37 degrees C with labeled estradiol, were analysed by Sephadex G-200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol-receptor complex: sedimentation coefficient 4.4 +/- 0.1 S; Stokes radius 4.12 +/- 0.02 nm; molecular weight 77000 and frictional ratio 1.47 (v = 0.73 ml g-1). Limited tryptic proteolysis of this extract gave an estradiol-binding fragment with molecular characteristics identical to the trypsin-modified cytoplasmic receptor. In addition, mild tryptic digestion of whole labeled nuclei allowed us to solubilize almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule thus obtained showed molecular parameters very similar to the 60000-dalton trypsin fragments obtained from high-salt cytoplasmic and nuclear extracts. These molecules were undistinguishable by gel electrophoresis analysis at six different acrylamide concentrations. These results in conjunction with those derived from dissociation kinetics experiments and ligand specificity studies indicate the cytosolic protein is a functional part of the nuclear receptor. Based upon these and other studies we suggest that proteolytic cleavage of the estradiol-receptor complex, which results in the removal of the estradiol-binding sites from the nuclear recognition sites of the molecule, could play a role in the inactivation of the estradiol receptor in vivo.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface; D014357:Trypsin; D014599:Uterus / Q000378:metabolism*
D009124:Muscle Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D004958:Estradiol; D014357:Trypsin