Limited Proteolysis of Cytoplasmic and Nuclear Uterine Estradiol Receptors Yields Identical Estradiol‐Binding Fragments

Abstract

Limited tryptic hydrolysis of the estradiol cytoplasmic receptor from calf uterus yielded 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; MW 60,000 (for an assumed .hivin.v value of 0.73 ml g-1) and frictional ratio 1.36. Nuclear KCl extracts, prepared from uteri preincubated at 37.degree. C with labeled estradiol, were analyzed by Sephadex G-200 chromatography and sucrose density gradient centrifugation. The following molecular parameters were found for the estradiol .cntdot. receptor complex: sedimentation coefficient 4.4 .+-. 0.1 S; Stokes radius 4.12 .+-. 0.02 nm; MW 77,000 and frictional ratio 1.47 (.hivin.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. Mild tryptic digestion of whole labeled nuclei permitted solubilizing almost quantitatively the nuclear [3H]estradiol in a macromolecular bound form. The molecule obtained showed molecular parameters very similar to the 60,000-dalton trypsin fragments obtained from high-salt cytoplasmic and nuclear extracts. These molecules were indistinguishable by gel electrophoresis analysis at 6 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. Proteolytic cleavage of the estradiol .cntdot. 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.