PROPERTIES OF THE MINERALOCORTICOID RECEPTOR IMMUNOPURIFIED FROM BOVINE KIDNEY

The mineralocorticoid receptor (MCR) from bovine kidney was purified o n an affinity column containing covalently linked polyclonal IgG raise d in the rabbit against rat kidney protein purified in the presence of RU 26752 that is specific to the MCR. The immune-affinity eluate was excluded as a single peak during gel permeation chromatography and cou ld be resolved as a single band of approximately 98 kDa by western blo t and gel electrophoresis. Immunohistochemistry revealed MCR-specific staining in bath the cortical and glomerular regions of bovine kidney. Interestingly, the purified MCR could not be activated in the presenc e of the specific ligand RU 26752 whereas binding to DNA-cellulose inc reased by 100% when crude cytosol was left at room temperature for 45 min. The binding of calcium to the MCR resulted in an increase in the fluorescence signal that could be partially reversed by EDTA. By a cal cium-specific fluorescence dye technique, 1.13 nM of ionized Ca2+ was bound per 0.01 nM MCR. The binding of ATP(32) to the immunopurified re ceptor was observed following chromatography on P-10 columns. The fluo rescence signal of etheno-ATP was maximally attenuated by the receptor at 1/1 stoichiometry of the ATP-MCR complex. Asparagine-linked comple x chain N-glycosylation of the purified MCR was also observed. Analysi s by far-UV circular dichroism spectra showed that MCR contains 33% al pha helices and 30% beta sheets, compatible with a relatively flat con formation of the native protein. These data provide experimental proof for the predicted computer simulation regarding the structural featur es of the steroid receptor superfamily and suggest crosstalk between s everal protein families.