Properties of the mineralocorticoid receptor immunopurified from bovine kidney.
The mineralocorticoid receptor (MCR) from bovine kidney was purified on an affinity column containing covalently linked polyclonal IgG raised in the rabbit against rat kidney protein purified in the presence of RU 26752 that is specific to the MCR. The immuno-affinity eluate was excluded as a single peak during gel permeation chromatography and could be resolved as a single band of approximately 98 kDa by western blot and gel electrophoresis. Immunohistochemistry revealed MCR-specific staining in both the cortical and glomerular regions of bovine kidney. Interestingly, the purified MCR could not be activated in the presence of the specific ligand RU 26752 whereas binding to DNA-cellulose increased 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 calcium-specific fluorescence dye technique, 1.13 nM of ionized Ca2+ was bound per 0.01 nM MCR. The binding of ATP32 to the immunopurified receptor was observed following chromatography on P-10 columns. The fluorescence signal of etheno-ATP was maximally attenuated by the receptor at 1/1 stoichiometry of the ATP-MCR complex. Asparagine-linked complex chain N-glycosylation of the purified MCR was also observed. Analysis by far-UV circular dichroism spectra showed that MCR contains 33% alpha helices and 30% beta sheets, compatible with a relatively flat conformation of the native protein. These data provide experimental proof for the predicted computer simulation regarding the structural features of the steroid receptor superfamily and suggest crosstalk between several protein families.
Pagano
M
M
Department of Biochemistry, Centre Universitaire des Cordeliers, Paris, France.
Mirshahi
M
M
Mirshahi
F
F
Monnot
M
M
Lazar
G
G
Chassaing
C G
CG
Fermandjian
S
S
Agarwal
M K
MK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D003198:Computer Simulation; D006031:Glycosylation; D007668:Kidney / Q000378:metabolism*; D015394:Molecular Structure; D018161:Receptors, Mineralocorticoid / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D018161:Receptors, Mineralocorticoid; D000255:Adenosine Triphosphate; D002118:Calcium