Purification and characterization of the activated mineralocorticoid receptor from rat myocardium.
Cardiac cytosol from adrenalectomized rats was radiolabelled with 10 nM tritiated RU 26752, R 5020 or aldosterone, to saturate the mineralocorticoid receptor (MCR) in the presence of 1 microM RU 38486 to block the glucocorticoid and progestin receptors. Free steroids were removed by charcoal treatment and the radiolabelled cytosol was passed through a phosphocellulose column. The MCR peak in the phosphocellulose eluate was activated at 25 degrees C for 45 min, adsorbed onto the DNA-cellulose and finally extracted once each with buffers containing 1 M potassium chloride or 25 mM magnesium chloride. The pooled DNA-cellulose extracts, reequilibrated with 10 nM [3H]RU 26752, were resolved as a single, homogeneous band of 78 kDa upon polyacrylamide gel electrophoresis. Ion-exchange analysis of the purified MCR on DEAE-cellulose-52 revealed a single peak in the 0.017 M sodium phosphate region with both RU 26752 and R 5020, but aldosterone dissociated during this procedure. Molecular filtration on Ultrogel AcA-44 columns revealed a major 145 kDa peak, with some smaller components of 40 and 80 kDa. These hydrodynamic properties of the purified MCR are at variance with those of the native receptor in crude myocardial cytosol, and suggest that some post-translational modifications in vivo may be required for the expression of MCR-mediated responses.
Lazar
G
G
Institute of Pathophysiology, Szeged Medical School, Hungary.
Pagano
M
M
Agarwal
M K
MK
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002845:Chromatography; D003600:Cytosol / Q000032:analysis; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D015636:Magnesium Chloride; D008297:Male; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis*; D011189:Potassium Chloride; D011397:Promegestone / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000302:isolation & purification* / Q000378:metabolism; D013148:Spironolactone / Q000031:analogs & derivatives / Q000378:metabolism
C033190:DNA-cellulose; D046911:Macromolecular Substances; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D015636:Magnesium Chloride; D013148:Spironolactone; D000450:Aldosterone; D011189:Potassium Chloride; C047175:7-propyl spirolactone; D002482:Cellulose; D004247:DNA; D011397:Promegestone