Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca2+-dependent elution from phenyl-sepharose.
Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two "intrinsic glycoproteins," while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.
D000818:Animals; D002118:Calcium; D002155:Calsequestrin / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange / Q000379:methods; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000032:analysis*; D009206:Myocardium / Q000032:analysis*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000032:analysis*; D012685:Sepharose / Q000031:analogs & derivatives
D002155:Calsequestrin; D009124:Muscle Proteins; C020927:phenyl-sepharose; D012685:Sepharose; D002118:Calcium