Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins.
Calmodulin binds quantitatively to phenyl-Sepharose through its Ca2+-induced hydrophobic binding region. Troponin C and S-100 protein, as well as several other proteins present in rat tissues, also bind to phenyl-Sepharose in a Ca2+-dependent manner. While the Ca2+-dependent binding of calmodulin to phenyl-Sepharose is not altered appreciably by monovalent cations, they do appear to compete for Ca2+ binding to most of the other proteins, including S-100 protein, which exhibit Ca2+-induced interaction with phenyl-Sepharose. The selective elution of these proteins from the phenyl-Sepharose column can be achieved with a 0.5 M concentration of monovalent cations such as K+, Na+, and NH4+ in the presence of a low (100 microM) Ca2+ concentration. Calmodulin-binding proteins associated with calmodulin in crude cell extracts can prevent the interaction of calmodulin with the phenyl-Sepharose, resulting in low recoveries of calmodulin from these tissues. The majority of these interfering proteins are heat labile so that heat treatment (boiling) of the cell extract for a limited time (5 min) negates any binding of these proteins to calmodulin and allows the quantitative recovery of calmodulin by hydrophobic interaction chromatography. This procedure allows the rapid and quantitative recovery of highly purified calmodulin from both cytosolic and Triton X-100-solubilized particulate fractions prepared from various rat tissues. Calmodulin isolated in this manner can be accurately and reliably quantitated by direct protein determination with Coomassie brilliant blue dye or fluorescamine or by the cyclic nucleotide phosphodiesterase stimulation assay.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000302:isolation & purification*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification; D002417:Cattle; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D011506:Proteins / Q000302:isolation & purification; D014336:Troponin / Q000302:isolation & purification; D019209:Troponin C
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D011506:Proteins; D014336:Troponin; D019209:Troponin C; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium