Calcium-dependent protein binding to phenothiazine columns.
Nonmuscle, smooth muscle, and striated muscle tissue extracts contain several proteins, in addition to calmodulin, which bind fluphenazine affinity columns in a calcium-dependent manner. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis shows four proteins in red blood cell lysates with Mr = 22,000, 12,000, 9,000, and 8,000. Rat brain tissue contains an 11,000-dalton peptide, while chicken gizzard and rabbit longissimus dorsi muscles have a similar set of peptides (67,000, 35,000, 33,000, and 11,000 daltons). These proteins all interact independently with fluphenazine and, except for the 22,000-dalton protein from red blood cells, do not bind to calmodulin-Sepharose affinity columns. Binding requires the presence of micromolar calcium, and dissociation occurs only by chelation of calcium with EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The muscle tissue proteins are present in ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid eluates from N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide- and phenothiazine-coupled Sepharose resins but are not bound to a Sepharose column without a coupled drug. These results suggest that the use of phenothiazines to indicate calmodulin action should be judiciously interpreted. These proteins may be functionally analogous to calmodulin in that they form a drug-binding site in a calcium-dependent manner.
D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002147:Calmodulin / Q000302:isolation & purification*; D002645:Chickens; D002846:Chromatography, Affinity; D005476:Fluphenazine; D009124:Muscle Proteins / Q000302:isolation & purification; D009130:Muscle, Smooth / Q000032:analysis; D009132:Muscles / Q000032:analysis; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats
D001798:Blood Proteins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009124:Muscle Proteins; D011506:Proteins; D005476:Fluphenazine; D002118:Calcium