Isolation and characterization of a new Mr 18,000 protein with calcium vector properties in amphioxus muscle and identification of its endogenous target protein.
A new Ca2+-binding protein, called CaVP, has been detected in muscle of the cephalochordate amphioxus and purified to electrophoretic homogeneity. The Mr 18,000 protein (pI = 4.9) binds 2 Ca2+ atoms in a noncooperative way with an intrinsic binding constant of 8.2 X 10(6) M-1. Ca2+, but not Mg2+, induces a 10% increase in alpha-helical content in the metal-free protein. CaVP does not interact with chlorpromazine, but forms a Ca2+-dependent complex with melittin. In situ, CaVP forms a high affinity Ca2+-dependent complex with an Mr 36,000 protein present in muscle extracts of amphioxus. This complex has been purified by gel filtration and ion exchange chromatography, and the target protein further purified after dissociation of the complex in the presence of Ca2+-chelating agents and 6 M urea. The nearly pure Mr 36,000 protein also forms a Ca2+-dependent complex with calmodulin which, however, is less stable during electrophoresis than the CaVP-Mr 36,000 protein complex. Amphioxus CaVP does not substitute for calmodulin in a specific enzyme assay nor for troponin C in restoring Ca2+ sensitivity to skinned muscle fibers. Its polyclonal antibody does not cross-react with the latter two activators. No immunological cross-reacting counterpart of CaVP was found in organs of fish and rat. Its relative abundance in amphioxus muscle indicates that CaVP must underlie an important new limb of Ca2+ regulation in this particular muscle.
D000818:Animals; D001923:Brain Chemistry; D002135:Calcium-Binding Proteins / Q000302:isolation & purification*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002746:Chlorpromazine / Q000378:metabolism; D003429:Cross Reactions; D005399:Fishes; D007526:Isoelectric Point; D008274:Magnesium / Q000378:metabolism; D008555:Melitten / Q000378:metabolism; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000032:analysis*; D051381:Rats; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet
C050084:CAVP protein, Amphioxus; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009124:Muscle Proteins; D008555:Melitten; D008274:Magnesium; D002746:Chlorpromazine