Isolation and properties of a sarcoplasmic calcium-binding protein from crayfish

Abstract

The sarcoplasmic Ca-binding protein from crayfish [Astacus leptodactylus] muscle was purified to homogeneity. The protein has a MW of 44,000, as determined by sedimentation equilibrium and Sephadex chromatography. It dissociates in the presence of sodium dodecyl sulfate, 8 M urea, or, after succinylation, into 2 subunits of 22,000 MW. The protein is free of carbohydrate and P but contains 4 g-atoms of Ca/44,000 at a free Ca concentration of 0.1 .mu.M. Of the polypeptide backbone N 45% appears .alpha.-helical. The amino acid composition reveals a high proportion of alanine and acidic amino acids, a normal content of aromatic amino acids and the absence of histidine. The isoelectric point as determined by isoelectric focusing, is 5.1. The protein contains a free threonyl NH2 terminal. Two thiols react rapidly in the native protein. 6 in the Ca-free form. Immunochemically, there is no difference between the protein from tail, claw and heart muscle. In these 3 crayfish tissues, the concentrations of Ca binding protein, as determined by rocket immunoelectrophoresis, are markedly different: 2.73 g/kg in tail, 0.72 in claw, and 0.073 in heart muscle. A functional analogy with the parvalbumins of vertebrates can be postulated.