Purification of a calcium-binding phosphoprotein from pig brain.

An acidic phosphoprotein has been isolated from adult pig brain and purified to apparent homogeneity. The purification procedure employed ammonium sulfate precipitation, ECTEOLA cellulose chromatography, gel filtration on Sephadex G-75, and chromatography on hydroxylapatite. This protein exhibits high affinity calcium binding (Kd = 2.5 X low5 M), binds 1 mole of calcium per mole of protein, and is one of two high affinity calcium-binding proteins found in pig brain. This calcium-binding protein is not present in either liver or kidney, suggesting that it may play a role in calcium function within nervous tissue. The calcium binding properties, phosphorus content and amino acid composition of the protein have been determined. The molecular weight of the protein, as determined by ultracentrifugation, is 11,500. The protein aggregates in the presence of calcium with the formation of multimers. Treatment with phosphatase results in loss of calcium binding activity, suggesting that calcium binds to the phosphate group of the protein. Each mole of protein contains 10 moles of phosphate.