Ca2+‐Binding Studies of the Phosphoprotein from Rat‐Incisor Dentine

Abstract

Rat incisor dentine was demineralized and extracted with 0.25 M EDTA containing protease inhibitors. The extract was purified by chromatography on DEAE‐cellulose and sulfonated polystyrene. The Ca2+‐binding properties of the phosphoprotein were studied by dynamic dialysis and by using a Ca2+‐selective electrode. Two different binding sites were detected with Kd= 0.9 × 10−7 M and 1.1 × 10−5 M and displaying a Ca2+‐binding capacity of 127 and 176 mol bound Ca2+/mol protein, respectively, assuming a molecular weight of 30 000. Upon enzymatic dephosphorylation of the phosphoprotein, the highest affinity sites disappeared and those with the lowest affinity were reduced The optimum for Ca2+ binding by the phosphoprotein occurred at pH 8.2. The specificity of the Ca2+ ion interaction with the phosphoprotein was investigated by studying the competitive nature of other divalent and monovalent cations. It was found that Ca2+ ions were to a large extent displaced from the phosphoprotein by other cations in physiological concentrations. Copyright © 1981, Wiley Blackwell. All rights reserved