Bennick A, McLaughlin A C, Grey A A, Madapallimattam G
J Biol Chem. 1981 May 25;256(10):4741-6.
The location of the calcium-binding sites in the human acidic proline-rich proteins, salivary proteins A and C, were determined by equilibrium dialysis of the tryptic peptides with buffers containing 45Ca. All the calcium-binding sites are located in the NH2-terminal tryptic peptide (TX peptide). The nature of the calcium binding sites in the TX peptide and native salivary proteins A and C, as well as dephosphorylated proteins were compared. Two types of sites can be distinguished in peptide TX. Type I sites have an apparent dissociation constant (K) of 38 microM and are responsible for the binding of 2.6 mol of Ca/mol of peptide. The corresponding figures for Type II sites are 780 microM and 5.3 mol of Ca/mol of peptide. In the native proteins, the amount of calcium bound at the type II sites decreases to 3.9 mol of Ca/mol of proteins A and C and K increases to 1100 microM. The amount of calcium bound at type I sites decreases to 1.5 mol/mol of protein A and 0.6 mol/mol of protein C, but there is no change in K. Dephosphorylation affects the calcium binding at both types of sites. The experiments indicate that the COOH-terminal parts of the native proteins affect the number and the nature of the protein calcium-binding sites. Proton and phosphorous NMR data demonstrate that beta-COOH in aspartic acid, as well as phosphoserine, are part of the calcium-binding sites. The difference in calcium binding to salivary proteins A and C may be due at least partially to differences in the environment of one or more aspartic acids.
通过用含45Ca的缓冲液对人富含酸性脯氨酸的蛋白质（唾液蛋白A和C）的胰蛋白酶肽段进行平衡透析，确定了钙结合位点在这些蛋白中的位置。所有的钙结合位点都位于NH2末端的胰蛋白酶肽段（TX肽段）中。对TX肽段、天然唾液蛋白A和C以及去磷酸化蛋白中的钙结合位点的性质进行了比较。在肽段TX中可区分出两种类型的位点。I型位点的表观解离常数（K）为38 microM，每摩尔肽段可结合2.6摩尔钙。II型位点的相应数值分别为780 microM和每摩尔肽段结合5.3摩尔钙。在天然蛋白质中，II型位点结合的钙量降至每摩尔蛋白A和C结合3.9摩尔钙，而K值增至1100 microM。I型位点结合的钙量降至每摩尔蛋白A结合1.5摩尔、每摩尔蛋白C结合0.6摩尔，但K值无变化。去磷酸化影响两种类型位点的钙结合。实验表明，天然蛋白质的COOH末端部分会影响蛋白质钙结合位点的数量和性质。质子和磷核磁共振数据表明，天冬氨酸中的β-COOH以及磷酸丝氨酸是钙结合位点的一部分。唾液蛋白A和C在钙结合上的差异可能至少部分归因于一个或多个天冬氨酸所处环境的差异。
