Association of phosphoprotein and collagen with calcification of dentin.

1.牙本质磷蛋白（Phosphophoryn）是牙本质中特有的磷蛋白，用生物素标记后，以胶原为结合底物，进行结合试验。磷酸化蛋白对胶原蛋白具有显著的亲和力。亲和力被认为是静电的，因为它在高离子强度下降低。以胶原蛋白的溴化氰肽段为结合底物，测定了胶原蛋白分子中磷配基的结合位点。标记的磷酸蛋白优先结合于α 1 CB 6肽段，该肽段对应于C-末端区域，标记的磷酸蛋白与重构的胶原纤维孵育，以亲和素-金为标记物进行电镜观察。磷配基优先结合到纤维的空穴区。磷酸化蛋白-胶原复合物的表征在牙本质成熟过程中，部分磷酸化蛋白被固定在胶原上，形成的磷酸化蛋白-胶原复合物用溴化氰消化不溶性胶原基质增溶，层析纯化。该复合物由磷酸化蛋白和胶原部分组成。这两个部分不能彼此分离，表明它们之间的缔合的共价性质。使该复合物与抗α 1CB 6肽的抗体反应。复合物的蛋白酶消化释放出衍生自α 1 CB 6肽的肽。推测磷酸化蛋白的共价结合位点为α 1CB 6肽.磷酸化蛋白与胶原蛋白的体外交联磷酸化蛋白与不溶性胶原蛋白孵育，以测定这两种组分之间的体内交联。磷酸化蛋白在孵育过程中被固定在胶原蛋白上，过量的胺抑制了这一过程，表明可能涉及脱氢丙氨酸衍生的交联。

1. Electrostatic interaction of phosphophoryn and collagenPhosphophoryn, a unique phosphoprotein of dentin, was rabeled with biotin and was subjected to binding assay using collagen as a binding substrate. Phosphophoryn had a significant affinity to collagen. The affinity is thought to be electrostatic, since it was reduced at high ionic strength. Binding site of phosphophoryn in a collagen molecule was determined using CNBr peptides of collagen as binding substrates. The labeled phosphophoryn was preferentially bound to an alpha 1 CB6 peptide, which is corresponding to the C-terminal region.The labeled phosphophoryn was incubated with reconstituted collagen fibril and was observed with electronmicroscope using avidin-gold as a marker. The phosphophoryn was preferentially bound to hole zones of the fibril.2. Characterization of phosphophoryn-collagen complexA part of phosphophoryn is immobilized to collagen in vivo during maturation of dentin.The phosphophoryn-collagen complex thus formed was solubilized by CNBr digestion ofinsoluble collagen matrix, and purified chromatographically. The complex was composed of phosphophoryn and collagen moieties. These two moieties could not be separated from each other, indicating the covalent nature of the association between them. The complex was reacted with an antibody against alpha 1 CB6 peptide. Proteinase digestion of the complex released a peptide which was derived from alpha 1 CB6 peptide. The covalent binding site of phosphophoryn is deduced to be alpha 1 CB6 peptide.3. In vitro crosslinking of phosphophoryn with collagenPhosphophoryn was incubated with insoluble collagen to minic the in vivo crosslinking between these two components. Phosphophoryn was immobilized to collagen with the incubation.The process was iinhibited by excessive amounts of amines, indicating a possible involvement of dehydroalanine-derived crosslinking.