Association of phosphoprotein and collagen with calcification of dentin.

1. phosphoryn是牙本质中一种独特的磷蛋白，用生物素对其进行了标记，并以胶原蛋白为结合底物进行了结合实验。磷酸化蛋白对胶原蛋白有明显的亲和力。这种亲和被认为是静电的，因为它在高离子强度下被还原。利用胶原蛋白的CNBr肽作为结合底物，确定了磷酸化蛋白在胶原分子中的结合位点。标记的磷酸化蛋白优先与α 1 CB6肽结合，该肽对应于c端区域。将标记的磷酸化蛋白与重组胶原纤维孵育，以亲和金为标记物，用电镜观察。磷酸化蛋白优先结合到原纤维的空穴区。在牙本质成熟过程中，部分磷酸化蛋白在体内固定在胶原蛋白上。形成的磷酸化蛋白-胶原复合物经CNBr消化不溶性胶原基质后溶解，并经层析纯化。该复合物由磷酸蛋白和胶原蛋白组成。这两个部分不能相互分离，表明它们之间的联系具有共价性质。该配合物与α 1 CB6肽抗体反应。蛋白酶消化该复合物释放出一种衍生自α 1 CB6肽的肽。磷酸化蛋白的共价结合位点推断为α 1 CB6肽。磷酸化蛋白与不溶性胶原在体外培养，以减少这两种成分之间的体内交联。磷酸化蛋白通过培养固定在胶原蛋白上。这一过程被过量的胺所抑制，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.