Changes of Intercellular Matrix proteins During Calcification in Hard Tissues

本研究的目的是验证工作假设，即骨和牙本质中的单个基质蛋白的功能独立以及合作进行定量和定性的变化，以调节钙化。首先，我们建立了从牛骨和牙本质中分离磷酸化蛋白、骨粘连蛋白、骨钙素、α-2 HS蛋白、62 K蛋白和24 K蛋白等主要非胶原蛋白的系统方法。系统方法包括用1.0 M NaAl和0.5 M EDTA/1 M NC 1（pH 7.4）提取，各种类型的常规色谱法和使用Asahipak GS-520的高效液相色谱法。通过这些方法，发现磷酸化蛋白的含量随着牙本质的发育而增加，这表明这种高度磷酸化的蛋白具有独特的功能。其次，我们发现EDTA/NaCl提取的蛋白质的蒸馏水溶液通过添加微量的钙离子（最终浓度小于2 mM）而沉淀，并且发现当钙离子添加至1 mM的最终浓度时，甚至在150 mM/5 mM Tris-HCl（pH7.4）的骨蛋白溶液中也发生沉淀。高效液相色谱分析表明，参与沉淀的蛋白质至少为骨粘连蛋白和62 K蛋白，在钙离子浓度低于250 μ M时，这两种蛋白结合形成高分子组分。这些骨蛋白的结合先于明显的钙诱导沉淀。结果表明，这两种蛋白质与钙离子相互作用，结合在一起，最终引起钙离子浓度的微小变化。结果提示，这两种蛋白可能在骨组织中起着固定初始钙离子和磷酸根离子簇，调节钙化的作用。

The purpose of this study is to verify the working hypothesis that the individual matrix protein in bone and dentin functions independently as well as cooperatively undergoing quantitative and qualitative changes in order to regulate calcification. First, we have established a systematic methods for the isolation of major noncollagenous proteins including phosphophoryn, osteonectin, osteocalcin, alpha-2HS pr otein, 62K protein and 24K protein from bovene bone and dentin. Systematic methods comprised the extraction with 1.0 M NaAl and 0.5 M EDTA/1 M NC1, pH 7.4, the various types of conventional chromatography and a high performance liquid chromatography using Asahipak GS-520. By these methods, phosphophoryn was found to increase its content with the development of dentin, which suggested an unique function of this highly phosphorylated protein. Secondly, we have found that the distilled water solution of EDTA/NaC1-extracted proteins was precipitated by the addition of trace amount of calcium ions ( less than 2 mM in final concentration ).Moreover, it was found that the precipitation occurred even in the 150 mM/5mM Tris-HC1 (pH7.4) solution of the bone proteins when the calcium ions were added to the final concentration of 1mM. High performance liquid chromatography revealed that the proteins involved in the precipitation were at least osteonectin and 62K proteins, and that these two proteins associated to form the higher molecular components at the calcium concentation of less than 250 uM. These association of bone proteins preceded the apparent calciuminduced precipitation. Thus it was shown that these two proteins interact with calcium ions, associate themselves and finaly prcipitate with a small change of calcium concenration. The results suggested that these two proteins may play a role of immobilization of the initial clusters of calcium and phosphate ions and regulation of the calcification if bone tissue.