Matrix Proteins Affecting De Novo Formation of Apatite

研究了骨GLA蛋白、骨连接蛋白和牙本质磷蛋白基质蛋白在多种磷酸钙溶液和存在胶原的β -甘油磷酸钙溶液中对磷灰石新生形成的影响。在每一种溶液中，当无定形磷酸钙、八磷酸钙或磷灰石作为可能的初始相沉淀时，每一种浓度低于1muM的蛋白质都会延缓沉淀、随后向磷灰石的转变和磷灰石的成熟晶体生长。与相同重量的蛋白相比，骨连接蛋白、骨玻璃蛋白和detin磷酸化蛋白的抑制活性依次增强。在使用β -甘油磷酸钙溶液的体系中，在碱性磷酸酶催化量存在的情况下，发现蛋白质抑制活性越高，磷酸钙沉淀所需的过饱和程度越高。重组或变性形式的胶原蛋白对蛋白质添加反应和无蛋白质反应没有影响，并且在我们的系统中没有观察到胶原原纤维和任何磷酸钙之间的结构相关性。溶液中游离钙水平的直接测量表明，由于与蛋白质络合而导致的钙活性降低很难解释蛋白质的抑制活性。相反，透射电镜观察表明，该蛋白抑制磷灰石形成的主要机制是阻止磷酸钙的生长位点未被清除。在抑制蛋白存在下形成的磷灰石显示出较少的x射线衍射模式，这在一定程度上解释了在蛋白质存在下形成的磷灰石晶体尺寸较小的原因。

Effects of matrix proteins of bone GLA protein, osteonectin and dentin phosphoprotein on de novo formation of apatite were studied in a wide range of calcium phosphate solutions and in calcium beta-glycerophosphate solutions in the presence of collagen. In every solution, from which amorphous calcium phosphate, octacalcium phosphate, or apatite precipitated as a possible initial phase, each protein at concentrations less than 1muM retarded the precipitation, subsequent transformation to apatite, and ripening crystal growth of apatite. The inhibitory activity increased in the order of osteonectin, bone Gla protein and detin phosphoprotein when compared at the same weight of the protein introduced. In the system employing the calcium beta-glycerophosphate solutions in the presence of catalytic quantity of alkaline phosphatase, it was found that the more the protein inhibitory activity the higher the degree of supersaturation was needed for calcium phosphate to precipitate.Collagen present as either reconstituted or denatured form had no effect on the protein-added reactions as well as the protein-free reactions and no structural correlation was observed between collagen fibrils and any of the calcium phosphates that appeared in our system. Direct measurement of free calcium levels in the solution suggested that the reduction in calcium activity due to complexing with the protein hardly explained the inhibitory activity of the protein. Instead, TEM observation suggested that the primary mechanism for the protein to inhibit the formation of apatite is to block growth sites of calciu phosphates uncleated. The apatite thus formed in the presence of the more inhibitory protein showed less resolved X-ray diffraction pattems, which accounts for in part smallness of crystal size of apatite formed in the presence of the protein as suggested by TEM.