Matrix Proteins Affecting De Novo Formation of Apatite

研究了骨GLA蛋白、骨连接素和牙本质磷蛋白等基质蛋白在广泛的磷酸钙溶液和β-甘油磷酸钙溶液中胶原存在下对磷灰石从头形成的影响。在每一种溶液中，无定形磷酸钙、磷酸八钙或磷灰石作为可能的初始相沉淀出来，每一种蛋白质在浓度低于1 μ M时都会延缓沉淀、随后向磷灰石的转化和磷灰石的成熟晶体生长。在相同的蛋白质导入量下，抑制活性依次为骨粘连蛋白、骨Gla蛋白和detin磷蛋白。在催化量的碱性磷酸酶存在下使用β-甘油磷酸钙溶液的系统中，发现蛋白质抑制活性越高，磷酸钙沉淀所需的过饱和度就越高。胶原蛋白以重组或变性形式存在，对蛋白质添加反应以及蛋白质-在胶原纤维和我们系统中出现的任何磷酸钙之间观察到自由反应和结构相关性。溶液中游离钙水平的直接测量表明，由于与蛋白质复合而导致的钙活性降低很难解释蛋白质的抑制活性。相反，TEM观察表明，蛋白质抑制磷灰石形成的主要机制是阻断无核磷酸钙的生长位点。在抑制性更强的蛋白质存在下形成的磷灰石显示出分辨率更低的X射线衍射图，这部分地解释了在蛋白质存在下形成的磷灰石的晶体尺寸小，如TEM所示。

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.