IDENTIFICATION OF HYDROXYAPATITE-BINDING SITES OF HYDROXYAPATITE-BINDING PROTEINS IN MINERALIZED TISSUES

1.利用蛋白水解分析鉴定羟基磷灰石结合位点我们通过蛋白水解消化吸附在羟基磷灰石晶体上的蛋白质来检测羟基磷灰石结合位点。检测的蛋白质是磷酸化蛋白，一种独特的牙本质磷蛋白，和骨粘连蛋白，一种酸性糖蛋白的骨。溶液中的磷酸化蛋白由于其高水平的磷酸化而对蛋白酶具有抗性。吸附在晶体上的磷酸蛋白被蛋白酶攻击，表明该蛋白在晶体表面的延伸结构。我们用蛋白酶消化了吸附在晶体上的骨连接素，发现即使在消化后，仍有一个碎片吸附在晶体上。该肽含有骨连接蛋白的结构域I和II。羟基磷灰石结合位点可能位于这些结构域中。羟基磷灰石亲和肽的合成在骨连接素的结构域I和II中存在一个富含Glu的序列。Glu或Asp的连续序列存在于骨和牙本质的其他羟基磷灰石结合蛋白中。我们合成了Glu_6和Asp_6肽作为这些序列的模型，并检测了它们与羟基磷灰石的亲和力。肽与晶体结合的结合常数为10^6M^<-1>。我们的结论是，这些连续的酸性氨基酸序列构成的羟基磷灰石结合位点。

1. Identification of hydroxyapatite-binding sites using proteolytic analysisWe examined hydroxyapatite-binding sites by proteolytic digestion of proteins adsorbed on hydroxyapatite crystals. Proteins examined were phosphophoryn, a unique phosphoprotein of dentin, and osteonectin, an acidic glycoprotein of bone. Phosphophoryn in solution was resistant to proteinases because of its high level of phophorylation. Phosphophoryn adsorbed on the crystals was attacked by the proteinases, indicating an extended structure of this protein on surface of the crystal. We digested osteonectin adsorbed on the crystal with a proteinase, and found that a ftagment was left adsorbed on the crystal even after the digestion. The peptide contained domains I and II of osteonectin. The hydroxyapatite-binding sites may be localized within these domains.2. Synthesis of peptides having affinity to hydroxyapatiteThere is a Glu-rich wequence in the domains I and II of osteonectin. Consecutive sequences of Glu or Asp are present in other hydroxyapatite-binding proteins of bone and dentin. We synthesized Glu_6 and Asp_6 peptides as models of these sequences, and examined their affinity to hydroxyapatite. The peptides were bound to the crystals with an association constant of 10^6M^<-1>. We conclude that these consecutive sequences of acidic amino acids constitute the hydroxyapatite-binding sites.