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

1.蛋白水解法鉴定羟基磷灰石结合位点我们通过蛋白水解法对吸附在羟基磷灰石晶体上的蛋白质进行了检测。被检测的蛋白质是牙本质中一种独特的磷蛋白--磷蛋白，以及骨的一种酸性糖蛋白--骨联素。溶液中的磷酸二氢钾具有较高的磷酸化水平，因此对蛋白水解酶具有抗性。吸附在晶体上的磷蛋白被蛋白酶攻击，表明该蛋白在晶体表面具有伸展结构。我们用蛋白酶消化吸附在晶体上的骨连蛋白，发现即使在消化后，晶体上仍然吸附着一种粘连蛋白。该多肽含有骨连蛋白结构域I和II。羟基磷灰石结合部位可能位于这些区域内。对羟基磷脂具有亲和力的多肽的合成在骨连接蛋白的结构域I和II中存在一个富含谷氨酸的序列。在骨和牙本质的其他羟基磷灰石结合蛋白中也存在Glu或Asp的连续序列。我们合成了Glu_6和Asp_6多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。多肽与晶体的结合常数为10^6m^&lt；-1&gt；。我们得出结论，这些连续的酸性氨基酸序列构成了羟基磷灰石的结合部位。

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.