Studies on the Chemical Structure of Enamel Proteins and the Mechanisms of Calcification

牙釉质是一种高度矿化的组织，几乎完全由磷灰石晶体和少量有机基质组成。然而，在牙釉质形成的早期阶段，可以发现高达25%-30%的蛋白质浓度。在牙釉质成熟过程中，蛋白质组分逐渐减少，并被磷灰石晶体所取代。釉原蛋白和釉质蛋白在釉质矿化过程中起着重要作用。1)用牛全长cDNA探针筛选噬菌体EMBL3中的人基因组DNA文库，成功地分离到一个含有编码人釉原蛋白的插入片段的克隆。从亚克隆到几个载体中的DNA片段的碱基序列中，推测出除前导序列、第一和第二个氨基酸外的釉原蛋白的一级结构。2)从发育中的牛牙釉质中分离到一种酸性最适的釉原蛋白。这一结果表明牙釉质基质中存在酸性条件。我们试图用颜色pH指示器测量发育中牙齿的pH，发现发育中的牙釉质中存在周期性的酸性和中性带。当用pH指示剂染色时，未萌发的牛牙釉质显示出几个交替的两种颜色的条纹，分别对应于酸性(pH 5.8-6.0)和中性(pH 7.0)反应。通过酸度计测量，确定了各区域釉质样品的pH值。深入检测了pH值的差异。中性条带对应于GBHA染色的红色条纹，酸性条带对应于未染色的白色区域。酸性和中性交替的酸性和中性区域与褶边和光端成釉细胞的功能有关，对于酸性和中性蛋白酶降解釉原基质以及羟基磷灰石晶体的生长和成熟具有重要意义。

Dental enamel is a highly mineralized tissue consisting almost exclusively of apatite crystals with a small amount of organic matrix. However, protein concentrations as high as 25-30 % can be found at an early stage of enamel formation. The protein components decrease gradually during the maturation of enamel and are replaced by the apatite crystals. The proteins present at the early stage of enamel formation, amelogenin and enamelin are thought to play important roles in the process of enamel mineralization.1) We screened human genomic DNA library in phage EMBL3 with a bovine full-length cDNA probe and succeeded in isolating one clone containing a insert which encodes the human amelogenin. From the base sequence of the DNA fragments subcloned into several vectors, the primary structure of amelogenin protein was deduced except leader sequence, 1st and 2nd amino acids. However, it was discovered that human mature amelogenin protein consists of 178 amino acids.2) We isolated an amelogenin-digestible enzyme having an acidic pH optimum from developing bovine enamel. This result suggested presence of acidic conditions in the enamel matrix. We tried to measure pH in developing teeth with color pH indicators and discovered the presence of cyclical acidic and neutral zones in developing enamel. When stained with pH indicators, unerupted bovine enamel manifested several alternating stripes of two colors corresponding to acidic (pH 5.8-6.0) and neutral (pH 7.0) reactions. Those pH values of the enamel samples from respective zone were confirmed by measuring with a ph-meter. The pH difference was detected in depth. Neutral bands corresponded to red stripes of GBHA-staining and acidic bands to unstained white zones. Importance of alternating acidic and neutral zones in relation to the functions of ruffle-ended and smooth-ended ameloblasts was confirmed for degradation of amelogenin matrix by acidic and neutral proteases and for growth and maturation of hydroxyapatite crystals.