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

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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.