Identification of four extracellular-matrix enamel proteins during embryonic-rabbit tooth-organ development

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Abstract
The proteins which characterize the ameloblast phenotype were studied to determine to what extent these extracellular-matrix proteins were degraded as a function of enamel matrix mineralization and maturation. The identification of enamel proteins was based on comparisons between the electrophoretic patterns of enamel-containg and non-enamel-containing matrix extracts isolated from specific regions within 26 day embryonic New Zealand white rabbit incisor and molar tooth organs. Since enamel proteins became mineralized on secretion, matrix specimens were demineralized in cold 5% (wt/vol) trichloroacetic acid, extracted with buffered 5 M urea and reduced with mercaptoethanol and then the solubilized proteins were fractionated by urea/polyacrylamide-gel electrophoresis. Three enamel-specific electrophoretic components were identified in newly enamel-matrix specimens and this number increased as a function of mineralization and maturation. Antibodies were prepared against embryonic rabbit extracellular matrix containing enamel. Comparison between immunoelectrophoretic patterns demonstrated that 2 of the 3 enamel components were antigenic. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate was used to identify 4 enamel proteins of MW (1) 65,000, (2) 58,000, (3) 22,000 and (4) 20,000, localized within enamel matrix. Enamel proteins (1) and (3) were phosphorylated, whereas (2) and (4) did not contain detectable phosphate. Labeled proline, leucine, tryptophan and glucosamine were incorporated into each of the 4 enamel proteins extracted from tooth explants incubated in the presence of radioactive precursors for 6 h. While 4 proteins were identified in newly secreted enamel matrix the concentrations of high MW proteins (1) and (2) decreased and the number (> 10) and concentration of low MW polypeptides increased as a function of advanced enamel-matrix mineralization and maturation.