Tooth enamel is secreted as a protein matrix which is removed as the enamel matures. The largest fraction of the protein matrix consists of amelogenins, which are selectively removed from the enamel as maturation progresses. This processing of amelogenins is important for regulating crystal growth in the mineralizing enamel matrix. The overall objective in this proposal is to better understand amelogenin processing by identifying and characterizing amelogeninases in the developing enamel matrix. The specific hypothesis to be tested is that a serine proteinase, expressed by ameloblasts, is responsible for the hydrolysis of amelogenin protein in the developing enamel matrix. The specific aims are: 1) to isolate and purify the serine proteinase in enamel which actively hydrolyzes amelogenin; 2) to characterize the amelogeninase for substrate specificity, pH optima and thermal stabilities; 3) to determine the specific cleavage sites of the amelogeninase by using purified 25,000 MW amelogenin as a susbstrate; 4) to amplify, clone and sequence the cDNA(s) from bovine enamel organ mRNA which contains the conserved active site for a serine proteinase; 5) to clone a full-length cDNA for the serine proteinase/amelogeninase from a bovine enamel organ lambda phage cDNA library and to begin in situ hybridization studies. Purification and characterization of the serine proteinase/amelogeninase will be completed by isoelectric focusing, gel electrophoresis and column chromatography. Specificity of the amelogeninase will be determined by hydrolysis of a purified amelogenin substrate. The cDNA for the serine proteinase will be cloned to allow comparisons between the initially expressed mRNA transcript and the active form of the proteinase present in the enamel matrix. These studies to identify and characterize the serine proteinase/amelogeninase in developing enamel will lead to a better understanding of the mechanisms by which amelogenesis occurs. They will also further our understanding of defects which occur in enamel formation, such as in enamel fluorosis.