This R29 application is designed to investigate the correlation between proteolytic processing of the enamel extracellular matrix protein amelogenin and growth of apatite crystals, using an in vitro system. The applicant identified a serine protease, designated ameloprotease-I, as a cleavage product of one of the larger non-amelogenin proteins. This protease was shown by the applicant to degrade amelogenin proteins in vitro. The hypotheses to be examined are: that the activity of ameloprotease-I correlates with formation and maturation of enamel apatite crystals; that ameloprotease-I is activated in the extracellular matrix through proteolytic cleavage of its precursors; and that adsorption and dissociation of both enzyme and substrate onto apatite crystals are involved in proteolytic processing.
Four specific aims are proposed: (I) To examine autocatalytic action of the precursor 89 kDa enamelin in release of the 32 kDA protease in the absence and presence of apatite crystals; (II) To investigate effects of calcium, phosphate, carbonate and fluoride on activity of ameloprotease-I; (III) To determine cleavage sites within amelogenin after proteolysis +/-apatite crystals, and to monitor adsorption of enzyme and substrate to the apatite; and (IV) To investigate cleavage of amelogenin by other serine proteases +/- apatite crystals, to determine site of enzyme accessibility on the protein surface.
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