The proposed study will focus on enzymes of matrix vesicles which play a role in the mineralization of teeth. Matrix vesicles are submicroscopic, extracellular, membrane-invested particles which serve as the initial loci of mineralization of dentin, and of other oral tissues including cartilage and bone. Our lab was involved in the original identification of matrix vesicles and in their subsequent isolation and partial characterization. We have provided evidence that matrix vesicle phosphatases (including ATPase, pyrophosphatase and alkaline phosphatase) are involved in triggering mineralization. Furthermore, these phosphatases reside on the vesicle membrane surface and can be solubilized by deoxycholate treatment with preservation of enzymatic activity and calcium-depositing ability. Two general types of studies with the solubilized enzyme are proposed: 1) chemical purification and characterization as to molecular size, inhibitors, substrate and cofactor requirements and 2) analysis and control of calcium accumulation by vesicle phosphatase including an electron microscopic study of this process. This is a fundamental study of the mechanism by which normal dentinal and other forms of mineralization are brought about. Techniques for tissue fractionation, radioisotopic labelling and counting, enzyme biochemistry and electron microscopy will be employed. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific disease states in which there is abnormal calcification.
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