BIOCHEMICAL STUDIES OF MINERALIZED TISSUE PROTEOGLYCANS

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Recently, several studies have been initiated to investigate the nature of the macromolecules of mineralized tissues in order to elucidate their roles in biological calcification. Proteoglycans (PGs) are one of the noncollagenous components of the extra-cellular matrix of bone and dentin. The aims of this proposal are to rigorously characterize the PGs of bone and to investigate the metabolic features of bone PGs which might be relevant to mineralization. Comparison of the characteristics of bone and dentin PGs may provide data regarding the features of PGs that are required for normal calcification. Also, various cell culture systems will be established and evaluated for their suitability as models for studying PG metabolism. These studies will use extensive precautions to avoid artifactual proteolytic degradation of PGs. Also, a sequential extraction procedure will be used which provides two pools of PGs: one from nonmineralized matrix and another from calcified areas of the tissue. PGs and constituent glycosaminoglycans (GAGs) will be characterized chemically by specific enzyme digestion in conjunction with various chromatographic and centrifugation procedures. Metabolic characteristics of bone PGs will be studied by using a specific radiolabeling technique in conjunction with the extraction and characterization methods above. Cell culture systems will also use radiolabeling procedures in pulse and pulse-chase experiments. Various metabolic inhibitors and effectors will be used to assess the biosynthetic features of the different systems. The data provided by these experiments will be used in designing sutdies to probe specific events in the alteration of bone and dentin PGs resulting from various nutritional insults. For example, fluoride and vitamin A are two nutrients which can have adverse affects on skeletal and dental structure.