The overall goal of this research is to characterize the process of biologic calcification. This proposal examines the mechanism of membrane-mediated proteolipid-dependent calcium hydroxyapatite (HA) by analyzing matrix vesicle proteolipid (MVP) structure, function, and metabolic regulation, using epiphyseal chondrocyte or odontoblast culture-derived matrix vesicles, matrix vesicles from chick growth cartilage and root incisor dentine, and synthetic proteoliposomes as models. The association of membranes and membrane components, particularly Pr and Ca-phospholipid-Pi complexes (CPLX), with initial HA formation in normal calcifying tissues, osseous induction, and hard tissue repair, as well as in dystrophic calcification, suggests that certain properties of membranes are conducive to HA deposition when sufficient Ca is available. This has important implications in the treatment of diseases like osteoporosis and periodontal diseases as well as for prevention and treatment of ectopic mineral deposits. Recent research indicates that Pr apoprotein structures phospholipid to interact with Ca and Pi to form CPLX and subsequent HA; however, the mechanism of this process is unknown. If the principals of membrane-associated calcification are constant and independent of tissue source, then normal mineral deposition must be regulated by the concentration of a particular apoprotein, and the nature of its phospholipid domain, as well as availability of mineral ions. This research described in this proposal will examine this hypothesis by chemically characterizing MVP (SDS-PAGE; Western blot, proteolytic digest maps, and N- and C- terminal amino acid analyses to determine aproprotein heterogeneity; purification of apoprotein subclasses by HPLC); examining its interaction with membrane lipids to form a calcifiable domain (construction of synthetic proteoliposomes to study protein-protein and protein-lipid interactions in CPLX and/or HA formation), determining whether MVP functions in transmembrane ion transport (H+, Ca++ or Pi transport in proteoliposomes and matrix vesicles), and examining its regulation by hormonal (vitamin D) and nutritional factors (Zn) influencing lipid metabolism. Tissue culture data, obtained in the absence of competing regulatory factors, will be compared to experiments with intact animals to ensure physiologic relevance.
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