In the proposed studies four aspects of osteoclast structure and function will be investigated. First, the function of several enzymes, two located at the ruffled border (Na,K-ATPase, H-ATPase) and one situated on the plasma membrane of the opposite side of the cell (Ca-ATPase), will be studied using enzyme activity assays and studies of ion-translocation into vesicles derived from osteoclast plasma membrane. Second, sites of acidity in and under osteoclasts at the ultrastructural level will be investigated. Third, studies of the distribution of cytoskeletal elements and the influence of calcium-regulating hormones on them will be continued. Of particular interest are the actin-associating proteins which link cytoskeletal elements to various organelles, including the plasma membrane. Immunoblotting, immunofluorescence, and ultrastructural immunocytochemistry will be employed. Fourth, the presence and distribution of calmodulin in osteoclasts will be studied. There are many calmodulin-regulated functions in cells, and it is important to establish its sites of action in osteoclasts. The several aspects of osteoclast structure, composition, and function we propose to investigate should provide further information for establishing a more rational basis in designing therapeutic measures directed at metabolic diseases of the skeleton.
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