Acidification is of critical importance in the process of bone resorption by osteoclasts (Baron et al. 1985; Baron et al. l988; Baron, 1989; Blair et al. l986; Blair et al. l989; Vaes, 1988). The lowering of the pH in the bone resorbing compartment, which underlies osteoclasts at their site of attachment to the bone matrix, results from the activity of H+-ATPases present in the osteoclast's membrane at the apical pole of the cell (ruffled-border). However, all cells express proton pumps in intracellular organelles (Nelson, 1991) and several cell types beside the osteoclast are specialized in acid secretion and express such proton pumps in their plasma membrane. Non-specific inhibition of proton transport would therefore lead to a systemic alteration of cellular physiology and acid base regulation. Observations by the applicant that the osteoclast - H+ATPase differs from other vacuolar ATPases in its pharmacology and, possibly, in its structure (Chatterjee et al. l992, 1993), together with the growing body of evidence that proton pumps actually differ between organelles and tissues, opens the possibility of specific inhibition of acid secretion in bone. Furthermore, and independently of the differences that may exist among vacuolar H+- ATPases, better understanding of the molecular mechanisms involved in proton transport across the osteoclast ruffled-border membrane may also lead to new developments in drug design. Two of the subunits composing the catalytic domain of the proton pump in chicken osteoclasts have been successfully cloned. The applicant has also purified and reconstituted the enzyme. The overall aim of this renewal application is therefore to further characterize the structure and properties of the osteoclast proton pump, comparing this ATPase with those of other tissues such as kidney and brain, and focusing mostly, but not exclusively, on the catalytic portion of the enzyme and the vanadate sensitive component of this pump.
Three specific aims are proposed. The first is to further characterize the catalytic domain of the enzyme at the molecular and biochemical level determining where each type of A and B subunit is expressed and the nature of the 63kD protein. The second specific aim is to determine the molecular basis for the increased nitrate and vanadate sensitivity of this proton pump. The third specific aim is to reconstitute the catalytic domain of the osteoclast H~ ATPase in yeast to analyze the relationship between primary structure and pharmacology of the enzyme. The work performed during the first two years, as well as the work proposed in the present renewal application, may aid in understanding the mechanisms by which osteoclasts acidify the bone surface, a process essential for bone resorption and may improve our ability to therapeutically interfere with this process in diseases such as osteoporosis, periodontal diseases and osteoarthritis.
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