Carbonic anhydrase (CA), has been postulated to be a pivotal enzyme in the destruction of calcified tissue by osteoclasts (OCs). This enzyme utilizes metabolic CO2 as a substrate for protein generation. These protons are then excreted into the resorptive cavity underneath the adhered OC. The combination of the acid secreted by the OC and the enhanced activity of lysosomal enzymes in this acidic microenvironment demineralizes and grades the extracellular matrix. This proposal is designed to characterize the mechanisms by which CA activity regulates OC resorption and the destruction of calcified tissue. There are Four Specific Aims. The First Specific Aim is to isolate a purified population of functional OCs from bone. The Second Specific Aim will explore the relationship between activation and changes in CA activity utilizing mass spectrometric techniques and employing nonradioactive isotopes of carbon, hydrogen and oxygen (O2). The experiments are planned to characterize effects of CA activation on OC acid secretion and intracellular pH. The Third Specific Aim is to develop the methodology to quantitate OC resorption. This methodology will then be used to relate CA activity to the resorptive process. The Fourth Specific Aim explores the shift in glucose utilization which occurs during the activation of OC resorption. These metabolic changes also alter the sources of CO2 for CA activity and will provide new insight into the role of CA in the regulation of bone resorption by osteoclasts.
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