Calcium ions are used by all eukaryotic cells as regulatory molecules to coordinate complex and disparate cellular activities. Calcium regulates components of cell division, secretion, motility, and energy metabolism. However, at high concentrations, calcium is cytotoxic, resulting in cell necrosis. In order to utilize calcium and maintain low intracellular free calcium concentrations, a variety of calcium-binding proteins have evolved. We have previously purified the ubiquitous, multifunctional calcium binding protein, calmodulin from Trypanosoma brucei rhodesiense. The following study is designed to systematically identify and characterize other calcium binding proteins from various life cycle stages of T.b.rhodesiense. Until recently, the methodologies for such a study did not exist. The specific goals of this project are to: 1) detect calcium-binding proteins in homogenates of T. brucei by Chelex 100 competitive binding assays, phenyl-Sepharose chromotography and calcium gel overlays; 2) purify the proteins by FPLC Mono S anion exchange, gel filtration, chromatofocusing and reverse phase chromatography; 3) structurally characterize the proteins by determining their i) affinity for calcium; ii) number of calcium binding sites, iii) amino acid composition, iv) amino terminal sequence and v) calcium induced conformational changes; 4) establish whether any of the proteins are developmentally regulated; 5) determine the site of calcium-binding protein activity by immunolocalization studies and 6) assay the proteins for kinase activity with emphasis on the calcium/phospholipid-dependent kinase C. Since calcium is both a critical regulatory component and cellular poison, the mechanisms by which cell control and utilize their intracellular cytosolic calcium content are essential for cell survival. Consequently identification of calcium-binding proteins is likely to reveal sensitive pathways that, in the long run can be targeted in the development of trypanocidal therapies.
