Calcium ions are used by all eukaryotic cells as regulatory molecules to coordinate complex and diverse cellular activities. Calcium-binding proteins (CaBPs) have evolved to mediate cellular responsiveness to calcium ions and to maintain low intracellular calcium concentrations. The following proposal is designed to systematically identify and characterize CaBPs in Trypanosoma brucei rhodesiense. Preliminary experiments have identified 8 CaBPs in T. b. rhodesiense. Four of these proteins, including a developmentally regulated CaBP have been purified. The specific goals of this project are to: 1) develop large scale preparative purification protocols for each of the CaBPs identified with emphasis on the four CaBPs that have already been purified on a small scale; 2) determine calcium binding properties of the purified proteins by equilibrium dialysis or by the gel filtration method of Hummel and Dreyer; 3) sequence portions of the purified proteins by automated Edman degredation and determine homologies with any other protein in the Protein Sequence Database of the Protein Identification Resource (1985,V5.0); 4) prepare polyclonal antibodies to selected proteins and use them in fluorescent and colloidal gold immunolocalization studies to identify the site of activity of the different CaBPs; 5) develop a gel overlay procedure to detect other proteins that associate with CaBPs; 6) detect the regulatory CaBP, protein kinase C, if it is present, by phosphorylase activity or as the (20-3H)phorbol 12,13 dibutyrate binding protein; 7) identify genes encoding the different CaBPs by screening an expression library with combinations of the following: i) polyclonal antibodies to different CaBPs, ii) synthetic oligonucleotides homologous with amino acid sequences and iii) 45 Ca using the same 45Ca gel overly procedure originally used to identify CaBPs in cell homogenates and 8) characterize the genes encoding the CaBPs in terms of: i) endonuclease maps, ii) nucleotide sequence, iii) chromosome localization and iv) corresponding mRNAs. Ultimately, this study will characterize major components of calcium-dependent regulatory pathways in African trypanosomes and will likely reveal sensitive processes that, in the long run, can be targeted in the development of trypanocidal therapies.
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