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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Tropical Medicine and Parasitology Study Section (TMP)
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Southern Methodist University
Schools of Arts and Sciences
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Ridgley, E L; Ruben, L (2001) Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1, sn-2 deacylation of phospholipids. Mol Biochem Parasitol 114:29-40
Ridgley, E; Webster, P; Patton, C et al. (2000) Calmodulin-binding properties of the paraflagellar rod complex from Trypanosoma brucei. Mol Biochem Parasitol 109:195-201
Parsons, M; Ruben, L (2000) Pathways involved in environmental sensing in trypanosomatids. Parasitol Today 16:56-62
Ridgley, E L; Xiong, Z H; Ruben, L (1999) Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei. Biochem J 340 ( Pt 1):33-40
Xiong, Z H; Ruben, L (1998) Trypanosoma brucei: the dynamics of calcium movement between the cytosol, nucleus, and mitochondrion of intact cells. Exp Parasitol 88:231-9
Eintracht, J; Maathai, R; Mellors, A et al. (1998) Calcium entry in Trypanosoma brucei is regulated by phospholipase A2 and arachidonic acid. Biochem J 336 ( Pt 3):659-66
Xiong, Z H; Ridgley, E L; Enis, D et al. (1997) Selective transfer of calcium from an acidic compartment to the mitochondrion of Trypanosoma brucei. Measurements with targeted aequorins. J Biol Chem 272:31022-8
Ruben, L; Akins, C D; Haghighat, N G et al. (1996) Calcium influx in Trypanosoma brucei can be induced by amphiphilic peptides and amines. Mol Biochem Parasitol 81:191-200
Ridgley, E L; Xiong, Z H; Kaur, K J et al. (1996) Genomic organization and expression of elongation factor-1 alpha genes in Trypanosoma brucei. Mol Biochem Parasitol 79:119-23
Xiong, Z H; Ruben, L (1996) Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin. Mol Biochem Parasitol 83:57-67

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