Purification of novel calcium binding proteins from Trypanosoma brucei: properties of 22-, 24- and 38-kilodalton proteins.
The present study was undertaken to systematically purify calcium binding proteins (CaBPs) from homogenates of Trypanosoma brucei. This work is important since CaBPs either serve as intracellular calcium buffers or mediate cellular response to calcium signals. Disruption of either process should be lethal to trypanosomes. We report that the 45Ca-gel overlay assay can be used to detect CaBPs following fractionation on DE-52, phenyl-Sepharose, Mono-Q, and Superose 12. Specific CaBPs of 22, 24, and 38 kDa were purified. Each of these proteins associated with 45Ca under denaturing and non-denaturing conditions. An approximate Kd for calcium of 8 microM was calculated for 22-kDa CaBP. None of the trypanosome CaBPs were related to known calcium binding protein families. They did not associate with hydrophobic interaction columns or cellular membranes in a calcium-dependent way, nor cross-react with 2 separate antibodies against annexin consensus sequences. A synthetic peptide corresponding to amino terminal residues 16-30 of 22-kDa CaBP was used to generate polyclonal antibodies. Immunoblots identified 22-kDa CaBP in African trypanosomes but not in other Kinetoplastidae or mammalian cells. Nonetheless, significant homology (58%) was observed between the amino terminal 37 residues of 22-kDa CaBP and the amino terminus of translationally controlled p21 from mammalian tumor cells. The present study is the first to apply systemic fractionation techniques to identify the complement of CaBPs in T. brucei. We conclude that novel CaBPs other than calmodulin and annexin family members contribute towards calcium pathways in these organisms.
Haghighat
N G
NG
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275.
Ruben
L
L
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D015800:Protozoan Proteins