PROTEIN TRANSLATION ELONGATION FACTOR-1-ALPHA FROM TRYPANOSOMA-BRUCEIBINDS CALMODULIN

The following study examines the calmodulin (CaM) branch of the calciu m signal pathway in the protozoan parasite Trypanosoma brucei. To acco mplish this goal, a subset of cytosolic CaM-binding proteins (CaMBPs) was partially purified by a combination of DE52 and CaM-Sepharose affi nity chromatography. Monoclonal antibodies (CBP-KK1) were used to clon e the cDNA for a 53-kDa CaMBP from a lambda ZAP expression library of the metacyclic stage of T. brucei. The deduced amino acid sequence of clone CaMBP-12B had 81% overall amino acid identity to the translation elongation factor-1 alpha (EF-1 alpha) from Euglena gracilis and 76% identity to the rabbit EF-1 alpha. Rabbit EF-1 alpha was recognized by CBP-KK1 and was shown to bind to CaM-Sepharose in a calcium-dependent manner. By contrast, the complex of EF-1 alpha beta gamma did not bin d CaM Sepharose. A heterobifunctional sulfhydryl derivative of CaM (N- succinimydyl 3-(2-pyridyldithio)propionate-CaM) formed reducible cross -links with EF-1 alpha in solution but not with the complex of EF-1 al pha beta gamma. Biotinylated CaM bound weakly to trypanosome and rabbi t EF-1 alpha in a gel overlay assay. This report demonstrates the dire ct interaction between CaM and the translation elongation factor EF-1 alpha.