Partial purification and characterization of microtubular protein from Trypanosoma brucei.
The tubulin proteins of the parasitic hemoflagellate Trypanosoma brucei brucei were purified and characterized. Cytoskeletal microtubules of trypanosomes do not disrupt under conditions used to solubilize brain tubulins. Trypanosomal tubulins, solubilized by extensive sonication, were partially purified from the crude cell extracts by taxol-mediated polymerization. Taxolinduced microtubules were identified by electron microscopy and analyzed biochemically. They consist predominantly of two proteins of about 52,000 and 56,000 Da. Their mobilities on sodium dodecyl sulfate gels differ slightly from those of bovine brain tubulins. Immunological cross-reactivity with antibodies raised against bovine brain tubulins confirmed the nature of the trypanosomal proteins. Peptide mapping of bovine and trypanosomal alpha- and beta-tubulins was performed by enzymatic digestion with staphylococcal protease V8 and chemical cleavage with N-chlorosuccinimide. In both cases, the peptide patterns generated from the trypanosomal alpha- and beta-tubulins were closely related to each other. This suggests that the trypanosomal alpha- and beta-tubulins may have remained more conserved during evolution than the tubulins from higher eukaryotes. The trypanosomal alpha-tubulin is post-translationally modified in vivo by the reversible addition of a tyrosine residue at its COOH terminus. As in higher eukaryotes, this reaction is completely specific for the alpha-polypeptide chain. Our observation represents the first documentation of the occurrence of COOH-terminal tyrosinolation of alpha-tubulin in an eukaryotic microorganism.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D014346:Trypanosoma brucei brucei / Q000032:analysis*; D014404:Tubulin / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014404:Tubulin