Purification and characterisation of a trypsin-like serine oligopeptidase from Trypanosoma congolense
Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. Here a similar enzyme from Trypanosoma congolense is described. This oligopeptidase, called OP-Tc, was purified using three-phase partitioning, and ion-exchange and affinity chromatography. OP-Tc is inhibited by alkylating agents, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumarin, 4-(2-aminoethyl) benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidyl chloromethyl ketones. Reducing agents such as dithiothreitol enhanced activity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino acid residues (Arg or Lys) in the P1 position. Potential substrates without a basic residue in P1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P2 and P3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower kcat/Km values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC (Km = 0.72 µM, kcat = 96 s-1). OP-Tc: preference for amino acids in the P2 position was (Gly,Lys,Arg) > Phe > Leu > Pro. The results also suggest that the P3-binding site has hydrophobic characteristics. OP-Tc may not be a naturally immunodominant molecule because neither IgG nor IgM anti-OP-Tc antibodies were detected in the blood of experimentally infected cattle.
| Main Authors: | , , , , |
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| Format: | article biblioteca |
| Language: | eng |
| Subjects: | L73 - Maladies des animaux, Trypanosoma congolense, peptide, purification, http://aims.fao.org/aos/agrovoc/c_27401, http://aims.fao.org/aos/agrovoc/c_5691, http://aims.fao.org/aos/agrovoc/c_6377, |
| Online Access: | http://agritrop.cirad.fr/510529/ http://agritrop.cirad.fr/510529/1/510529.pdf |
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