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 k(cat)/Km values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC (Km = 0.72 microM, k(cat) = 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.
Morty
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RE
Department of Biochemistry, University of Natal, Scottsville, South Africa.
Authié
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Troeberg
L
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Lonsdale-Eccles
J D
JD
Coetzer
T H
TH
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D002417:Cattle; D004789:Enzyme Activation; D008297:Male; D010447:Peptide Hydrolases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014348:Trypanosoma congolense / Q000201:enzymology* / Q000472:pathogenicity; D014353:Trypanosomiasis, African / Q000276:immunology / Q000469:parasitology / Q000662:veterinary; D014354:Trypanosomiasis, Bovine / Q000276:immunology / Q000469:parasitology; D014357:Trypsin / Q000378:metabolism
D000913:Antibodies, Protozoan; D015842:Serine Proteinase Inhibitors; D010447:Peptide Hydrolases; C028759:oligopeptidase; D014357:Trypsin