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Trypanosoma cruzi: partial characterization of minor cruzipain isoforms non-adsorbed to Concanavalin A–Sepharose

Experimental Parasitology
Publication Date
DOI: 10.1016/j.exppara.2003.08.001
  • Biology


Abstract The present paper reports the partial characterization of a subset of atypical cruzipain molecules which do not bind to Concanavalin A–Sepharose column. They are present in different strains of epimastigote forms of Trypanosoma cruzi and represent a 2–4% of total cruzipain. They were purified by affinity chromatography on Cystatin–Sepharose, recognized by the polyclonal anti-cruzipain serum, and their activity in gelatin-containing gels was completely abolished by E-64, TLCK, leupeptin, and aprotinin but not by PMSF, pepstatin A, EDTA or 1,10-phenantroline. These cysteine proteinases, as well as cruzipain showed to be endoproteinases able to hydrolize azocasein, hemoglobin, and bovine serum albumin at acidic pHs. However, evidences are presented indicating that this subset of cruzipain isoforms were also able to use the same blocked chromogenic peptidyl substrates than cruzipain at similar optimal alkaline pH values although with a different order of preference. Moreover, they showed a different oligosaccharide pattern after enzymatic treatment by high pH anion exchange chromatography, suggesting that this structural difference may account for the atypical behaviour in the lectin columm. Index Descriptors and Abbreviations: Trypanosoma cruzi; Cysteine proteinases; Cruzipain. CPs, Cysteine proteinases; Cr, cruzipain; C-T, C-terminal extension; HPLC, high performance liquid chromatography; SDS–PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; ConA, concanavalin A; NACrI, non-adsorbed cruzipain isoforms; DEAE, diethylaminoethyl; FPLC, fast protein liquid chromatography; TCA, trichloroacetic acid; PMSF, phenyl methyl sulfonyl fluoride; TLCK, tosyl lisyl chloromethyl ketone; E-64, trans-epoxysuccinyl- l-leucylamido-(4-guanidine) butane; Bz, Benzoyl; CBZ-, N-carbobenzoxy; BOC, N- tert-butoxycarbonyl; OMe, Methoxy-; p-NA, p-nitroanilide; β-NAm, β-naphthylamide; BSA, bovine serum albumin; EndoH, EndoH-β- N-acetylglycosaminidase; PNGase F, Protein N-glycanase F; HPAEC-PAD, high pH anion exchange chromatography with pulse amperometric detection

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