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Trypanosoma cruzi: Isolation and characterization of aspartyl proteases |
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| Authors: | Rosa T. Pinho Carlos R. Alves Salvatore G. De-Simone |
| Affiliation: | a Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Imunologia Clínica, Av. Brasil 4365, 21040-900, Rio de Janeiro, RJ, Brazil b Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil c Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Bioquímica de Proteínas e Peptídeos, Av. Brasil 4365, 21040-900, Rio de Janeiro, RJ, Brazil d Universidade de São Paulo, Instituto de Física de São Carlos, Departamento de Física e Informática, Av. Trabalhador Sãocarlense 400, 13566-590, São Paulo, SP, Brazil e Universidade Federal Fluminense, Instituto de Biologia, Departamento de Biologia Celular e Molecular, Outeiro São João Batista S/N, 24030-000, Niterói, RJ, Brazil |
| Abstract: | Two aspartyl proteases activities were identified and isolated from Trypanosoma cruzi epimastigotes: cruzipsin-I (CZP-I) and cruzipsin-II (CZP-II). One was isolated from a soluble fraction (CZP-II) and the other was solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CZP-I). The molecular mass of both proteases was estimated to be 120 kDa by HPLC gel filtration and the activity of the enzymes was detected in a doublet of bands (56 and 48 kDa) by substrate-sodium dodecyl sulphate-polyacrylamide-gelatin gel electrophoresis. Substrate specificity studies indicated that the enzymes consistently hydrolyze the cathepsin D substrate Phe-Ala-Ala-Phe (4-NO2)-Phe-Val-Leu-O4MP but failed to hydrolyze serine and other protease substrates. Both proteases activities were strongly inhibited by the classic inhibitor pepstatin-A (?68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (?80%). These findings show that both proteases are novel T. cruzi acidic proteases. The physiological function of these enzymes in T. cruzi has under investigation. |
| Keywords: | Trypanosoma cruzi Proteases Cathepsin D Pepstatin-A Chromatographic |
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