title: Trypanosoma cruzi: isolation and characterization of aspartyl proteases.
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 (> or =68%) and the aspartic active site labeling agent, 1,2-epoxy-3-(phenyl-nitrophenoxy) propane (> or =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.
authors.affiliation: Fundação Oswaldo Cruz, Instituto Oswaldo Cruz, Laboratório de Imunologia Clínica, Av. Brasil 4365, Rio de Janeiro, RJ 21040-900, Brazil.
authors.forename: Rosa T
authors.initials: RT
authors.lastname: Pinho
authors.forename: Leila M
authors.initials: LM
authors.lastname: Beltramini
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Alves
authors.forename: Salvatore G
authors.initials: SG
authors.lastname: De-Simone
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002863:Chromogenic Compounds / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
chemical_list: D002863:Chromogenic Compounds; D004791:Enzyme Inhibitors; D016282:Aspartic Acid Endopeptidases