Trypanosoma cruzi: Isolation and characterization of aspartyl proteases

• Published in: Experimental parasitology Vol. 122; no. 2; pp. 128 - 133
• Main Authors: Pinho, Rosa T., Beltramini, Leila M., Alves, Carlos R., De-Simone, Salvatore G.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 01.06.2009; Elsevier
• Subjects: Animals; Aspartic Acid Endopeptidases - antagonists & inhibitors; Aspartic Acid Endopeptidases - chemistry; Aspartic Acid Endopeptidases - isolation & purification; Aspartic Acid Endopeptidases - metabolism; Biological and medical sciences; Cathepsin D; Chromatographic; Chromatography, High Pressure Liquid; Chromogenic Compounds - metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Life cycle. Host-agent relationship. Pathogenesis; Molecular Weight; Pepstatin-A; Proteases; Protozoa; Substrate Specificity; Trypanosoma cruzi; Trypanosoma cruzi - enzymology; Cathepsin D; Chromatographic; Proteases; Pepstatin-A; Trypanosoma cruzi; Kinetoplastida; Protozoa; Enzyme; Parasite; Peptidases; Pepstatin; Hydrolases; Isolation; Aspartic endopeptidases
• ISSN: 0014-4894; 1090-2449
• DOI: 10.1016/j.exppara.2009.02.005

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-NO 2)-Phe-Val-Leu-O 4MP 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.