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Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates

  • N. Healy (a1), S. Greig (a1), H. Enahoro (a1), H. Roberts (a1), L. Drake (a1), E. Shaw (a2) and F. Ashall (a1)...

Summary

Detergent extracts of Trypanosoma cruzi epimastigotes catalysed the hydrolysis of a range of amino-acyl and peptidyl p-nitro-anilides and aminomethylcoumarins. At least three enzymes were detected that cleave Z–Phe–Arg–MCA. Two of these were optimally active at alkaline pH, the other at pH 4·0. Of the two enzymes with alkaline pH optima, one was a cysteine peptidase and was unable to cleave Bz–Arg–MCA readily, whilst the other cleaved Bz–Arg–MCA and was inhibited by diisopropyl fluorophosphate. The acidic enzyme was similar to cathespin L of other eukayrotes with respect to its pH profile, substrate-specificity and inhibitor-sensitivity. Evidence was presented that epimastigotes contain a cysteine-type dipeptidyl aminopeptidase, one or more aminopeptidases, and a serine peptidase that cleaves Boc–Ala–Ala–pNA. Digitonin solubilization of the activities from cells supports the hypothesis that the cathespin L-like enzyme and the dipeptidyl aminopeptidase are lysosomal, whilst the Bz–Arg–MCA hydrolase, the aminopeptidases and the Boc–Ala–Ala–pNA serine peptidase are cytosolic.

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Ashall, F. (1990). Characterisation of an alkaline peptidase of Trypanosoma cruzi and other trypanosomatids. Molecular and Biochemical Parasitology 38, 7788.
Ashall, F., Angliker, H. & Shaw, E. (1990 a). Lysis of trypanosomes by peptidyl fluoromethyl ketones. Biochemical and Biophysical Research Communications 170, 923–9.
Ashall, F., Harris, D., Roberts, H., Healy, N. & Shaw, E. (1990 b). Substrate specificity and inhibitor sensitivity of a trypanosomatid alkaline peptidase. Biochimica et Biophysica Acta 1035, 293–9.
Avila, J. L., Perez-Kepp, R. & Bretana, A. (1983). A minimal medium for the cultivation of infective Trypanosoma cruzi epimastigotes. Journal of General Microbiology 129, 285–91.
Barrett, A. J. & Kirschke, H. (1981). Cathespin B, cathepsin H and cathepsin L. Methods in Enzymology 80, 535–61.
Bongertz, V. & Hungerer, K. D. (1978). Trypanosoma cruzi: isolation and characterisation of a protease. Experimental Parasitology 45, 818.
Bontempi, E., Franke De Cazzulo, B. M., Ruiz, A. M. & Cazzulo, J. J. (1984). Purification and properties of an acidic protease from epimastigotes of Trypanosoma cruzi. Comparative Biochemistry and Physiology 77B, 599604.
Bontempi, E., Martinez, J. & Cazzulo, J. J. (1989). Subcellular localization of a cysteine proteinase from Trypanosoma cruzi. Molecular and Biochemical Parasitology 33, 43–8.
Bradford, M. M. (1976). A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–54.
Cazzulo, J. J., Cazzulo Franke, M. C., Martinez, J. & Franke De Cazzulo, B. M. (1990). Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi. Biochimica et Biophysica Acta 1037, 186–91.
Davis, M. H. (1987). Hormonal regulation of dipeptidyl-aminopeptidase I activity in cultured human fibroblasts. Archives of Biochemistry and Biophysics 254, 498503.
Erlanger, B. F., Kokowsky, N. & Cohen, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics 95, 271–8.
Etges, R., Bouvier, J. & Bordier, C. (1986). The major surface protein of Leishmania promastigotes is a protease. Journal of Biological Chemistry 261, 9098–101.
Greig, S. & Ashall, F. (1990). Electrophoretic characterisation of Trypanosoma cruzi peptidases. Molecular and Biochemical Parasitology 39, 31–8.
Hui, K.-S. (1988). A novel dipeptidyl aminopeptidase in rat brain membranes. Journal of Biological Chemistry 263, 6613–18.
Itow, & Camargo, E. P. (1977). Proteolytic activities in cell extracts of Trypanosoma cruzi. Journal of Protozoology 24, 591–5.
Kam, C. M., Fujikawa, K. & Powers, J. C. (1988). Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants. Biochemistry 27, 2547–57.
Kirshke, H., Wikstrom, P. & Shaw, E. (1988). Active center differences between cathepsins L and B: the S1 binding region. FEBS Letters 228, 128–30.
Lonsdale-Eccles, J. D. & Grab, D. J. (1987). Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei. European Journal of Biochemistry 169, 467–75.
Piras, M. M., Henriquez, D. & Piras, R. (1985). The effect of proteolytic enzymes and protease inhibitors on the interaction Trypanosoma cruzi-fibroblasts. Molecular and Biochemical Parasitology 14, 151–63.
Rangel, H. A., Araujo, P. M. F., Repka, D. & Costa, M. G. (1981). Trypanosoma cruzi: isolation and characterisation of a proteinase. Experimental Parasitology 52, 199209.
Rauber, P., Angliker, H., Walker, B. & Shaw, E. (1986). The synthesis of peptidylfluoromethanes and their properties as inhibitors of serine proteinases and cysteine proteinases. The Biochemical Journal 239, 633–40.
Schechter, I. & Berger, A. (1967). On the size of the active site in proteases. I. Papain. Biochemical and Biophysical Research Communications 27, 157–62.
Silva, L. H. P. & Nussenzweig, V. (1963). Sobre uma cepa de Trypanosoma cruzi altamente virulenta para o camundongo branco. Folia Clinica Biologica 20, 191203.

Keywords

Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates

  • N. Healy (a1), S. Greig (a1), H. Enahoro (a1), H. Roberts (a1), L. Drake (a1), E. Shaw (a2) and F. Ashall (a1)...

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