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

Published online by Cambridge University Press:  06 April 2009

N. Healy
Affiliation:
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
S. Greig
Affiliation:
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
H. Enahoro
Affiliation:
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
H. Roberts
Affiliation:
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
L. Drake
Affiliation:
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
E. Shaw
Affiliation:
Friedrich Miescher-Institut, Basle, Switzerland
F. Ashall
Affiliation:
Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK

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.

Type
Research Article
Copyright
Copyright © Cambridge University Press 1992

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