Detection of peptidases in Trypanosoma cruzi epimastigotes using chromogenic and fluorogenic substrates.
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 eukaryotes 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.
Healy
N
N
Department of Biology, Imperial College of Science, Technology and Medicine, London, UK.
Greig
S
S
Enahoro
H
H
Roberts
H
H
Drake
L
L
Shaw
E
E
Ashall
F
F
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000032:analysis / Q000378:metabolism; D000818:Animals; D002863:Chromogenic Compounds; D003374:Coumarins / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D002863:Chromogenic Compounds; D003374:Coumarins; D004151:Dipeptides; D005456:Fluorescent Dyes; D011480:Protease Inhibitors; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D010447:Peptide Hydrolases; D000626:Aminopeptidases