Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi.
A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.
Instituto de Investigaciones Bioquimicas Fundación Campomar, Universidad de Buenos Aires-CONICET, Argentina.
J J
JJ
Cazzulo
M C
MC
Cazzulo Franke
J
J
Martínez
B M
BM
Franke de Cazzulo
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D002863:Chromogenic Compounds / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D002364:Caseins; D002863:Chromogenic Compounds; D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; C026018:azocasein; D003546:Cysteine Endopeptidases; C063214:cruzipain; D007930:Leucine; C024974:E 64