Labelling of four distinct trophozoite falcipains of Plasmodium falciparum by a cystatin-derived probe.
Trophozoite cysteine protease (TCP) activity, isolated from Plasmodium falciparum soluble 100,000 g extracts, displayed native falcipain-1 kinetic parameters towards peptidyl substrates. The labelling of either isolated TCP or soluble 100,000 g extracts by a cystatin-derived probe (biotinyl-Leu-Val-Gly-CHN2) revealed a single band of ca. 30 kDa by SDS-PAGE, which was resolved into four spots displaying isoelectric points (pI) from 4.7 to 5.3 after two-dimensional separation. The molecular mass and pI correspond to those of falcipain-3, falcipain-2, falcipain-2' and falcipain-1, respectively. The two central spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry as falcipain-2 and falcipain-2'. This activity-based probe represents a potential tool for profiling active falcipains in parasites.
Florent
Isabelle
I
Biologie Fonctionnelle des Protozoaires, Département Régulation, Dévelopement, Diversité Moléculaire, Muséum National d'Histoire Naturelle, 61, rue Buffon, F-75005 Paris, France. florent@mnhn.fr
Lecaille
Fabien
F
Montagne
Jean-Jacques
JJ
Gauthier
Francis
F
Schrével
Joseph
J
Lalmanach
Gilles
G
D000818:Animals; D015891:Cystatins / Q000032:analysis / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism*; D015335:Molecular Probes / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000254:growth & development*
D015891:Cystatins; D015335:Molecular Probes; D010446:Peptide Fragments; D003546:Cysteine Endopeptidases; C416224:falcipain 2; C443101:falcipain 3