title: Specific calpain activity evaluation in Plasmodium parasites.
abstract: In the intraerythrocytic trophozoite stages of Plasmodium falciparum, the calcium-dependent cysteine protease calpain (Pf-calpain) has an important role in the parasite calcium modulation and cell development. We established specific conditions to follow by confocal microscopy and spectrofluorimetry measurements the intracellular activity of Pf-calpain in live cells. The catalytic activity was measured using the fluorogenic Z-Phe-Arg-MCA (where Z is carbobenzoxy and MCA is 4-methylcoumaryl-7-amide). The calmodulin inhibitor calmidazolium and the sarcoplasmic reticulum calcium ATPase inhibitor thapsigargin were used for modifications in the cytosolic calcium concentrations that persisted in the absence of extracellular calcium. The observed calcium-dependent peptidase activity was greatly inhibited by specific cysteine protease inhibitor E-64 and by the selective calpain inhibitor ALLN (N-acetyl-l-leucyl-l-leucyl-l-norleucinal). Taken together, we observed that intracellular Pf-calpain can be selectively detected and is the main calcium-dependent protease in the intraerythrocytic stages of the parasite. The method described here can be helpful in cell metabolism studies and antimalarial drug screening.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, São Paulo, SP, Brazil; Departamento de Biociências, Universidade Federal de São Paulo, Santos, SP, Brazil.
authors.forename: Mayrim M
authors.initials: MM
authors.lastname: Gomes
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Budu
authors.affiliation: Departamento de Biociências, Universidade Federal de São Paulo, Santos, SP, Brazil.
authors.forename: Priscilla D S
authors.initials: PD
authors.lastname: Ventura
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Piero
authors.initials: P
authors.lastname: Bagnaresi
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Simone S
authors.initials: SS
authors.lastname: Cotrin
authors.affiliation: Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, SP, Brazil.
authors.forename: Rodrigo L O R
authors.initials: RL
authors.lastname: Cunha
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Departamento de Biociências, Universidade Federal de São Paulo, Santos, SP, Brazil. Electronic address: marcos.gazarini@unifesp.br.
authors.forename: Marcos L
authors.initials: ML
authors.lastname: Gazarini
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D016788:Plasmodium chabaudi / Q000201:enzymology*; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D015800:Protozoan Proteins; C063261:acetylleucyl-leucyl-norleucinal; D002154:Calpain; D002118:Calcium
keywords: Calcium signaling; Calmodulin; Calpain; Plasmodium; Proteases