Specific calpain activity evaluation in Plasmodium parasites

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Gomes, Mayrim Machado [UNIFESP]
Budu, Alexandre [UNIFESP]
Ventura, Priscilla Dantas de Souza [UNIFESP]
Bagnaresi, Piero [UNIFESP]
Cotrin, Simone Silva [UNIFESP]
Cunha, Rodrigo Luiz Oliveira Rodrigues
Carmona, Adriana Karaoglanovic [UNIFESP]
Juliano, Luiz [UNIFESP]
Gazarini, Marcos Leoni [UNIFESP]
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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-t-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. (C) 2014 Elsevier Inc. All rights reserved.
Analytical Biochemistry. San Diego: Academic Press Inc Elsevier Science, v. 468, p. 22-27, 2015.