Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates

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Caroselli, Élide Escolástico [UNIFESP]
Assis, Diego Magno [UNIFESP]
Barbiéri, Clara Lúcia [UNIFESP]
Júdice, Wagner Alves de Souza [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Gazarini, Marcos Leoni [UNIFESP]
Juliano, Luiz [UNIFESP]
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In this study we investigated the peptidase activity in Leishmania (L) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fiuorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. it was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA. Z-RR-MCA) or inhibitors (E64. TLCK) containing positively charged groups. the peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. the effects of temperature and different salts were also included in this study. the hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. the pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at R A bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite. (C) 2012 Elsevier B.V. All rights reserved.
Molecular and Biochemical Parasitology. Amsterdam: Elsevier B.V., v. 184, n. 2, p. 82-89, 2012.