Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates
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- 作者：Elide E. Caroselli ; Diego M. Assis ; Clara L. Barbi¨¦ri ; Wagner A.S. J¨²dice ; Maria A. Juliano ; Marcos L. Gazarini ; Luiz Juliano
- 关键词：DTT ; 1 ; 4-dithiothreitol ; MCA ; [7-amino-4-methyl] coumarin ; E64 ; 1-[l-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane ; Abz ; ortho-aminobenzoic acid ; EDDnp[ ; N-(2 ; 4-dinitrophenyl) ethylenediamine] ; TLCK ; tosyl-lysine chloromethyl ketone ; FRET ; fluores
- 刊名：Molecular and Biochemical Parasitology
- 出版年：2012
- 出版时间：August, 2012
- 年：2012
- 卷：184
- 期：2
- 页码：82-89
- 全文大小：1097 K
文摘
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 fluorophore 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 RA 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.