title: Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates.
abstract: 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.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil.
authors.forename: Elide E
authors.initials: EE
authors.lastname: Caroselli
authors.forename: Diego M
authors.initials: DM
authors.lastname: Assis
authors.forename: Clara L
authors.initials: CL
authors.lastname: Barbiéri
authors.forename: Wagner A S
authors.initials: WA
authors.lastname: Júdice
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Marcos L
authors.initials: ML
authors.lastname: Gazarini
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002951:Citrates / Q000737:chemistry; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007891:Leishmania / Q000166:cytology / Q000201:enzymology*; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008647:Mesocricetus; D009842:Oligopeptides / Q000737:chemistry; D010436:Pepstatins / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D059748:Proteolysis; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D054712:Salinity; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D000077559:Sodium Citrate; D013431:Sulfates / Q000737:chemistry; D013696:Temperature; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology
chemical_list: D002951:Citrates; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D010436:Pepstatins; D015800:Protozoan Proteins; D015842:Serine Proteinase Inhibitors; D013431:Sulfates; C012036:sodium sulfate; D000077559:Sodium Citrate; D014107:Tosyllysine Chloromethyl Ketone; D010447:Peptide Hydrolases; D007930:Leucine; C024974:E 64; C031375:pepstatin