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Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
Morgado-Díaz, José Andrés; Silva-Lopez, Raquel Elisa da; Alves, Carlos Roberto; Soares, Maurilio José; Corte-Real, Suzana; De Simone, Salvatore Giovanni.
  • Morgado-Díaz, José Andrés; Instituto Nacional de Câncer. Centro de Pesquisas. Divisão de Biologia Celular. Grupo de Biologia Estrutural. Rio de Janeiro. BR
  • Silva-Lopez, Raquel Elisa da; Departamento de Bioquímica e Biologia Molecular. Laboratório de Bioquímica de Proteínas e Peptídios. Rio de Janeiro. BR
  • Alves, Carlos Roberto; Departamento de Bioquímica e Biologia Molecular. Laboratório de Bioquímica de Proteínas e Peptídios. Rio de Janeiro. BR
  • Soares, Maurilio José; Instituto Oswaldo Cruz-Fiocruz. Departamento de Ultra-estrutura e Biologia Celular. Rio de Janeiro. BR
  • Corte-Real, Suzana; Instituto Oswaldo Cruz-Fiocruz. Departamento de Ultra-estrutura e Biologia Celular. Rio de Janeiro. BR
  • De Simone, Salvatore Giovanni; Universidade Federal Fluminense. Instituto de Biologia. Departamento de Biologia Celular e Molecular. Niterói. BR
Mem. Inst. Oswaldo Cruz ; 100(4): 377-383, July 2005. ilus, tab
Article Dans Anglais | LILACS | ID: lil-405992
RESUMO
Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.
Sujets)
Texte intégral: Disponible Indice: LILAS (Amériques) Sujet Principal: Leishmania mexicana / Serine endopeptidases Limites du sujet: Animaux langue: Anglais Texte intégral: Mem. Inst. Oswaldo Cruz Thème du journal: Médecine tropicale / Parasitologie Année: 2005 Type: Article Pays d'affiliation: Brésil Institution/Pays d'affiliation: Departamento de Bioquímica e Biologia Molecular/BR / Instituto Nacional de Câncer/BR / Instituto Oswaldo Cruz-Fiocruz/BR / Universidade Federal Fluminense/BR

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Texte intégral: Disponible Indice: LILAS (Amériques) Sujet Principal: Leishmania mexicana / Serine endopeptidases Limites du sujet: Animaux langue: Anglais Texte intégral: Mem. Inst. Oswaldo Cruz Thème du journal: Médecine tropicale / Parasitologie Année: 2005 Type: Article Pays d'affiliation: Brésil Institution/Pays d'affiliation: Departamento de Bioquímica e Biologia Molecular/BR / Instituto Nacional de Câncer/BR / Instituto Oswaldo Cruz-Fiocruz/BR / Universidade Federal Fluminense/BR