Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Detalhes bibliográficos
Autor(a) principal: Morgado-Díaz,José Andrés
Data de Publicação: 2005
Outros Autores: Silva-Lopez,Raquel Elisa da, Alves,Carlos Roberto, Soares,Maurilio José, Corte-Real,Suzana, De Simone,Salvatore Giovanni
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Memórias do Instituto Oswaldo Cruz
Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762005000400007
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.
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spelling Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotesLeishmaniaserine proteasesubcellular localizationtrypanosomatidHere 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.Instituto Oswaldo Cruz, Ministério da Saúde2005-07-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762005000400007Memórias do Instituto Oswaldo Cruz v.100 n.4 2005reponame:Memórias do Instituto Oswaldo Cruzinstname:Fundação Oswaldo Cruzinstacron:FIOCRUZ10.1590/S0074-02762005000400007info:eu-repo/semantics/openAccessMorgado-Díaz,José AndrésSilva-Lopez,Raquel Elisa daAlves,Carlos RobertoSoares,Maurilio JoséCorte-Real,SuzanaDe Simone,Salvatore Giovannieng2020-04-25T17:49:24Zhttp://www.scielo.br/oai/scielo-oai.php0074-02761678-8060opendoar:null2020-04-26 02:13:10.909Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruztrue
dc.title.none.fl_str_mv Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
title Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
spellingShingle Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
Morgado-Díaz,José Andrés
Leishmania
serine protease
subcellular localization
trypanosomatid
title_short Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
title_full Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
title_fullStr Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
title_full_unstemmed Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
title_sort Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes
author Morgado-Díaz,José Andrés
author_facet Morgado-Díaz,José Andrés
Silva-Lopez,Raquel Elisa da
Alves,Carlos Roberto
Soares,Maurilio José
Corte-Real,Suzana
De Simone,Salvatore Giovanni
author_role author
author2 Silva-Lopez,Raquel Elisa da
Alves,Carlos Roberto
Soares,Maurilio José
Corte-Real,Suzana
De Simone,Salvatore Giovanni
author2_role author
author
author
author
author
dc.contributor.author.fl_str_mv Morgado-Díaz,José Andrés
Silva-Lopez,Raquel Elisa da
Alves,Carlos Roberto
Soares,Maurilio José
Corte-Real,Suzana
De Simone,Salvatore Giovanni
dc.subject.por.fl_str_mv Leishmania
serine protease
subcellular localization
trypanosomatid
topic Leishmania
serine protease
subcellular localization
trypanosomatid
dc.description.none.fl_txt_mv 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.
description 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.
publishDate 2005
dc.date.none.fl_str_mv 2005-07-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762005000400007
url http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762005000400007
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S0074-02762005000400007
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Instituto Oswaldo Cruz, Ministério da Saúde
publisher.none.fl_str_mv Instituto Oswaldo Cruz, Ministério da Saúde
dc.source.none.fl_str_mv Memórias do Instituto Oswaldo Cruz v.100 n.4 2005
reponame:Memórias do Instituto Oswaldo Cruz
instname:Fundação Oswaldo Cruz
instacron:FIOCRUZ
reponame_str Memórias do Instituto Oswaldo Cruz
collection Memórias do Instituto Oswaldo Cruz
instname_str Fundação Oswaldo Cruz
instacron_str FIOCRUZ
institution FIOCRUZ
repository.name.fl_str_mv Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruz
repository.mail.fl_str_mv
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