title: Identification and characterization of an ecto-pyrophosphatase activity in intact epimastigotes of Trypanosoma rangeli.
abstract: In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H(+)-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H(+)-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl2. In the presence of MgCl2, this activity was inhibited by millimolar concentrations of CaCl2. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.
authors.affiliation: Laboratório de Terapia e Fisiologia Celular e Molecular, Centro Universitário Estadual da Zona Oeste, Rio de Janeiro, RJ, Brazil.
authors.forename: André Luiz
authors.initials: AL
authors.lastname: Fonseca-de-Souza
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ, Brazil.
authors.forename: Anita Leocadio
authors.initials: AL
authors.lastname: Freitas-Mesquita
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ, Brazil.
authors.forename: Lisvane Paes
authors.initials: LP
authors.lastname: Vieira
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: David
authors.initials: D
authors.lastname: Majerowicz
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Nathalia
authors.initials: N
authors.lastname: Daflon-Yunes
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ, Brazil; Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Lia Carolina Almeida
authors.initials: LC
authors.lastname: Soares-de-Medeiros
authors.affiliation: Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ, Brazil; Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Kildare
authors.initials: K
authors.lastname: Miranda
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, RJ, Brazil.
authors.forename: Katia Calp
authors.initials: KC
authors.lastname: Gondim
authors.affiliation: Institutode Bioquímica Médica Leopoldo de Meis, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ, Brazil.
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Meyer-Fernandes
mesh_terms: D049109:Cell Proliferation; D011756:Diphosphates / Q000378:metabolism; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000378:metabolism*; D008018:Life Cycle Stages; D057509:Trypanosoma rangeli / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development*
chemical_list: D011756:Diphosphates; D043564:Inorganic Pyrophosphatase