title: A Mg(2+)-dependent ecto-phosphatase activity on the external surface of Trypanosoma rangeli modulated by exogenous inorganic phosphate.
abstract: In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).
authors.affiliation: Laboratório de Bioquímica Celular, Instituto de Bioquímica Medica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, UFRJ, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, Brazil.
authors.forename: André L
authors.initials: AL
authors.lastname: Fonseca-de-Souza
authors.forename: Claudia Fernanda
authors.initials: CF
authors.lastname: Dick
authors.forename: André Luiz Araújo
authors.initials: AL
authors.lastname: Dos Santos
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Meyer-Fernandes
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003470:Culture Media; D015971:Gene Expression Regulation, Enzymologic; D007287:Inorganic Chemicals / Q000494:pharmacology*; D008274:Magnesium / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects* / Q000378:metabolism; D013379:Substrate Specificity; D014345:Trypanosoma / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development
chemical_list: D003470:Culture Media; D007287:Inorganic Chemicals; D010710:Phosphates; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium; D002118:Calcium