A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms.
In this work, we describe how living cells of Trypanosoma brucei procyclic forms were able to hydrolyze extracellular p-nitrophenylphosphate (pNPP). These intact parasites, which had their viability determined by motility and the Trypan blue method, presented a low level of pNPP hydrolysis in the absence of any divalent metal (0.72+/-0.07 nmol pNP/mg min). Interestingly, in the presence of 5mM MgCl(2), ectophosphatase activity of 1.91+/-0.21 nmol pNP/mg min was observed. The ectophosphatase activity was also stimulated by MnCl(2), CoCl(2) and CuCl(2) but not by CaCl(2) and CdCl(2) and was inhibited by ZnCl(2). The addition of Mg(2+), Mn(2+), Co(2+) and Cu(2+) to extracellular medium increased the ectophosphatase activity in a dose-dependent manner. At 5mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39+/-0.05 mM MgCl(2), 0.33+/-0.03 mM MnCl(2), 1.63+/-0.12 mM CoCl(2) and 2.04+/-0.33 mM CuCl(2). In the absence of any divalent metal (basal activity) the apparent K(m) for pNPP was 0.66+/-0.09 mM, while at saturating MgCl(2) concentrations the corresponding apparent K(m) for pNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.27+/-0.03 mM. The Mg(2+)-stimulated pNPP hydrolysis was strongly inhibited by ZnCl(2) and vanadate, while the metal-independent basal phosphatase activity was less inhibited by these phosphotyrosyl phosphatase inhibitors.
Fernandes
Eloise Cedro
EC
Centro de Genética, Biologia Molecular e Fitoquímica-Instituto, Agronomico de Campinas, 13075-630 Campinas, SP, Brazil.
Granjeiro
José Mauro
JM
Aoyama
Hiroshi
H
Fonseca
Fábio Vasconcelos
FV
Meyer-Fernandes
José Roberto
JR
Vercesi
Aníbal Eugênio
AE
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D002413:Cations, Divalent; D008670:Metals; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D003035:Cobalt; D008345:Manganese; D003300:Copper; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium