A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms

Abstract

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 5 mM 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 5 mM 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.