Leishmania amazonensis: Characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70 ± 1.17 nmol Pi × h−1 × 10−7 cells. The dependence on p-NPP concentration shows a normal Michaelis–Menten kinetics for this ecto-phosphatase activity present a Vmax of 31.93 ± 3.04 nmol Pi × h−1 × 10−7 cells and apparent Km of 1.78 ± 0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the Ki value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the Ki values of 0.33 μM, 0.36 μM and 0.25 μM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with Ki 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1 mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.