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 x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) 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 K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 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 1mM 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.
de Almeida-Amaral
Elmo Eduardo
EE
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro-UFRJ, CCS, Bloco H sala H2-013, Cidade Universitária, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brazil.
Belmont-Firpo
Rodrigo
R
Vannier-Santos
Marcos André
MA
Meyer-Fernandes
José Roberto
JR
D000818:Animals; D002712:Chlorides / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D007894:Leishmania mexicana / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D016774:Leishmaniasis, Diffuse Cutaneous / Q000469:parasitology; D008982:Molybdenum / Q000494:pharmacology; D009596:Nitrophenols / Q000378:metabolism; D017711:Nonlinear Dynamics; D009942:Organometallic Compounds / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012044:Regression Analysis; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology*; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D009596:Nitrophenols; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D010618:Phenanthrolines; D010710:Phosphates; D017967:Zinc Compounds; C008644:nitrophenylphosphate; D014638:Vanadates; C102120:bisperoxo(1,10-phenanthroline)oxovanadate(1-); D008982:Molybdenum; C016837:zinc chloride; D010744:Phosphoric Monoester Hydrolases; D005978:Glutathione; D003545:Cysteine; C022175:ammonium molybdate