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Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes

Published online by Cambridge University Press:  30 July 2002

E. S. COIMBRA ,
S. C. GONÇALVES-DA-COSTA ,
S. CORTE-REAL ,
F. G. R. DE FREITAS ,
A. C. DURÃO ,
C. S. F. SOUZA ,
M. I. SILVA-SANTOS  and
E. G. VASCONCELOS
E. S. COIMBRA
Affiliation:
Laboratório de Imunomodulação, Departamento de Protozoologia, Instituto Oswaldo Cruz – FIOCRUZ – C.P. 926, 21045–900 Rio de Janeiro, RJ, Brasil
S. C. GONÇALVES-DA-COSTA
Affiliation:
Laboratório de Imunomodulação, Departamento de Protozoologia, Instituto Oswaldo Cruz – FIOCRUZ – C.P. 926, 21045–900 Rio de Janeiro, RJ, Brasil
S. CORTE-REAL
Affiliation:
Laboratório de Biologia Celular, Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz – FIOCRUZ – C.P. 926, 21045–900 Rio de Janeiro, RJ, Brasil
F. G. R. DE FREITAS
Affiliation:
Departamento de Bioquímica, ICB, Universidade Federal de Juiz de Fora, 36015-400 Juiz de Fora, MG, Brasil
A. C. DURÃO
Affiliation:
Departamento de Bioquímica, ICB, Universidade Federal de Juiz de Fora, 36015-400 Juiz de Fora, MG, Brasil
C. S. F. SOUZA
Affiliation:
Laboratório de Imunomodulação, Departamento de Protozoologia, Instituto Oswaldo Cruz – FIOCRUZ – C.P. 926, 21045–900 Rio de Janeiro, RJ, Brasil
M. I. SILVA-SANTOS
Affiliation:
Laboratório de Imunomodulação, Departamento de Protozoologia, Instituto Oswaldo Cruz – FIOCRUZ – C.P. 926, 21045–900 Rio de Janeiro, RJ, Brasil
E. G. VASCONCELOS
Affiliation:
Departamento de Bioquímica, ICB, Universidade Federal de Juiz de Fora, 36015-400 Juiz de Fora, MG, Brasil
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Abstract

An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6·5–8·5 were examined using ATP or p-NPP as substrate. At pH 7·4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5–10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.

Keywords

ATP diphosphohydrolaseapyraseLeishmania amazonensispromastigote
Type
Research Article
Information
Parasitology , Volume 124 , Issue 2 , February 2002 , pp. 137 - 143
Copyright
2002 Cambridge University Press

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