Characterization and immunolocalization of an NTP diphosphohydrolase of Trypanosoma cruzi.
An ecto-NTP diphosphohydrolase (NTPDase) activity, insensitive to inhibitors of ATPases and phosphatases, was characterized on the surface of live Trypanosoma cruzi intact parasites. The enzyme exhibits broad substrate specificity, typical of NTPDases, and a high hydrolysis rate for GTP. A 2282 bp message encoding a full-length NTPDase was cloned by RT-PCR using epimastigote mRNA. A single protein was immunoprecipitated from [(35)S]methionine-labeled parasites using antibodies against Toxoplasma gondii NTPase I. This antibody localized an NTPDase on the external surface of all forms of T. cruzi, as seen by confocal immuno-fluorescence microscopy. The NTPDase could be part of the parasite's purine salvage pathway. Additionally, trypomastigotes (infective form) presented a 2:1 ATP/ADP hydrolysis ratio, while epimastigotes (non-infective form) presented a 1:1 ratio, suggesting a possible role for the NTPDase in the parasite's virulence mechanisms.
Fietto
Juliana L R
JL
Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, MG, Brazil.
DeMarco
Ricardo
R
Nascimento
Ivan P
IP
Castro
Ieso M
IM
Carvalho
Técia M U
TM
de Souza
Wanderley
W
Bahia
Maria T
MT
Alves
Maria J M
MJ
Verjovski-Almeida
Sergio
S
D000818:Animals; D003001:Cloning, Molecular; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011755:Pyrophosphatases / Q000032:analysis* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017421:Sequence Analysis; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development
D011755:Pyrophosphatases; C092064:ectonucleotide pyrophosphohydrolase