Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity.
In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.
Fonseca
Fábio Vasconcelos
FV
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21541-590, Rio de Janeiro, RJ, Brazil.
Fonseca de Souza
André Luíz
AL
Mariano
Ana Claudia
AC
Entringer
Peter F
PF
Gondim
Katia C
KC
Meyer-Fernandes
José Roberto
JR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015703:Antigens, CD / Q000187:drug effects / Q000378:metabolism*; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015636:Magnesium Chloride / Q000494:pharmacology*; D013498:Suramin / Q000494:pharmacology; D014344:Trypanocidal Agents / Q000494:pharmacology; D014345:Trypanosoma / Q000201:enzymology*
D015703:Antigens, CD; D014344:Trypanocidal Agents; D015636:Magnesium Chloride; D013498:Suramin; D000255:Adenosine Triphosphate; D001081:Apyrase; C067869:CD39 antigen; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid