Trypanosoma brucei brucei: biochemical characterization of ecto-nucleoside triphosphate diphosphohydrolase activities.
In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.
de Souza Leite
Milane
M
Instituto de Bioquímica Médica, CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ, Brazil.
Thomaz
Rachel
R
Fonseca
Fábio Vasconcelos
FV
Panizzutti
Rogério
R
Vercesi
Anibal E
AE
Meyer-Fernandes
José Roberto
JR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology; D013997:Time Factors; D014344:Trypanocidal Agents / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D017895:Manganese Compounds; D014344:Trypanocidal Agents; D017967:Zinc Compounds; D015636:Magnesium Chloride; D013498:Suramin; C016837:zinc chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D002122:Calcium Chloride; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C025340:manganese chloride