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 × 10−7 cells × h−1). The ATP hydrolysis was stimulated by MgCl2 and the Mg-dependent ecto-ATPase activity was 27.15 ± 2.91 nmol Pi × 10−7 cells × h−1. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2. CaCl2 and ZnCl2 were also able to stimulate the ATPase activity, although less than MgCl2. The apparent Km 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 P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-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.