Trypanosoma brucei brucei: effects of ferrous iron and heme on ecto-nucleoside triphosphate diphosphohydrolase activity.

Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.

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.