Ectonucleotide Diphosphohydrolase Activities in Entamoeba histolytica

Abstract

In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol Pi/h/105 cells). Interestingly, in the presence of 5 mM MgCl2 an ecto-ATPase activity of 300 nmol Pi/h/105 cells was observed. The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl2. Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2 but not by SrCl2, ZnCl2, or FeCl3. In fact, FeCl3 inhibited both Mg2+-dependent and Mg2+-independent ecto-ATPase activities. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.4 and 8.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5′-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg2+ (basal activity), the apparent Km for ATP4− was 0.053 ± 0.008 mM, whereas at saturating MgCl2 concentrations, the corresponding apparent Km for Mg-ATP2− for Mg2+-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM ± 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P2 purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4′-4′-diisothiocyanostylbenzene-2′-2′-disulfonic acid. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg2+-dependent ecto-ATPase but not the Mg2+-independent ATPase activity.