Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica

In this work, we describe the ability of living cells of Entamoeba histolyt ica to hydrolyze extracellular ATP. In these intact parasites, whose viabil ity was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol P-i/h/10(5) cells). Inter estingly, in the presence of 5 mM MgCl2 an ecto-ATPase activity of 300 nmol P-i/h/10(5) 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 a t least 1 h. The ecto-ATPase activity was also stimulated by MnCl2 and CaCl 2 but not by SrCl2, ZnCl2, or FeCl3. In fact, FeCl3 inhibited both Mg2+-dep endent and Mg2+-independent ecto-ATPase activities. The Mg2+-independent AT Pase activity was unaffected by pH in the range between 6.4 and 8.4, in whi ch the cells were viable. However, the Mg2+-dependent ATPase activity was e nhanced concomitantly with the increase in pH. In order to discard the poss ibility that the ATP hydrolysis observed was due to phosphatase or 5'-nucle otidase activities, several inhibitors for these enzymes were tested. Sodiu m orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg2+ (basal activity), the apparent K-m for ATP(4-) was 0.053 +/- 0.008 mM, whereas at saturating MgCl2 concentrations, the corresponding apparent K-m for Mg-ATP(2-) for Mg2 +-dependent ecto-ATPase activity (difference between total and basal ecto-A TPase activity) was 0.503 mM +/- 0.062. Both ecto-ATPase activities were hi ghly 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 P-2 purinoreceptors and an in hibitor of some ecto-ATPases, as well as the impermeant agent 4'-4'-diisoth iocyanostylbenzene-2'-2'-disulfonic acid. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different exte nts, and the inhibition by both agents was prevented by ATP. A comparison a mong the ecto-ATPase activities of three amoeba species showed that the non invasive E. histolytica and the free-living E. moshkovskii were less effici ent than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytic a is known to have a galactose-specific lectin on its surface, which is rel ated to the pathogenesis of amebiasis, galactose was tested for an effect a n ecto-ATPase activities. It stimulated the Mg2+-dependent ecto-ATPase but not the Mg2+-independent ATPase activity. (C) 2000 Academic Press.