Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica.
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 P(i)/h/10(5) cells). Interestingly, in the presence of 5 mM MgCl(2) an ecto-ATPase activity of 300 nmol P(i)/h/10(5) cells was observed. The addition of MgCl(2) 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 MgCl(2). Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2) but not by SrCl(2), ZnCl(2), or FeCl(3). In fact, FeCl(3) inhibited both Mg(2+)-dependent and Mg(2+)-independent ecto-ATPase activities. The Mg(2+)-independent ATPase activity was unaffected by pH in the range between 6.4 and 8. 4, in which the cells were viable. However, the Mg(2+)-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 Mg(2+) (basal activity), the apparent K(m) for ATP(4-) was 0.053 +/- 0.008 mM, whereas at saturating MgCl(2) concentrations, the corresponding apparent K(m) for Mg-ATP(2-) for Mg(2+)-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 P(2) 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 Mg(2+)-independent and the Mg(2+)-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 Mg(2+)-dependent ecto-ATPase but not the Mg(2+)-independent ATPase activity.
Barros
F S
FS
Departamento de Bioquímica Médica, Instituto de Biofísica Carlos Chagas Filho, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, RJ, Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundã.
De Menezes
L F
LF
Pinheiro
A A
AA
Silva
E F
EF
Lopes
A H
AH
De Souza
W
W
Meyer-Fernandes
J R
JR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D009597:4-Nitrophenylphosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004747:Entamoeba / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004748:Entamoeba histolytica / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000472:pathogenicity; D004789:Enzyme Activation / Q000187:drug effects; D005690:Galactose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013498:Suramin / Q000494:pharmacology
D002413:Cations, Divalent; D013498:Suramin; D000255:Adenosine Triphosphate; D009597:4-Nitrophenylphosphatase; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D008274:Magnesium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D005690:Galactose