Characterization of an ecto-ATPase of Tritrichomonas foetus.
In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl(2) to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46mM MgCl(2). The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2), but not by SrCl(2). The Mg(2+)-dependent ATPase presents two apparent K(m) values for Mg-ATP(2-) (K(m1)=0.03 mM and K(m2)=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg(2+)-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg(2+)-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg(2+)-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2',2'-disulfonic acid (DIDS) 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. This ecto-ATPase was stimulated by more than 90% by 50mM D-galactose. Since previous results showed that D-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg(2+)-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.
Jesus
José B
JB
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21541-590, Rio de Janeiro, RJ, Brazil.
Lopes
Angela H C S
AH
Meyer-Fernandes
José R
JR
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000135:Acid Phosphatase / Q000037:antagonists & inhibitors; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D005690:Galactose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology*; D013379:Substrate Specificity; D013498:Suramin / Q000494:pharmacology; D016835:Tritrichomonas foetus / Q000187:drug effects / Q000201:enzymology* / Q000472:pathogenicity; D014344:Trypanocidal Agents / Q000494:pharmacology
D014344:Trypanocidal Agents; D015636:Magnesium Chloride; D013498:Suramin; D000255:Adenosine Triphosphate; D000135:Acid Phosphatase; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D005690:Galactose