Characterization of an ecto-ATPase of Tritrichomonas foetus

Abstract

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 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46 mM 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 50 mM 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.