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 MgCl2 to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 nM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46 nM MgCl2. The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2, but not by SrCl2. The Mg2+-dependent ATPasc 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 low reaction rate and ADP was not a substrate for this enzyme. The Mg2+-dependent ecto-ATPase activity was insensitive to inhibitors of other ATPasc and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A[, ouabain, furoscmide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60-70% of the Mg2+-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 Mg2+-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 Mg2+-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 Mg2+-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity. (C) 2002 Elsevier Science B.V. All rights reserved.