A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible rolein adenosine acquisition and virulence

The plasma membrane of cells contains enzymes whose active sites face the e xternal medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using living cells. In this wor k we describe the ability of living promastigotes of Leishmania amazonensis to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of an y divalent metal (5.39 +/- 0.71 nmol P-i/h x 10(7) cells). The ATP hydrolys is was stimulated by MgCl2 and the Rig-dependent ecto-ATPase activity was 3 0.75 +/- 2.64 nmol P-i/h x 10(7) cells. The Mg-dependent ecto-ATPase activi ty was linear with cell density and with time for at least 60 min. The addi tion of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydr olysis was obtained with 1.21 mM MgCl2. This stimulatory activity was also observed when MgCl2 was replaced by MnCl2, but not by CaCl2 or SrCl2. The a pparent K-m for Mg-ATP(2-) was 0.98 mM and free Mg2+ did not increase the e cto-ATPase activity. In the pH range from 6.8 to 8.4, in which the cells we re viable, the acid phosphatase activity decreased, while the Mg2+-dependen t ATPase activity increased. This ecto-ATPase activity was insensitive to i nhibitors of other ATPase and phosphatase activities, such as oligomycin, s odium azide, bafilomycin A(1), ouabain, furosemide, vanadate, molybdate, so dium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, 4,4'-diisothioc yanostylbene 2',2'-disulfonic acid as well as suramin, an antagonist of P-2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inh ibited the Mg2+-dependent ATPase activity in a dose dependent manner. A com parison between the Mg2+-dependent ATPase activity of virulent and avirulen t promastigotes showed that avirulent promastigotes were less efficient tha n the virulent promastigotes in hydrolyzing ATP. (C) 2001 Academic Press.