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dos Passos Lemos A, de Sa Pinheiro AA, de Berredo-Pinho M, Fonseca de Souza L, Motta CM, de Souza W, Meyer-Fernandes R: Ectonucleotide diphosphohydrolase activity in Crithidia deanei. Parasitol Res. 2002 Oct;88(10):905-11. Epub 2002 Jun 19.
In this work we describe the ability of living Crithidia deanei to hydrolyze extracellular ATP. In intact cells at pH 7.2, a low level of ATP hydrolysis was observed in the absence of any divalent metal (0.41+/-0.13 nmol P (i) h (-1) 10 (7) cells (-1)). The ATP hydrolysis was stimulated by MgCl (2) and the Mg (2+)-dependent ecto-ATPase activity was 4.05+/-0.17 nmol P (i) h (-1) 10 (7) cells (-1). Mg (2+)-dependent ecto-ATPase activity increased linearly with cell density and with time for at least 60 min. The addition of MgCl (2) to 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 0.93+/-0.26 mM MgCl (2). This stimulatory activity was also observed when MgCl (2) was replaced by MnCl (2), but not CaCl (2) or SrCl (2). The apparent K (m) for Mg-ATP (2-) was 0.26+/-0.03 mM. ATP was the best substrate for this enzyme; other nucleotides, such as ITP, GTP, UTP and CTP, produced lower reaction rates. In the pH range from 6.6 to 8.4, in which the cells were viable, the acid phosphatase activity also present in this cell decreased, while the Mg (2+)-dependent ATPase activity did not change. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A (1), ouabain, vanadate, molybdate, sodium fluoride and tartrate. To confirm that this Mg (2+)-dependent ATPase was an ecto-ATPase, we used the impermeant inhibitor 4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid 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. The cell surface location of the ATP-hydrolyzing site was also confirmed by cytochemical analysis. |
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