No abstract

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable of hydrolyzing nucleoside di- and triphosphates. Although a number of possible physiological roles have been proposed for ATPDases, detailed studies on structure-function relationships have generally been hampered by the lack of specific inhibitors of these enzymes. We have previously characterized a Ca2+-activated ATPDase on the external surface of the tegument of Schistosoma mansoni, the etiologic agent of human schistosomiasis. In the present work, we have examined the effects of thapsigargin, a sesquiterpene lactone known as a high affinity inhibitor of sarco-endoplasmic reticulum calcium transport (SERCA) ATPase, on ATPDase activity. Whereas other lactones tested had little or no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (K(i) approximately 20 microM). Interestingly, hydrolysis of ADP was not inhibited by thapsigargin. The lack of inhibition of ATPase activity by orthovanadate, a specific inhibitor of P-type ATPases, and the inhibition of the Mg2+-stimulated ATP hydrolysis by thapsigargin ruled out the possibility that the observed inhibition of the ATPDase by thapsigargin could be due to the presence of contaminating SERCA ATPases in our preparation. Kinetic analysis indicated that a single active site in the ATPDase is responsible for hydrolysis of both ATP and ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigargin was little or not affected by changes in free Ca2+ or Mg2+ concentrations. These results suggest that interaction of thapsigargin with the S. mansoni ATPDase prevents binding of ATP or its hydrolysis at the active site, while ADP can still undergo catalysis.

Hydrolysis of ATP or ADP catalyzed by the ATP diphosphohydrolase of Schistosoma mansoni tegument was measured in the presence of different cations. ATP diphosphohydrolase was stimulated by micromolar concentrations of either Ca 2ϩ or Mg 2ϩ , Ca 2ϩ producing threefold higher maximal activities than Mg 2ϩ . Kinetic studies indicated that Ca 2ϩ and Mg 2ϩ compete for the same binding site on the enzyme. The effect of covalent labeling of ATP diphosphohydrolase with the ATP analog fluorosulfonylbenzoyl adenosine (FSO 2BzAdo) was studied. Schistosome tegument was passed through with Sephadex G-50 filtration centrifugation columns to remove endogenous nucleotides, and this was followed by labeling with FSO 2 BzAdo. Incubation of ATP diphosphohydrolase with 1 mM FSO 2 BzAdo for 1 h inhibited ATPase or ADPase activities by 60% and 50%, respectively. Addition of ATP together with FSO 2BzAdo provided greater than 90% protection against FSO 2 BzAdo inactivation, indicating that FSO 2 BzAdo binds to an ATP-binding site on the ATP diphosphohydrolase. Furthermore, addition of FSO 2 BzAdo to a medium containing intact worms caused 30% and 50% inhibition of ATPase and ADPase activities, respectively, indicating that the ATP-binding site of diphosphohydrolase is accessible to FSO 2 BzAdo from the external surface of S. mansoni worms.

No abstract

The mechanism by which adenosine (Ado) and 2-cloroadenosine (Cl-Ado) inhibit platelet aggregation is not clear. In order to get some insight into the mode of action of these compounds, we studied the effect of Cl-Ado on the uptake of Ado by intact platelets, the effect of these compounds on the endogenous phosphorylation of specific plasma membrane proteins, and its effect on the carboxymethylation pattern of plasma membrane proteins in intact platelets. Cl-Ado does not modify the uptake of Ado by intact platelets, nor is itself incorporated into the platelet’s pool of nucleotides. Phosphorylation of plasma membrane proteins is not affected by Cl-Ado; however, Ado produces a selective increase in the phosphorylation of one plasma membrane component of glycoproteic nature. As has been reported, phosphorylation of this glycoprotein is also modulated by cAMP (BBA, 455:371, 1976). Although the electrophoretic pattern of carboxymethylated plasma membranes is unaffected by Ado or Cl-Ado, it was found that the former markedly increases the label of all the susceptible proteins, while Cl-Ado selectively protects a single membrane component. Electrophoretically, this component seems to be related to the above mentioned glycoprotein. The results reported suggest that Ado and Cl-Ado interact with different components of the plasma membrane, impairing platelet aggregation through different mechanisms. In the case of Ado, two ways seem operative: a) A cAMP-like stimulation of a specific membrane glycoprotein and b) A more general perturbation of the membrane structure, perhaps through an Ado-carrier complex (Acta Med. Scand. 525:169, 1971). Cl-Ado seems to interact solely on the external surface of the plasma membrane, suggesting that the transmembrane phospho-glycoprotein previously described is in some way closely related to the ADP-receptor of the platelet plasma membrane.