Inhibition of the Ecto-ATPdiphosphohydrolase of Schistosoma mansoni by Thapsigargin

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.

Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides

“…Our data show that, as for other apyrases, the SmATPDase1 enzyme is not intrinsically inhibitable by thapsigargin in a dose dependent manner. The inhibition reported (Martins, Torres & Ferreira, 2000) is likely related to the use of tegument preparations rather than recombinant enzyme.…”

“…The fact that SmATPDase1 is a calcium-activated plasma membrane-bound enzyme again confirms it as a member of the apyrase family. Earlier substrate competition experiments (ATP versus ADP) involving schistosome tegument extracts, as well as comparative heat inactivation profiles for ATP versus ADP hydrolytic activities using these extracts, led to the hypothesis that a single enzyme in the tegument was responsible for degrading both ATP and ADP (Martins, Torres & Ferreira, 2000; Vasconcelos et al, 1993). Our work confirms this hypothesis.…”

“…One difference is apparent, however; the ATPase activity of the tegument preparation (but not its ADPase activity) has been reported to be inhibited by thapsigargin in a dose dependent manner (Martins, Torres & Ferreira, 2000). Inhibition of ∼70% was seen with 100 µM thapsigargin (Martins, Torres & Ferreira, 2000).…”

“…One difference is apparent, however; the ATPase activity of the tegument preparation (but not its ADPase activity) has been reported to be inhibited by thapsigargin in a dose dependent manner (Martins, Torres & Ferreira, 2000). Inhibition of ∼70% was seen with 100 µM thapsigargin (Martins, Torres & Ferreira, 2000). This finding was a surprise since thapsigargin is best known as a specific inhibitor of sarco/endoplasmic reticulum Ca ++ (SERCA) ATPases and not of apyrases (Rogers et al, 1995).…”

Peer Review #1 of "Schistosome tegumental ecto-apyrase (SmATPDase1) degrades exogenous pro-inflammatory and pro-thrombotic nucleotides (v0.1)"

“…The fact that SmATPDase1 is a calcium-activated plasma membranebound enzyme again confirms it as a member of the apyrase family. Earlier substrate competition experiments (ATP versus ADP) involving schistosome tegument extracts, as well as comparative heat inactivation profiles for ATP versus ADP hydrolytic activities using these extracts, led to the hypothesis that a single enzyme in the tegument was responsible for degrading both ATP and ADP (Martins et al 2000;Vasconcelos et al 1993). Our work confirms this hypothesis.…”