Vacuolar proton pyrophosphatase activity and pyrophosphate (PP(i)) in Toxoplasma gondii as possible chemotherapeutic targets

Claudia O. Rodrigues, David A. Scott, Brian N. Bailey, Wanderley De Souza, Marlene Benchimol, Ben Moreno, Julio A. Urbina, Eric Oldfield, Silvia N.J. Moreno

Research output: Contribution to journalArticlepeer-review

57 Scopus citations

Abstract

The addition of PP(i) promoted the acidification of a subcellular compartment in cell homogenates of Toxoplasma gondii tachyzoites, implying the presence of a proton-translocating pyrophosphatase. The proton gradient was collapsed by addition of the K+/H+ antiporter nigericin, and was also inhibited by addition of the PP(i) analogue aminomethylenediphosphonate (AMDP). Both proton transport and PP(i) hydrolysis were dependent upon K-, but Na+ caused partial inhibition of these activities. PP(i) hydrolysis was sensitive in a dose-dependent manner to AMDP, imidodiphosphate, NaF and to the thiol reagent N-ethylmaleimide. This activity was unaffected by common inhibitors of phosphohydrolases, except that NaO3V (sodium orthovanadate) stimulated the activity by 87%. Immunofluorescence microscopy, using antisera raised against conserved peptide sequences of a plant vacuolar pyrophosphatase, suggested that the pyrophosphatase in T. gondii tachyzoites was located in the plasma membrane and intracellular vacuoles of the parasite. High-field 31P-NMR spectroscopy showed that PP(i) was more abundant than ATP in tachyzoites. Bisphosphonates (PP(i) analogues), drugs that are used in the treatment of bone diseases, inhibited proton transport and PP(i) hydrolysis in tachyzoite homogenates, and also inhibited intracellular proliferation of tachyzoites in tissue culture cells.

Original languageEnglish (US)
Pages (from-to)737-745
Number of pages9
JournalBiochemical Journal
Volume349
Issue number3
DOIs
StatePublished - Aug 1 2000
Externally publishedYes

Keywords

  • P NMR
  • Acidocalcisome
  • Apicomplexan
  • Bisphosphonates

ASJC Scopus subject areas

  • Biochemistry

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