Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets.
نویسندگان
چکیده
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 NaO(3)V (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 (31)P-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.
منابع مشابه
Proton pumping inorganic pyrophosphatase of endoplasmic reticulum-enriched vesicles from etiolated mung bean seedlings.
Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata) were successfully isolated using Ficoll gradient and two-phase (polyethylene glycol-dextran) partition. The ER-enriched vesicles contained inorganic pyrophosphate (PPi) hydrolysis and its associated proton translocating activities. Antiserum prepared against vacuolar H+-pyrophosphatase ...
متن کاملKinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors.
The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H(+)-PPase) from oat (Avena sativa L.) roots to changes in Mg(2+) and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indi...
متن کاملContribution of PPi-Hydrolyzing Function of Vacuolar H+-Pyrophosphatase in Vegetative Growth of Arabidopsis: Evidenced by Expression of Uncoupling Mutated Enzymes
The vacuolar-type H(+)-pyrophosphatase (H(+)-PPase) catalyzes a coupled reaction of pyrophosphate (PPi) hydrolysis and active proton translocation across the tonoplast. Overexpression of H(+)-PPase improves growth in various plant species, and loss-of-function mutants (fugu5s) of H(+)-PPase in Arabidopsis thaliana have post-germinative developmental defects. Here, to further clarify the physiol...
متن کاملHeterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.
The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H(+)-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H(+)-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H(+)-pyrophosphat...
متن کاملA vacuolar-H(+) -pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii.
The vacuolar proton pyrophosphatase (H(+) -PPase) of Toxoplasma gondii (TgVP1), a membrane proton pump, localizes to acidocalcisomes and a novel lysosome-like compartment termed plant-like vacuole (PLV) or vacuolar compartment (VAC). We report the characterization of a T. gondii null mutant for the TgVP1 gene. Propagation of these mutants decreased significantly because of deficient attachment ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The Biochemical journal
دوره 349 Pt 3 شماره
صفحات -
تاریخ انتشار 2000