Toxoplasma gondii tachyzoites were fractionated by modification of an
iodixanol density gradient method previously used for acidocalcisome isolation from Trypanosoma cruzi epimastigotes. Fractions were characterized using electron microscopy, x-ray microanalysis, and enzymatic markers, and it was demonstrated that the heaviest (pellet) fraction contains electron-dense vacuoles rich in
phosphorus,
calcium, and
magnesium, as found before for acidocalcisomes. Staining with
4',6-diamidino-2-phenylindole (
DAPI) indicated that poly-
phosphate (
polyP) was preferentially localized in this fraction together with
pyrophosphate (PP(i)). Using an
enzyme-based method, millimolar levels (in terms of P(i) residues) of
polyP chains of less than 50 residues long and micromolar levels in
polyP chains of about 700-800 residues long were found to be preferentially localized in this fraction. The fraction also contained the
pyrophosphatase and
polyphosphatase activities characteristic of acidocalcisomes. Western blot analysis using
antibodies against
proteins from micronemes, dense granules, rhoptries, and plasma membrane showed that the acidocalcisomal fraction was not contaminated by these other organelles. T. gondii
polyP levels rapidly decreased upon exposure of the parasites to a
calcium ionophore (
ionomycin), to an inhibitor of the V-
H(+)-ATPase (
bafilomycin A(1)), or to the
alkalinizing agent NH(4)Cl. These changes were in parallel to an increase in intracellular Ca(2+) concentration, suggesting a close association between
polyP hydrolysis and Ca(2+) release from the acidocalcisome. These results provide a useful method for the isolation and characterization of acidocalcisomes, showing that they are distinct from other previously recognized organelles present in T. gondii, and provide evidence for the role of
polyP metabolism in response to cellular stress.