bafilomycin-a and iodixanol

The mass-dense granules of Dictyostelium discoideum were shown to contain large amounts of phosphorus, magnesium, and calcium, as determined by x-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation. The high phosphorus content was due to the presence of pyrophosphate and polyphosphate, which were also present in the contractile vacuoles. Both organelles also possessed a vacuolar H(+)-ATPase, an H(+)-pyrophosphatase, and a Ca(2+)-ATPase, as determined by biochemical methods or by immunofluorescence microscopy. The H(+)-pyrophosphatase activity of isolated mass-dense granules was stimulated by potassium ions and inhibited by the pyrophosphate analogs aminomethylenediphosphonate and imidodiphosphate and by KF and N-ethylmaleimide in a dose-dependent manner. The mass-dense granules and the contractile vacuole appeared to contact each other when the cells were submitted to hyposmotic stress. Acetazolamide inhibited the carbonic anhydrase activity of the contractile vacuoles and prolonged their contraction cycle in a dose-dependent manner. Similar effects were observed with the anion exchanger inhibitor 4,4' -diisothiocyanatodihydrostilbene-2, 2' -disulfonic acid and the vacuolar H(+)-ATPase inhibitor bafilomycin A(1). Together, these results suggest that the mass-dense granules of D. discoideum are homologous to the acidocalcisomes described in protozoan parasites and are linked to the function of the contractile vacuole.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acetazolamide; Animals; Anti-Bacterial Agents; Blotting, Western; Calcium; Carrier Proteins; Contractile Proteins; Cross-Linking Reagents; Dictyostelium; Diphosphonates; Dose-Response Relationship, Drug; Electrons; Enzyme Inhibitors; Ethylmaleimide; Hydrogen-Ion Concentration; Hydrolysis; Inorganic Pyrophosphatase; Ions; Macrolides; Magnesium; Microscopy, Confocal; Microscopy, Electron; Organelles; Osmosis; Phosphorus; Protein Binding; Pyrophosphatases; Stress, Physiological; Triiodobenzoic Acids; Vacuoles; X-Rays

The acidocalcisome is an acidic calcium store in trypanosomatids with a vacuolar-type proton-pumping pyrophosphatase (V-H(+)-PPase) located in its membrane. In this paper, we describe a new method using iodixanol density gradients for purification of the acidocalcisome from Trypanosoma cruzi epimastigotes. Pyrophosphatase assays indicated that the isolated organelle was at least 60-fold purified compared with the large organelle (10,000 x g) fraction. Assays for other organelles generally indicated no enrichment in the acidocalcisome fraction; glycosomes were concentrated 5-fold. Vanadate-sensitive ATP-driven Ca(2+) uptake (Ca(2+)-ATPase) activity was detectable in the isolated acidocalcisome, but ionophore experiments indicated that it was not acidic. However, when pyrophosphate was added, the organelle acidified, and the rate of Ca(2+) uptake increased. Use of the indicator Oxonol VI showed that V-H(+)-PPase activity generated a membrane potential. Use of sulfate or nitrate in place of chloride in the assay buffer did not affect V-H(+)-PPase activity, but there was less activity with gluconate. Organelle acidification was countered by the chloride/proton symport cycloprogidiosin. No vacuolar H(+)-ATPase activity was detectable in isolated acidocalcisomes. However, immunoblots showed the presence of at least a membrane-bound V-H(+)-ATPase subunit, while experiments employing permeabilized epimastigotes suggested that vacuolar H(+)-ATPase and V-H(+)-PPase activities are present in the same Ca(2+)-containing compartment.

Topics: Acids; Acridine Orange; Adenosine Triphosphate; Animals; Anions; Anti-Bacterial Agents; Biological Transport, Active; Calcium; Cell Fractionation; Diphosphates; Indoles; Ionophores; Macrolides; Membrane Potentials; Organelles; Proton Pumps; Proton-Translocating ATPases; Pyrroles; Triiodobenzoic Acids; Trypanosoma cruzi; Vacuolar Proton-Translocating ATPases; Vanadates