pyrophosphate and iodixanol

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PP

Topics: Calcium Signaling; Cell Fractionation; Centrifugation, Density Gradient; Diphosphates; Enzyme Assays; Hydrogen-Ion Concentration; Microscopy, Electron; Organelles; Polyphosphates; Protozoan Proteins; Triiodobenzoic Acids; Trypanosoma brucei brucei

Acidocalcisomes are acidic, calcium storage compartments with a H(+) pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens. In this work, we report that the H(+)-pyrophosphatase (H(+)-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H(+)-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane. polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light. Taken together, these results suggest that the H(+)-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes.

Topics: Bacterial Chromatophores; Blotting, Western; Calcium; Cell Membrane; Cellular Structures; Diphosphates; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Inorganic Pyrophosphatase; Magnetic Resonance Spectroscopy; Microscopy, Confocal; Microscopy, Electron; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Microscopy, Immunoelectron; Perchlorates; Polyphosphates; Protons; Pyrophosphatases; Rhodospirillum rubrum; Triiodobenzoic Acids; 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