Characterization of isolated acidocalcisomes of Trypanosoma cruzi.
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.
Scott
D A
DA
Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA. d-scott1@uiuc.edu
Docampo
R
R
D000143:Acids; D000165:Acridine Orange / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D011756:Diphosphates / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007476:Ionophores; D018942:Macrolides; D008564:Membrane Potentials; D015388:Organelles / Q000378:metabolism*; D017494:Proton Pumps; D006180:Proton-Translocating ATPases / Q000032:analysis; D011758:Pyrroles / Q000494:pharmacology; D014283:Triiodobenzoic Acids; D014349:Trypanosoma cruzi / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases; D014638:Vanadates / Q000494:pharmacology
D000143:Acids; D000838:Anions; D000900:Anti-Bacterial Agents; D011756:Diphosphates; D007211:Indoles; D007476:Ionophores; D018942:Macrolides; D017494:Proton Pumps; D011758:Pyrroles; D014283:Triiodobenzoic Acids; C057620:bafilomycin A; D014638:Vanadates; C108328:cycloprodigiosin; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D000165:Acridine Orange; C044834:iodixanol; D002118:Calcium