Ca2+ transport in isolated mitochondrial vesicles from Leishmania braziliensis promastigotes.

Leishmania braziliensis maintained very low (50 +/- 20 nM) intracellular concentrations of calcium ions under normal conditions, as shown by the fluorimetric indicator QUIN2. Digitonin-permeabilized cells liberated large amounts of calcium ions in the presence of the ionophore A23187, indicating the presence of a large intracellular reservoir for this ion. Given the extraordinary extension of the single giant mitochondrion of Kinetoplastida and the known capacity of mitochondria from other sources to accumulate calcium, we tested the capacity of this organelle to accumulate calcium ions in Leishmania. Coupled mitochondrial vesicles, five-fold enriched in succinate-cytochrome c oxidoreductase, were obtained from promastigotes by gentle grinding (45 s) with glass beads in hypertonic buffer solution, followed by differential centrifugation. These vesicles had a respiratory control ratio of 1.82 +/- 0.15, and two phosphorylation sites (sites II and III) using succinate as electron donor, and were capable of calcium uptake in the presence of several respiratory substrates; this uptake was enhanced in the presence of ADP and Pi and was blocked by classical electron transport inhibitors. Uncouplers such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and the calcium ionophore A23187 released previously accumulated calcium ions, suggesting that the driving force for the calcium uptake by the vesicles is the respiratory generated electrochemical potential gradient of protons. A study of the affinity of this system for calcium showed that even at 90 microM free calcium, succinate-induced calcium uptake is not saturated while approaching a level of 200 nmol min-1 (mg protein)-1, indicating a low-affinity, large-capacity system.(ABSTRACT TRUNCATED AT 250 WORDS)