Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes

Inorganic pyrophosphate promoted the acidification of an intracellular comp artment in permeabilized procyclic trypomastigotes of Trypanosoma bruccei, as measured by acridine orange uptake. The proton gradient generated by pyr ophosphate was collapsed by addition of nigericin or NH4Cl. Pyrophosphate-d riven proton translocation was stimulated by potassium ions and inhibited b y KF, by the pyrophosphate analogs imidodiphosphate and aminomcthylenedipho sphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at conce ntrations similar to those that inhibit the plant vacuolar H+-pyrophosphata se (PPase), The proton translocation activity had a pH optimum around 7.5 a nd was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-19-diazole (10 mu M) and unaffected by bafilomycin A(1) (40 nM), concanamycin A (5 nM), sodiu m o-vanadate (500 mu M), oligomycin (1 mu M), N-ethylmaleimide (100 mu M), and KNO3. AMDP-sensitive pyrophosphate hydrolysis was detected in both proc yclic and bloodstream trypomastigotes. Measurements of acridine orange upta ke in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H+-ATPase, H+-PPase, an d (ADP-dependent) H+/Na+ antiport activity in the same compartment. Separat ion of bloodstream and procyclic trypomastigote extracts on Percoll gradien ts yielded fractions that contained H+-PPase (both stages) and N+/Na+ excha nger (procyclics) activities but lacked markers for mitochondria, glycosome s, and lysosomes. The organelles in these fractions were identified by elec tron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles), These results provide further evidence for the unique nature of acidocaleisomes in comparison with other, previously described, organelles.