Leishmania plasma membrane Mg2+-ATPase is a H+/K+-antiporter involved in glucose symport - Studies with sealed ghosts and vesicles of opposite polarity

Experiments from other laboratories conducted with Leishmania donovani prom astigote cells had earlier indicated that the plasma membrane Mg2+-ATPase o f the parasite is an extrusion pump for H+. Taking advantage of the pellicu lar microtubular structure of the plasma membrane of the organism, we repor t procedures for obtaining sealed ghost and sealed everted vesicle of defin ed polarity. Rapid influx of H+ into everted vesicles was found to be depen dent on the simultaneous presence of ATP (1 mM) and Mg2+ (1 mM). Excellent correspondence between rate of H+ entry and the enzyme activity clearly dem onstrated the Mg2+-ATPase to be a true H+ pump. HC entry into everted vesic le was strongly inhibited by SCH28080 (IC50 = similar to 40 muM) and by ome prazole (IC50 = similar to 50 muM), both of which are characteristic inhibi tors of mammalian gastric H+,K+ -ATPase. H+ influx was completely insensiti ve to ouabain (250 muM), the typical inhibitor of Na+,K+-ATPase. Mg2(+)-ATP ase activity could be partially stimulated with Kt (20 mM) that was inhibit able (>85%) with SCH28080 (50 Cat). ATP-dependent rapid efflux of Rb-86(+) from preloaded vesicles was completely inhibited by preincubation with omep razole (150 muM) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mM), an inh ibitor of the enzyme. Assuming Rb+ to be a true surrogate for Kt, an ATP-de pendent, electroneutral stoichiometric exchange of Hf and K+ (1:1) was esta blished. Rapid and 10-fold active accumulation of [U-C-14]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior a lkaline) was imposed. Rapid efflux of [U-14C]D-glucose from preloaded evert ed vesicles could also be initiated by activating the enzyme, with ATP. Tak en together, the plasma membrane Mg2(+)-ATPase has been identified as an el ectroneutral H+/K+ antiporter with some properties reminiscent of the gastr ic H+,K+- ATPase. This enzyme is possibly involved in active accumulation o f glucose via a H+-glucose symport system and in KC accumulation.