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

Abstract

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg²⁺-ATPase of the parasite is an extrusion pump for H⁺. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H⁺into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg²⁺ (1 mm). Excellent correspondence between rate of H⁺ entry and the enzyme activity clearly demonstrated the Mg²⁺-ATPase to be a true H⁺ pump. H⁺ entry into everted vesicle was strongly inhibited by SCH28080 (IC₅₀ = ~40 μm) and by omeprazole (IC₅₀ = ~50 μm), both of which are characteristic inhibitors of mammalian gastric H⁺,K⁺-ATPase. H⁺ influx was completely insensitive to ouabain (250 μm), the typical inhibitor of Na⁺,K⁺-ATPase. Mg²⁺-ATPase activity could be partially stimulated with K⁺ (20 mm) that was inhibitable (>85%) with SCH28080 (50 μm). ATP-dependent rapid efflux of ⁸⁶Rb⁺ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 μm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb⁺ to be a true surrogate for K⁺, an ATP-dependent, electroneutral stoichiometric exchange of H⁺ and K⁺ (1:1) was established. Rapid and 10-fold active accumulation of [U-¹⁴C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-¹⁴C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg²⁺-ATPase has been identified as an electroneutral H⁺/K⁺ antiporter with some properties reminiscent of the gastric H⁺,K⁺-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H⁺-glucose symport system and in K⁺accumulation.