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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 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), 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 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) 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-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]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.
Highlights
Kala-azar or visceral leishmaniasis is a major public health problem in many parts of the tropical and subtropical world
Extensive cytological studies had earlier shown that these microtubular beads are closely associated with the inner lamina of the plasma membrane of intact cells [28]. This arrangement provides scaffolding and rigidity for the membrane structure and can be very conveniently used for determining polarity of closed structures. This is evident from the thin section electron photomicrograph of a typical sealed ghost that has an array of microtubules at regular intervals in the inner side of the ghost (Fig. 1A)
Taking advantage of the comparative rigidity provided by the pellicular microtubular structure to the plasma membrane of L. donovani, we could quite conveniently prepare sealed ghosts and everted vesicles of defined polarity that are free from cytoplasmic marker enzymes and internal metabolites (Fig. 1 and Methods)
Summary
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؉. 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. The functional capability, if any, of the Mg2ϩ-ATPase to act as an ion translocator needs to be demonstrated clearly and unambiguously This is necessary to analyze the related problem of energy coupling in glucose transport across the plasma membrane [10]. In this paper, using sealed ghosts and vesicles of opposite polarity, we demonstrate that the plasma membrane Mg2ϩ-ATPase of Leishmania is an electroneutral Hϩ/Kϩ antiporter. The second role is important in view of the uncertain status of Naϩ,Kϩ-ATPase in this organism, as will be discussed later
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