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 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.
Mukherjee
T
T
Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India.
Mandal
D
D
Bhaduri
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002414:Cations, Monovalent / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007893:Leishmania donovani / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D027961:Potassium-Hydrogen Antiporters; D012413:Rubidium / Q000378:metabolism
D017920:Antiporters; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; D007093:Imidazoles; D027961:Potassium-Hydrogen Antiporters; C035235:Sch 28080; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D005947:Glucose; D012413:Rubidium; D011188:Potassium