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Leishmania Plasma Membrane Mg21-ATPase Is a H1/K1-Antiporter Involved in Glucose Symport

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

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Title Leishmania Plasma Membrane Mg21-ATPase Is a H1/K1-Antiporter Involved in Glucose Symport
 
Creator Mukherjee, Tanmoy
Mandal, Debjani
Bhaduri, Amar
 
Subject Chemistry
 
Description Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg21-ATPase of the parasite is an extrusion pump for H1. 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 H1 into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mM) and Mg21 (1 mM). Excellent correspondence between rate of H1 entry and the enzyme activity clearly demonstrated the Mg21-ATPase to be a true H1 pump. H1 entry into everted vesicle was strongly inhibited by SCH28080 (IC50 5 ;40 mM) and by omeprazole (IC50 5 ;50 mM), both of which are characteristic inhibitors of mammalian gastric H1,K1-ATPase. H1 influx was completely insensitive to ouabain (250 mM), the typical inhibitor of Na1,K1-ATPase. Mg21- ATPase activity could be partially stimulated with K1 (20 mM) that was inhibitable (>85%) with SCH28080 (50 mM). ATP-dependent rapid efflux of 86Rb1 from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 mM) and by 5,5*-dithiobis-(2- nitrobenzoic acid) (1 mM), an inhibitor of the enzyme. Assuming Rb1 to be a true surrogate for K1, an ATP-dependent, electroneutral stoichiometric exchange of H1 and K1 (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 Mg21-ATPase has been identified as an electroneutral H1/K1 antiporter with some properties reminiscent of the gastric H1,K1- ATPase. This enzyme is possibly involved in active accumulation of glucose via a H1-glucose symport system and in K1 accumulation.
 
Date 2001
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/896/1/JOURNAL_OF_BIOLOGICAL_CHEMISTRY%2C__276(_8)%2C__5563%2C2001[56].pdf
Mukherjee, Tanmoy and Mandal, Debjani and Bhaduri, Amar (2001) Leishmania Plasma Membrane Mg21-ATPase Is a H1/K1-Antiporter Involved in Glucose Symport. The Journal of Biological Chemistry, 276 (8). pp. 5563-5569.
 
Relation http://dx.doi.org/10.1074/jbc.M008469200
http://www.eprints.iicb.res.in/896/