Entamoeba histolytica: ouabain-insensitive Na(+)-ATPase activity.
ABSTRACT Our aim was to determine the presence of sodium pumps in Entamoeba histolytica. It is shown through the measurement of ouabain-sensitive ATPase activity and immunoblotting that E. histolytica does not express (Na(+)+K(+))ATPase. On the other hand, we observed a Na(+)-ATPase with the following characteristics: (1) stimulated by Na(+) or K(+), but these effects are not addictive; (2) the apparent affinity is similar for Na(+) and K(+) (K(0.5) = 13.3 +/- 3.7 and 15.4 +/- 3.1mM, respectively), as well as the V(max) (24.9 +/- 1.5 or 27.5 +/- 1.6 nmol Pi mg(-1)min(-1), respectively); (3) insensitive up to 2mM ouabain; and (4) inhibited by furosemide with an IC(50) of 0.12 +/- 0.004 mM. Furthermore, this enzyme forms a Na(+)- or K(+)-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate.
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ABSTRACT: The K(+) and Na(+) concentrations in living cells are strictly regulated at almost constant concentrations, high for K(+) and low for Na(+). Because these concentrations correspond to influx-efflux steady states, K(+) and Na(+) effluxes and the transporters involved play a central role in the physiology of cells, especially in environments with high Na(+) concentrations where a high Na(+) influx may be the rule. In eukaryotic cells two P-type ATPases are crucial in these homeostatic processes, the Na,K-ATPase of animal cells and the H(+)-ATPase of fungi and plants. In fungi, a third P-type ATPase, the ENA ATPase, was discovered nineteen years ago. Although for many years it was considered to be exclusively a fungal enzyme, it is now known to be present in bryophytes and protozoa. Structurally, the ENA (from exitus natru: exit of sodium) ATPase is very similar to the sarco/endoplasmic reticulum Ca(2+) (SERCA) ATPase, and it probably exchanges Na(+) (or K(+)) for H(+). The same exchange is mediated by Na(+) (or K(+))/H(+) antiporters. However, in eukaryotic cells these antiporters are electroneutral and their function depends on a DeltapH across the plasma membrane. Therefore, the current notion is that the ENA ATPase is necessary at high external pH values, where the antiporters cannot mediate uphill Na(+) efflux. This occurs in some fungal environments and at some points of protozoa parasitic cycles, which makes the ENA ATPase a possible target for controlling fungal and protozoan parasites. Another technological application of the ENA ATPase is the improvement of salt tolerance in flowering plants.Biochimica et Biophysica Acta 10/2010; 1798(10):1841-53. DOI:10.1016/j.bbamem.2010.07.009 · 4.66 Impact Factor
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ABSTRACT: The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na(+) influx, causing [Na(+)] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na(+)] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na(+) against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na(+)-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na(+) homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na(+) regulation. Taken together, our results are consistent with PfATP4 being a Na(+) efflux ATPase and a target of the spiroindolones.Cell host & microbe 02/2013; 13(2):227-37. DOI:10.1016/j.chom.2012.12.006 · 13.02 Impact Factor
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ABSTRACT: Transepithelial Na(+) transport is mediated by passive Na(+) entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na(+)/K(+) pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na(+)/K(+)-ATPase and the ouabain-insensitive, furosemide-inhibitable Na(+)-ATPase, respectively. Over the last 40 years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na(+)-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na(+)- and Na(+)/K(+)-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na(+)-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na(+)-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na(+)-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na(+) transport and cellular Na(+) homeostasis leads us to reconsider its role in health and disease.Pflügers Archiv - European Journal of Physiology 04/2012; 463(6):755-77. DOI:10.1007/s00424-012-1101-3 · 4.87 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.