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Query: EC:3.6.3.1 (Mg2+-ATPase)
 1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transmembrane asymmetry has been extensively studied in eukaryotic cells. It is as yet only clearly demonstrated in the plasma membrane of a few cells. Subcellular organelles have evidence of lipid asymmetry, but very little consistent quantitative data exist. Proteins involved in transmembrane passage of lipids comprise enzymes of lipid metabolism and also the so-called phospholipid flippases that are either passive or active putative lipid transporters. The aminophospholipid translocase that pumps amino-phospholipids from the outer to the inner monolayer of the plasma membrane of eukaryotes is a Mg(2+)-ATP dependent protein with a high lipid selectivity. Lipid asymmetry provides an asymmetrical environment for membrane enzymes. Thus, PS (and PE) reorientation could be a way of controlling or triggering specific enzymes. Also, the asymmetrical distribution of phospholipids most likely determines the fusion-competent membranes and/or which sides of membranes should fuse. Finally, the lipid pump as well as all enzymes responsible for the net transmembrane flux of phospholipids may provide the driving force for membrane bending, notably during the formation of endocytic vesicles. Clearly, real progress in this area will be made only if the proteins of the flippase family are purified and antibodies obtained that will permit the recognition and localization of these proteins in various cells. Also, specific inhibitors as well as mutants would allow one to infer more directly what are the real functions of these proteins. At a late stage, the protein purification will eventually permit speculation on the mechanism of action of a pump that must transport simultaneously hydrophilic and hydrophobic groups through a membrane.
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PMID:Protein involvement in transmembrane lipid asymmetry. 152 72

The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport of phospholipid takes place in some organelles such as chromaffin granules. On the other hand, the phospholipid flippase of rat liver endoplasmic reticulum does not require ATP and has a low lipid specificity. The biological implications of these phospholipid flippases are discussed.
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PMID:Control of the transmembrane phospholipid distribution in eukaryotic cells by aminophospholipid translocase. 228 6

The Ca2+-dependent binding of annexin proteins to secretory granule membranes seems to be involved in the early stage of exocytosis. Binding studies have shown that these proteins have a specificity for phosphatidylserine (PtdS) interfaces. Furthermore, aminolipids are necessary for contact and fusion between lipid vesicles or between liposomes and chromaffin granules. Thus, PtdS must be present on the granule outer (cytoplasmic) monolayer. We report here that chromaffin granules possess a mechanism to maintain PtdS orientation, comparable to the ATP-dependent aminophospholipid translocase from human erythrocytes. The translocase, in granules, selectively transports PtdS from the luminal to the cytoplasmic monolayer, provided the incubation medium contains ATP. As this protein shares several properties with the granule vanadate-sensitive ATPase II, we infer that this ATPase, of relative molecular mass 115,000, is the protein responsible for aminophospholipid translocation. This is the first evidence for an ATP-dependent specific phospholipid 'flippase' in intracellular organelles.
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PMID:Control of transmembrane lipid asymmetry in chromaffin granules by an ATP-dependent protein. 254 8

Erythrocyte membrane Mg(2+)-ATPase activity was stimulated by echinocytogenic agents (2,4-dinitrophenol and salicylate), a stomatocytogenic agent Triton X-100 and other membrane-disturbing agents including hydrophobic organic anions, alcohols and detergents. Various possible mechanisms of the stimulation are possible but apparently most probable one consists in induction of membrane phospholipid scrambling by the compounds studied (as demonstrated for DNP) and of aminophospholipid translocase (flippase) activity.
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PMID:Stimulation of erythrocyte membrane Mg(2+)-ATPase activity by dinitrophenol and other membrane-disturbing agents. 783 29

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na(+)-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-D-glucose and oligomycin. In cells dialyzed in the presence of ATP, no "run-down" was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at approximately 5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or "flippase.". The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATP gamma S were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In contrast, GTP gamma S was ineffective. We conclude that ATP is the only soluble factor necessary for optimal activity of the NHE-1 isoform of the antiporter. Mg2+ does not appear to be essential for the stimulatory effect of ATP. We propose that two mechanisms mediate the activation of the antiporter by ATP: one requires hydrolysis and is likely an energy-dependent event. The second process does not involve hydrolysis of the gamma-phosphate, excluding mediation by protein or lipid kinases. We suggest that this effect is due to binding of ATP to an as yet unidentified, nondiffusible effector that activates the antiporter.
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PMID:ATP dependence of Na+/H+ exchange. Nucleotide specificity and assessment of the role of phospholipids. 904 42

Uncouplers of mitochondrial oxidative phosphorylation, dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), were found to stimulate Mg(2+)-ATPase activity of human erythrocyte membranes in a manner competitive with respect to 2,4-dinitrophenyl-S-glutathione (DNP-SG) which suggested that these compounds may also be substrates of the glutathione-S-conjugate pump. We confirm that the stimulation of erythrocyte membrane ATPase activity by DNP and by another uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), is competitive with respect to DNP-SG. However, we found no evidence for active transport of DNP and CCCP out of erythrocytes and demonstrate that they inhibit the low-affinity component of DNP-SG transport noncompetitively while stimulating the high-affinity DNP-SG transport (mediated by multidrug resistance-associated protein, MRP1). Implications of these findings may indicate the electrogenic nature of MRP1-mediated transport of glutathione-S conjugates and stimulation of aminophospholipid translocase (flippase) rather than the glutathione-S-conjugate pump by the uncouplers.
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PMID:Uncouplers of mitochondrial oxidative phosphorylation are not substrates of the erythrocyte glutathione-S-conjugate pump. 943 89

ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.
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PMID:Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function. 1060 36

The appearance of phosphatidylserine (PS) on the outer surface of red cells is an important signal for their uptake by macrophages. We report for the first time that procaspase 3 present in the anucleated mature human erythrocyte is activated under oxidative stress induced by t-butylhydroperoxide leading to impairment of the aminophospholipid translocase, PS externalization and increased erythrophagocytosis. This is the first report linking caspase 3 activation to inhibition of flippase activity and uptake of red cells by macrophages.
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PMID:Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes. 1190 47

ATPase II (a Mg2+-ATPase) is also believed to harbor aminophospholipid translocase (APTL) activity, which is responsible for the translocation of phosphatidylserine (PS) from the outer leaflet of the plasma membrane to the inner. To test this hypothesis we overexpressed the mouse ATPase II cDNA in the neuronal HN2 cells. In addition to a dramatic increase in APTL activity, we also made the unexpected observation that expression of the mouse ATPase II cDNA from the vector pCMV6 resulted in the appearance of calcium current. Although the hybrid cell line HN2 or a line (HN2V32) obtained by expressing a heterologous gene from the same expression vector showed no calcium current, both ATPase II-overexpressing clones (HN2A12 and HN2A22) showed significant barium conductance. This current was due to calcium channels because it was blocked almost completely by 100 microM CdCl2 and it had a significant N-type component since it was blocked by 38.5% in the presence of 5 microM omega-conotoxin (omega-CTX). Western blot analysis using an antibody against the N-type calcium-channel alpha1B subunit revealed a dramatic increase in expression of this protein in the HN2A12 and HN2A22 cell lines. Our results suggest that ATPase II also harbors APTL activity. In view of the prior knowledge that APTL activity is inhibited by an increase in calcium, our results also suggest that APTL expression exerts a negative feedback regulation on itself by inducing expression of channels that cause an influx of calcium ions. The mechanism of this regulation could reveal important information on a possible cross-regulation between these two families of proteins in neuronal cells.
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PMID:Appearance of voltage-gated calcium channels following overexpression of ATPase II cDNA in neuronal HN2 cells. 1455 44

Cytotoxicity by unconjugated bilirubin involves disturbances of membrane structure, excitotoxicity and cell death. These events were reported to trigger elevated free radicals production and impairment of calcium homeostasis, and to result in loss of cell membrane integrity. Therefore, this study was designed to investigate whether interaction of clinically relevant concentrations of free unconjugated bilirubin with synaptosomal membrane vesicles could be linked to oxidative stress, cytosolic calcium accumulation and perturbation of membrane function. Synaptosomal vesicles were prepared from gerbil cortical brain tissue and incubated with purified bilirubin (<or=1 microM), for 4 h at 37 degrees C. Intracellular concentrations of reactive oxygen species (ROS) and calcium were determined by dichlorofluorescin and BAPTA fluorescent probes, respectively. Membrane protein and lipid oxidation were evaluated by immunocytochemistry and phosphatidylserine exposure by annexin V binding. Levels of reduced and oxidized glutathione (GSH and GSSG, respectively), as well as activities of Mg(2+)-ATPase aminophospholipid translocase (flippase) and Na(+),K(+)-ATPase, were also measured. Our results showed that bilirubin induced oxidative stress, due to a rise in lipid (>or=10%, P<0.05) and protein oxidation (>or=20%, P<0.01), ROS content (approximately 17%, P<0.01), and a decrease in GSH/GSSG ratio (>30%, P<0.01). In addition, synaptosomes exposed to bilirubin exhibited increased externalization of phosphatidylserine (approximately 10%, P<0.05), together with decreased flippase and NA(+),K(+)-ATPase (>or=15%, P<0.05) activities, events that were accompanied by enhanced intracellular calcium levels ( approximately 20%, P<0.01). The data obtained point out that interaction of unconjugated bilirubin with synaptosomal membrane vesicles leads to oxidative injury, loss of membrane asymmetry and functionality, and calcium intrusion, thus potentially contributing to the pathogenesis of encephalopathy by hyperbilirubinemia.
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PMID:A link between hyperbilirubinemia, oxidative stress and injury to neocortical synaptosomes. 1547 95


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