Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A Mg2(+)-ATPase-enriched fraction was obtained from solubilized human erythrocyte membranes by ammonium sulphate precipitation and anion-exchange chromatography. The solubilized enzyme, of apparent molecular weight 120 kDa, requires phosphatidylserine to be fully active. Phosphatidylethanolamine but not other anionic phospholipids can only partially restore the activity. The Mg-ATPase has a low affinity for Mg2(+)-ATP and is inhibited by fluoride, vanadate, vanadyl and calcium ions. From these characteristics, we infer that this Mg2(+)-ATPase is the same protein as the aminophospholipid translocase which regulates the membrane phospholipid transverse distribution in human erythrocytes by actively transporting aminophospholipids from the outer to the inner monolayer.
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PMID:Partial purification and characterization of the human erythrocyte Mg2(+)-ATPase. A candidate aminophospholipid translocase. 214 4

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

Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg2+)-dependent adenosine triphosphatase (Mg(2+)-ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg(2+)-ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg(2+)-ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg(2+)-ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase.
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PMID:Enhanced Mg(2+)-ATPase activity in ghosts from HS erythrocytes and in normal ghosts stripped of membrane skeletal proteins may reflect enhanced aminophospholipid translocase activity. 778 96

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidylcholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by these membranes.
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PMID:Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes. 797 87

The platelet procoagulant response involves an increase in surface-exposed phosphatidylserine, which allows binding and assembly of enzyme complexes of the coagulation pathway resulting in acceleration of the clotting process. This response essentially requires the presence of extracellular Ca2+, and varies in extent with the type of agonist used. In the present paper we demonstrate that the moderate procoagulant response of human platelets caused by thrombin is strongly amplified by the presence of thapsigargin, an inhibitor of the microsomal Ca(2+)-ATPase. Thapsigargin, like thrombin, has only a weak effect on procoagulant activity. The large increase in procoagulant activity observed with the combined action of these two agonists is associated with increased shedding of microvesicles from the platelet plasma membrane as well as with inhibition of transport of a fluorescent-labeled analog of phosphatidylserine from the outer to the inner leaflet of the plasma membrane by the aminophospholipid translocase. The latter two observations support current concepts regarding the mechanism of development of procoagulant activity. Although the synergistic effect of thapsigargin on thrombin-induced procoagulant activity is at least in part due to the high levels of intracellular [Ca2+] evoked by these agonists, the data clearly indicate that a rise of the intracellular [Ca2+] is insufficient to completely explain this response. The present findings suggest that additional factors control expression of procoagulant activity upon stimulation of platelets by thrombin.
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PMID:Thapsigargin amplifies the platelet procoagulant response caused by thrombin. 816 95

Phospholipids are normally asymmetrically distributed between leaflets of the plasma membrane, due to the activity of aminophospholipid translocase (APT), a putative plasma membrane Mg2(+)-ATPase which is thought to selectively transport phosphatidylserine (PS) and other aminophospholipids from outer to inner membrane leaflet. Although several candidate proteins have been proposed to serve this function, positive identification awaits demonstration of their capacity to restore APT activity to a cell line that is deficient in this process. This study describes a simple and rapid protocol for the production and selection of mutant cell lines that are defective in APT activity and suitable for expression cloning of cDNAs coding for candidate APT enzymes. By flow cytometry, we demonstrate the time-dependent uptake of NBD-labeled PS, but not phosphatidylcholine (PC), by the mouse fibroblast cell line SV-T2. This uptake was inhibited by known inhibitors of APT, including o-vanadate and N-ethylmaleimide, and by ATP-depletion. SV-T2 cells were mutagenized with ethyl methanesulfonate, and APT-deficient cells were isolated by fluorescence activated cell sorting using NBD-PS as substrate. From a total of 7.2 x 10(6) cells passed through the flow cytometer, 98 clones exhibited APT activity that was less than 50% of that observed for wild-type SV-T2 cells. One clone which exhibited < or = 25% of that observed for wild-type cells, mutant M2711, was further characterized. The defect in mutant M2711 was specific for NBD-PS, and cellular ATP was unchanged, suggesting that the defect in APT activity was not due to a decrease in cellular ATP levels. Mutant M2711 exhibited a growth pattern indistinguishable from that of wild-type SV-T2 cells, and SV-40 large T antigen, which is needed for efficient episomal replication of plasmids containing the SV40 origin of replication, was unchanged. Finally, transfection of M2711 with cDNAs for marker membrane proteins consistently resulted in the same high level of protein expression as that observed for identically-transfected wild-type SV-T2. Thus, flow cytometry can be used for rapid identification of mutants with defects in phospholipid transport that are suitable for functional reconstitution by transfection with candidate APT cDNAs.
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PMID:Production and characterization of a mutant cell line defective in aminophospholipid translocase. 920 75

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

The transmembrane distribution of phospholipids in the membranes of eukaryotic cells depends on specific proteins (called flippases). The aminophospholipid translocase is responsible for the sequestration of phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet of plasma membranes. Several laboratories are presently working on the identification, purification and cloning of this Mg-ATPase, first recognized in the human red cell membrane. In accordance with the 1992 hypothesis of Higgins and Gottesman, proteins of the MDR1 family appear to be able to translocate certain phospholipids from the inner to the outer monolayer of the plasma membrane. It has been reported in particular that expression of the human MDR3 and mouse mdr2 genes promote translocation of long chain phosphatidylcholine, while expression of the MDR1 gene stimulates the outward motion of phospholipids possessing at least one short chain. ATP-independent flippases activities were recognized not only in microsomes but also in Golgi membranes.
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PMID:Aminophospholipid translocase and proteins involved in transmembrane phospholipid traffic. 946 21

The yeast DRS2 gene, which is required for growth at 23 degreesC or below, encodes a member of a P-type ATPase subgroup reported to transport aminophospholipids between the leaflets of the plasma membrane. Here, we evaluated the potential role of Drs2p in phospholipid transport. When examined by fluorescence microscopy, a drs2 null mutant showed no defect in the uptake or distribution of fluorescent-labeled 1-palmitoyl-2[6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl (NBD))aminocaproyl]phosphatidylserine) or 1-myristoyl-2[6-NBD-aminocaproyl]phosphatidylethanolamine. Quantification of the amount of cell-associated NBD fluorescence using flow cytometry indicated a significant decrease in the absence of Drs2p, but this decrease was not restricted to the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) and was dependent on culture conditions. Furthermore, the absence of Drs2p had no effect on the amount of endogenous PE exposed to the outer leaflet of the plasma membrane as detected by labeling with trinitrobenzene sulfonic acid. The steady state pool of Drs2p, which was shown to reside predominantly in the plasma membrane, increased upon shift to low temperature or exposure to various divalent cations (Mn2+, Co2+, Ni2+, and Zn2+ but not Ca2+ or Mg2+), conditions that also inhibited the growth of a drs2 null mutant. The data presented here call into question the identification of Drs2p as the exclusive or major aminophospholipid translocase in yeast plasma membranes (Tang, X., Halleck, M. S., Schlegel, R. A., and Williamson, P. (1996) Science 272, 1495-1497).
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PMID:Loss of Drs2p does not abolish transfer of fluorescence-labeled phospholipids across the plasma membrane of Saccharomyces cerevisiae. 985 6


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