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1.
Annexin V staining due to loss of membrane asymmetry can be reversible and precede commitment to apoptotic death. 总被引:7,自引:0,他引:7
Signal-induced apoptosis is a normal phenomenon in which cells respond to changes in their environment through a cascade of intracellular biochemical changes culminating in cell death. However, it is not clear at what point in this process the cell becomes committed to die. An early biochemical change characteristic of cells undergoing apoptosis is the loss of plasma membrane asymmetry, such that high levels of phosphatidylserine become exposed on the outside cell surface. These cells can be recognized by staining with Annexin V, which binds to phosphatidylserine with high affinity. To investigate the mechanisms controlling signal-induced apoptosis we have examined the response of a B cell lymphoma to crosslinking of the membrane immunoglobulin (mIg) receptor. We have found that many of the cells that stain positive for Annexin V are viable and can resume growth and reestablish phospholipid asymmetry once the signal is removed. These results indicate that Annexin V staining, and thus loss of membrane asymmetry, precedes commitment to apoptotic death in this system. 相似文献
2.
Pavone B Bucci S Sirolli V Merlini G Del Boccio P Di Rienzo M Felaco P Amoroso L Sacchetta P Di Ilio C Federici G Urbani A Bonomini M 《Molecular bioSystems》2011,7(3):651-658
The exposure of the aminophospholipid phosphatidylserine on the external leaflet of red blood cell plasma membrane can have several pathophysiological consequences with particular regard to the processes of cell phagocytosis, haemostasis and cell-cell interaction. A significant increase in phosphatidylserine-exposing erythrocytes has been reported in chronic haemodialysis patients and found to be strongly influenced by the uraemic milieu. To identify uraemic compound(s) enhancing phosphatidylserine externalization in erythrocytes, we fractionated by chromatographic methods the ultrafiltrate obtained during dialysis, and examined by flow cytometry the effect of the resulting fractions on phosphatidylserine exposure in human red cells. Chromatographic procedures disclosed a homogeneous fraction able to increase erythrocyte phosphatidylserine exposure. The inducer of such externalization was identified by monodimensional gel electrophoresis and mass spectrometry investigations as beta2-microglobulin. To confirm the beta2-microglobulin effect and to examine the influence of protein glycation (as it occurs in uraemia) on phosphatidylserine erythrocyte exposure, erythrocytes from normal subjects were incubated with recombinant beta2-microglobulin (showing no glycation sites at mass analysis), commercial beta2-microglobulin (8 glycation sites), or with in vitro glycated recombinant beta2-microglobulin (showing multiple glycation sites). Elevated concentrations of beta2-microglobulin (corresponding to plasma levels reached in dialysis patients) increased slightly but significantly the protein's ability to externalize phosphatidylserine on human erythrocytes. Such an effect was markedly enhanced by glycated forms of the protein. Beta2-microglobulin is recognized as a surrogate marker of middle-molecule uraemic toxins and represents a key component of dialysis-associated amyloidosis. Our study adds further evidence to the potential pathophysiologic consequences of beta2-microglobulin accumulation in chronic uraemic patients. 相似文献
3.
Kristy M. DiVittorio Frank T. Hofmann James R. Johnson Lica Abu-Esba Bradley D. Smith 《Bioorganic & medicinal chemistry》2009,17(1):141-148
A series of 16 synthetic scramblase candidates were prepared from a tris(aminoethyl)amine (TREN) scaffold and evaluated for ability to facilitate translocation of fluorescent phospholipid probes across vesicle membranes and endogenous phosphatidylserine across the plasma membrane of nucleated cells. More than half of the compounds were found to greatly accelerate phospholipid translocation in vesicles. However, they were generally unable to induce large increases in the amount of phosphatidylserine on the surface of nucleated mammalian cells, which contrasts with previous results using erythrocytes. Fluorescence microscopy showed that the synthetic scramblases are rapidly trafficked out of the cell plasma membrane and into the membranes of internal organelles. Future molecular designs of synthetic scramblases should focus on structures that are more amphiphilic, a structural feature that is expected to increase plasma membrane residence time. 相似文献
4.
Nelson J Francom LL Anderson L Damm K Baker R Chen J Franklin S Hamaker A Izidoro I Moss E Orton M Stevens E Yeung C Judd AM Bell JD 《Biochimica et biophysica acta》2012,1818(5):1196-1204
Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. 相似文献
5.
Phospholipid localization in the plasma membrane of Friend erythroleukemic cells and mouse erythrocytes 总被引:2,自引:0,他引:2
The distribution of phospholipids over outer and inner layers of the plasma membranes of Friend erythroleukemic cells (Friend cells) and mature mouse erythrocytes has been determined. The various techniques which have been applied to establish the phospholipid localization include the following: phospholipase A2, phospholipase C, and sphingomyelinase C treatment, fluorescamine labeling of phosphatidylethanolamine, and a phosphatidylcholine transfer protein mediated exchange procedure. The data obtained with these different techniques were found to be in good agreement with each other. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol were found to be distributed symmetrically over both layers of the plasma membrane of Friend cells. In contrast, sphingomyelin was found to be enriched in the outer layer of the membrane (80-85%), and phosphatidylserine appeared to be present mainly in the inner layer (80-90%). From these results, it was calculated that the outer and inner layers accounted for 46% and 54%, respectively, of the total phospholipid complement of that membrane. Analogous studies on the plasma membrane of mature mouse erythrocytes showed that the transbilayer distribution of the total phospholipid mass appeared to be the same as in the plasma membrane of the Friend cell, namely, 46% and 54% in outer and inner layers, respectively. The outer layer of this membrane contains 57% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 85% of the sphingomyelin, and 42% of the phosphatidylinositol, and none of the phosphatidylserine was present.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Significance of capacitative Ca2+ entry in the regulation of phosphatidylserine expression at the surface of stimulated cells. 总被引:1,自引:0,他引:1
M C Martínez S Martin F Toti E Fressinaud J Dachary-Prigent D Meyer J M Freyssinet 《Biochemistry》1999,38(31):10092-10098
The transverse redistribution of plasma membrane phosphatidylserine is one of the hallmarks of cells undergoing apoptosis and also occurs in cells fulfilling a more specialized function, such as platelets after appropriate activation. Although an increase in intracellular Ca2+ is required to trigger the remodeling of the plasma membrane, little information regarding intracellular signals leading to phosphatidylserine externalization has been provided. Scott syndrome is an extremely rare inherited disorder of the migration of phosphatidylserine toward the exoplasmic leaflet of the plasma membrane of stimulated blood cells. We have studied here the intracellular Ca2+ mobilization and Ca2+ entry involved in tyrosine phosphorylation in Epstein Barr virus (EBV)-infected B cells derived from a patient with Scott syndrome, her daughter, and control subjects. An alteration of Ca2+ entry through the plasma membrane and subsequent tyrosine phosphorylation induced by Ca2+ were observed in Scott EBV-B cells, but the release of Ca2+ from intracellular stores was normal. Furthermore, phosphatidylserine externalization at the surface of stimulated cells does not depend on tyrosine kinases. These results suggest that the defect of phosphatidylserine exposure in Scott syndrome cells is related to the alteration of a particular way of Ca2+ entry, referred to as capacitative Ca2+ entry, although some differences may be related to the cell type. Hence, this genetic mutant testifies to the prime significance of Ca2+ signaling in the regulation of phosphatidylserine expression at the surface of stimulated cells. 相似文献
7.
N Kotagal J R Colca D Buscetto M L McDaniel 《Archives of biochemistry and biophysics》1985,238(1):161-169
Studies were undertaken to determine whether factors which affect insulin secretion may exert their effects by altering the activity of an islet-cell plasma membrane Ca2+ extrusion pump. The insulin secretagogue, D-glucose, and a variety of phosphorylated hexoses, glucose 6-P, glucose 1,6-P, fructose 6-P, and fructose 2,6-P, were evaluated for their effect on an islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase and were found to be ineffective in altering enzyme activity. D-Glucose also did not alter the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Similarly, cAMP, the catalytic subunit of cAMP-dependent protein kinase, arachidonic acid, or prostaglandin E2 did not affect either the plasma membrane (Ca2+ + Mg2+)-ATPase or the rate of ATP-dependent Ca2+ uptake into plasma membrane vesicles. Whereas previous studies have suggested that D-glucose and/or cAMP may inhibit ATPase activities in islets, these results indicate that the agents, i.e., D-glucose and cAMP, which stimulate and/or potentiate insulin secretion from the islet cell, do not modify Ca2+ fluxes by directly regulating the islet-cell plasma membrane (Ca2+ + Mg2+)-ATPase. In contrast, the acidic phospholipids, phosphatidic acid and phosphatidylserine, stimulated the enzyme activity in a concentration-dependent manner whereas phosphatidylcholine had only a minimal effect. The diacylglycerol, dilinolein, stimulated the (Ca2+ + Mg2+)-ATPase activity in the presence of phosphatidylserine, but not in the absence of phospholipids. These effects were independent of phospholipid-stimulated protein phosphorylation in the islet-cell plasma membrane under the conditions of the ATPase assay. 相似文献
8.
Jennifer Nelson Lyndee L. Francom Lynn Anderson Kelly Damm Ryan Baker Joseph Chen Sarah Franklin Amy Hamaker Izadora Izidoro Eric Moss Mikayla Orton Evan Stevens Celestine Yeung Allan M. Judd John D. Bell 《生物化学与生物物理学报:生物膜》2012,1818(5):1196-1204
Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A2 but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A2. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A2. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A2, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine. 相似文献
9.
The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties. 相似文献
10.
D R Voelker 《Microbiological reviews》1991,55(4):543-560
The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly. 相似文献
11.
Evaluation of the electrostatic field strength at the site of exocytosis in adrenal chromaffin cells.
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K Rosenheck 《Biophysical journal》1998,75(3):1237-1243
Exocytosis in secretory cells consists of release from intracellular storage granules directly into the extracellular space via fusion of the granule membrane with the plasma membrane of the cell. It is considered here as comprising two distinct processes. One is the close apposition of granule and plasma membranes. The other arises from interactions between the two membranes during the process of apposition, leading to the formation of a fusion pore. In the following it is shown for the case of the adrenal medullary chromaffin cell that the fusion pore can be ascribed to electroporation of the granule membrane, triggered by the strong electric field existing at the site of exocytosis. Based on an electric surface charge model of the cytoplasmic side of the plasma membrane, resulting from the negatively charged phosphatidylserine groups, it is found that the electrostatic field strength at the site of exocytosis reaches values on the order of 10(8) V/m at small intermembrane distances of 3 nm and lower. The field strength increases with the size of the disc-shaped plasma membrane region generating the electric field, reaching an approximate limit for a radius of 10 nm, at a surface charge density of 5.4 x 10(-2) C/m2. According to previous experimental evaluations of threshold field strength, this field is sufficiently strong to cause membrane electroporation. This step is a precondition for the subsequent membrane fusion during the ongoing process of apposition, leading to secretion. 相似文献
12.
Phosphatidylserine exposure during early primary necrosis (oncosis) in JB6 cells as evidenced by immunogold labeling technique 总被引:5,自引:0,他引:5
Krysko O De Ridder L Cornelissen M 《Apoptosis : an international journal on programmed cell death》2004,9(4):495-500
Apoptotic cell death is characterized by the early exposure of phosphatidylserine (PS) at the outer surface of the plasma membrane. The aim of the present study was to examine whether PS exposure also occurs during oncosis (early primary necrosis) and to localize PS at the subcellular level, applying a pre-embedding immunogold labeling technique with biotin conjugated annexin V. The issue was addressed by using caspase-8 deficient, Bcl-2 overexpressing JB6 cells, which die by oncosis when stimulated with synthetic dsRNA. We observed by fluorescence microscopy that oncotic cells with preserved plasma membrane integrity showed PS exposure (annexin+/propidium iodide-). The data was confirmed on the ultrastructural level and PS was localized in oncosis at the outer leaflet of the continuous plasma membrane with preserved trilamellar structure. In postoncotic necrotic cells the immunogold labels were found on the plasma membrane and on the intracellular membranes of the cells, which underwent plasma membrane disruption. In conclusion, this study reveals that PS externalization occurs not only in apoptosis but also in oncosis at least in our cell model system. 相似文献
13.
Israr-ul H. Ansari Melissa J. Longacre Coen C. Paulusma Scott W. Stoker Mindy A. Kendrick Michael J. MacDonald 《The Journal of biological chemistry》2015,290(38):23110-23123
The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075–11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis. 相似文献
14.
Isolation and characterization of plasma membranes from Friend erythroleukaemic cells. A study with sphingomyelinase C 总被引:2,自引:0,他引:2
A J Rawyler B Roelofsen J A Op den Kamp L L Van Deenen 《Biochimica et biophysica acta》1983,730(1):130-138
Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (I) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6-7% of phospholipid, 8-9% of cholesterol and 12-15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15-20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C (S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer. 相似文献
15.
We have determined the asymmetric distribution of two aminophospholipids phosphatidylethanolamine and phosphatidylserine in the plasma membrane of chick embryo fibroblast and myoblasts. Right-side-out membrane preparations were incubated with two different amidating reagents, trinitrobenzenesulfonate and isethionylacetimidate, under nonpenetrating conditions. Inside-out membranes were incubated with trinitrobenzenesulfonate. In fibroblasts, the similar plateau values suggested that 35% of the phosphatidylethanolamine and 20% of the phosphatidylserine is externally disposed. These values agree with previous measurements on fibroblast plasma membranes. In myoblasts, however, labelling plateaux were achieved which suggested that 65% of the phosphatidylethanolamine and 45% of the phosphatidylserine is externally disposed. This represents a 2-3-fold increase in potentially fusogenic lipids on the external leaflet of the plasma membrane. This unique distribution of aminophospholipids in myoblasts extends through the stage of development during which myoblasts become competent to fuse and form myotubes in culture. Two inferences may be drawn from these results. First, the external concentration of aminophospholipids in myoblasts is enriched significantly over that of fibroblasts or erythrocytes. This orientation may contribute to its fusion competence. Second, although large amounts of externally disposed aminophospholipid may be necessary for myoblast fusion, they do not confer fusion competence. 相似文献
16.
The internalization of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine following their insertion into the plasma membrane of cultured Chinese hamster fibroblasts was examined. When liposomes containing the fluorescent lipid 1,2-(palmitoyl-N-4-nitrobenzo-2-oxa-1,3-diazole-amino-caproyl) phosphatidylserine [palmitoyl-C6-NBD)-PS), were incubated with monolayer cell cultures at 2 degrees C, spontaneous transfer of the fluorescent lipid from the liposomes to the cells occurred, resulting in prominent labeling of the plasma membrane. However, if the cells were washed and warmed to 7 degrees C for 30 min, the (palmitoyl-C6-NBD)-PS also labeled numerous intracellular membranes. Evidence is presented suggesting that this internalization was not due to endocytosis, but was the result of transmembrane movement of the (palmitoyl-C6-NBD)-PS at the plasma membrane followed by translocation of lipid monomers from the plasma membrane to internal membranes. This transmembrane movement was reversibly inhibited by depletion of cellular ATP levels and was blocked by treatment with structural analogs of the lipid or by pretreatment of cells with glutaraldehyde or N-ethyl-maleimide. A fluorescent analog of phosphatidylethanolamine [palmitoyl-C6-NBD)-PE), which also exhibits transmembrane movement at the plasma membrane at 7 degrees C (Sleight, R. G., and Pagano, R. E. (1985) J. Biol. Chem. 260, 1146-1154), was further studied. Its transmembrane movement was also inhibited by depletion of cellular ATP levels, or by pretreatment of cells with N-ethylmaleimide. The transmembrane movement of the fluorescent phosphatidylserine and phosphatidylethanolamine analogs was inhibited when the unnatural D-isomers of these lipids were used, further suggesting that this process was stereospecific and therefore likely to have been protein-mediated. 相似文献
17.
G. Poste N. C. Lyon P. Macander C. W. Porter P. Reeve H. Bachmeyer 《Experimental cell research》1980,129(2)
Cultured mouse 3T3 cells treated with phosphatidylserine or phosphatidylserine/phosphatidylcholine (3: 7 mole ratio) liposomes containing ortho- and paramyxovirus envelope glycoproteins become susceptible to killing by virus-specific cytotoxic T lymphocytes indicating that the liposome-derived glycoproteins have been inserted into the cellular plasma membrane. Cells incubated with liposomes of similar lipid composition containing viral antigens plus a dinitrophenylated lipid hapten were killed by both virus- and hapten-specific T lymphocytes indicating that both protein and lipid components are inserted into the plasma membrane. We consider that assimilation of liposome-derived antigens into the plasma membrane results from fusion of liposomes with the plasma membrane. Cells incubated with phosphatidylcholine liposomes containing lipid haptens and viral glycoproteins were not killed by cytotoxic lymphocytes indicating that liposomes of this composition do not fuse with the plasma membrane. Liposome-derived paramyxovirus glycoproteins inserted into the plasma membrane retain their functional activity as shown by their ability to induce cell fusion. These experiments demonstrate the feasibility of using liposomes as carriers for introducing integral membrane (glyco)proteins into the plasma membrane of cultured cells and establish a new approach for studying the role of individual (glyco)proteins in the expression of specific cell surface properties. 相似文献
18.
Layden BT Abukhdeir AM Malarkey C Oriti LA Salah W Stigler C Geraldes CF Mota de Freitas D 《Biochimica et biophysica acta》2005,1741(3):339-349
Li(+) binding in subcellular fractions of human neuroblastoma SH-SY 5 Y cells was investigated using (7)Li NMR spin-lattice (T(1)) and spin-spin (T(2)) relaxation measurements, as the T(1)/T(2) ratio is a sensitive parameter of Li(+) binding. The majority of Li(+) binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li(+) binding constants and the values of the T(1)/T(2) ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by (31)P NMR spectroscopy the effects of chronic Li(+) treatment for 4--6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li(+) binding occurs predominantly to membrane domains, and that chronic Li(+) treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li(+) treatment of bipolar patients results in irregularities in Li(+) binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li(+) action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed. 相似文献
19.
Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis 总被引:4,自引:0,他引:4
Van den Eijnde SM Boshart L Reutelingsperger CP De Zeeuw CI Vermeij-Keers C 《Cell death and differentiation》1997,4(4):311-316
The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis. 相似文献
20.
A. S. Fomin O. A. Koval D. V. Semenov M. O. Potapenko E. V. Kuligina Yu. Ya. Kit V. A. Richter 《Russian Journal of Bioorganic Chemistry》2012,38(1):77-82
Earlier, a pro-apoptotic peptide lactaptin was isolated from human milk and characterized and its recombinant analogue RL2
was generated. Both peptides were demonstrated to be capable of apoptotic cell death induction in human breast adenocarcinoma
MCF-7 cells. In the work, biochemical markers of RL2-induced MCF-7 apoptosis are analyzed. Activation of initiator and effector
caspases, as well as apoptotic changes in mitochondrial membrane potential and cytoplasm membrane composition in cells treated
with RL2, were analyzed using flow cytometry and Western blot techniques. MCF-7 cell death induced by RL2 was found to be
associated with phosphatidylserine exposure on the surface of the plasma membrane. Also, RL2 was demonstrated to induce dissipation
of the mitochondrial membrane potential and activation of initiator caspases 8 and 9 and an effector caspase 7. 相似文献