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1.
On the mechanism of transbilayer transport of phosphatidylglycerol in response to transmembrane pH gradients 总被引:9,自引:0,他引:9
Previous work [Hope et al. (1989) Biochemistry 28, 4181-4187] has shown that asymmetric transmembrane distributions of phosphatidylglycerol (PG) in PG-phosphatidylcholine (PC) large unilamellar vesicles can be induced in response to transbilayer pH gradients (delta pH). Here the mechanism of PG transport has been investigated. It is shown that PG movement in response to delta pH is consistent with permeation of the uncharged (protonated) form and that the half-time for transbilayer movement of the uncharged form can be on the order of seconds at 45 degrees C. This can result in rapid pH-dependent transmembrane redistributions of PG. The rate constant for transbilayer movement exhibits a large activation energy (31 kcal/mol) consistent with transport of neutral dehydrated PG where dehydration of the (protonated) phosphate presents the largest barrier to transmembrane diffusion. It is shown that acyl chain saturation, chain length, and the presence of cholesterol modulate the rate constants for PG transport in a manner similar to that observed for small nonelectrolytes. 相似文献
2.
Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm. 相似文献
3.
Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients 总被引:10,自引:0,他引:10
The influence of membrane pH gradients on the transbilayer distribution of some common phospholipids has been investigated. We demonstrate that the transbilayer equilibrium of the acidic phospholipids egg phosphatidylglycerol (EPG) and egg phosphatidic acid (EPA) can be manipulated by membrane proton gradients, whereas phosphatidylethanolamine, a zwitterionic phospholipid, remains equally distributed between the inner and outer monolayers of large unilamellar vesicles (LUVs). Asymmetry of EPG is examined in detail and demonstrated by employing three independent techniques: ion-exchange chromatography, 13C NMR, and periodic acid oxidation of the (exterior) EPG headgroup. In the absence of a transmembrane pH gradient (delta pH) EPG is equally distributed between the outer and inner monolayers of LUVs. When vesicles composed of either egg phosphatidylcholine (EPC) or DOPC together with 5 mol % EPG are prepared with a transmembrane delta pH (inside basic, outside acidic), EPG equilibrates across the bilayer until 80-90% of the EPG is located in the inner monolayer. Reversing the pH gradient (inside acidic, outside basic) results in the opposite asymmetry. The rate at which EPG equilibrates across the membrane is temperature dependent. These observations are consistent with a mechanism in which the protonated (neutral) species of EPG is able to traverse the bilayer. Under these circumstances EPG would be expected to equilibrate across the bilayer in a manner that reflects the transmembrane proton gradient. A similar mechanism has been demonstrated to apply to simple lipids that exhibit weak acid or base characteristics [Hope, M. J., & Cullis, P. R. (1987) J. Biol. Chem 262, 4360-4366] 相似文献
4.
The ability of lipid asymmetry to regulate Ca(2+)-stimulated fusion between large unilamellar vesicles has been investigated. It is shown that for 100-nm-diameter LUVs composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylinositol, and dioleoylphosphatidic acid (DOPC/DOPE/PI/DOPA; 25:60:5:10) rapid and essentially complete fusion is observed by fluorescent resonance energy transfer techniques when Ca2+ (8 mM) is added. Alternatively, for LUVs with the same lipid composition but when DOPA was sequestered to the inner monolayer by incubation in the presence of a pH gradient (interior basic), little or no fusion is observed on addition of Ca2+. It is shown that the extent of Ca(2+)-induced fusion correlates with the amount of exterior DOPA. Further, it is shown that LUVs containing only 2.5 mol % DOPA, but where all the DOPA is in the outer monolayer, can be induced to fuse to the same extent and with the same rate as LUVs containing 5 mol % DOPA. These results strongly support a regulatory role for lipid asymmetry in membrane fusion and indicate that the fusogenic tendencies of lipid bilayers are largely determined by the properties of the monolayers proximate to the fusion interface. 相似文献
5.
Influence of transbilayer area asymmetry on the morphology of large unilamellar vesicles. 总被引:2,自引:2,他引:0
The morphological consequences of differences in the monolayer surface areas of large unilamellar vesicles (LUVs) have been examined employing cryoelectron microscopy techniques. Surface area was varied by inducing net transbilayer transport of dioleoylphosphatidylglycerol (DOPG) in dioleoylphosphatidylcholine (DOPC):DOPG (9:1, mol:mol) LUVs in response to transmembrane pH gradients. It is shown that when DOPG is transported from the inner to the outer monolayer, initially invaginated LUVs are transformed to long narrow tubular structures, or spherical structures with one or more protrusions. Tubular structures are also seen in response to outward DOPG transport in DOPC:DOPG:Chol (6:1:3, mol:mol:mol) LUV systems, and when lyso-PC is allowed to partition into the exterior monolayer of DOPC:DOPG (9:1, mol:mol) LUVs in the absence of DOPG transport. Conversely, when the inner monolayer area is expanded by the transport of DOPG from the outer monolayer to the inner monolayer of non-invaginated LUVs, a reversion to invaginated structures is observed. The morphological changes are well described by an elastic bending theory of the bilayer. Identification of the difference in relaxed monolayer areas and of the volume-to-area ratio of the LUVs as the shape-determining factors allows a quantitative classification of the observed morphologies. The morphology seen in LUVs supports the possibility that factors leading to differences in monolayer surface areas could play important roles in intracellular membrane transport processes. 相似文献
6.
7.
Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus
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Valcarcel CA Dalla Serra M Potrich C Bernhart I Tejuca M Martinez D Pazos F Lanio ME Menestrina G 《Biophysical journal》2001,80(6):2761-2774
Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA > PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure. 相似文献
8.
We have investigated the influence of transmembrane pH gradients across large unilamellar vesicle membranes on the transbilayer distributions of simple lipids with weak base and weak acid characteristics. Trinitrobenzenesulfonic acid labeling results consistent with a rapid and complete migration of stearylamine and sphingosine to the inner monolayer of the large unilamellar vesicles are observed when the large unilamellar vesicles' interior is acidic. Alternatively, when the vesicle interior is basic, oleic and stearic acid cannot be removed by external bovine serum albumin, indicating a localization in the inner monolayer. Moreover, effects corresponding to the decrease in external surface charge predicted upon the migration of stearylamine or stearic acid to the inner monolayer are readily detected employing ion exchange chromatography. These results are consistent with transbilayer distributions of these agents dictated by a Henderson-Hasselbach equilibrium. The possible implications for metabolic regulation by pH gradients, as well as factors giving rise to phospholipid transbilayer asymmetry, are discussed. 相似文献
9.
Dopamine accumulation in large unilamellar vesicle systems induced by transmembrane ion gradients 总被引:1,自引:0,他引:1
M B Bally L D Mayer H Loughrey T Redelmeier T D Madden K Wong P R Harrigan M J Hope P R Cullis 《Chemistry and physics of lipids》1988,47(2):97-107
Transmembrane movement of dopamine in response to K+ or H+ ion gradients has been investigated. It is shown that dopamine can accumulate rapidly into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine exhibiting either a K+ diffusion potential (delta psi; negative inside) or a pH gradient (inside acidic). This can result in entrapped dopamine concentrations of 30-40 mM and inside-outside concentration gradients of nearly 300-fold. The transmembrane dopamine gradients formed in LUV systems exhibiting delta pH (inside acidic) indicate that the transport process can be dictated by movement of the neutral form of dopamine which redistributes according to a simple Henderson-Hasselbach equilibrium. The mechanism of dopamine transport in response to a valinomycin-induced K+ potential is more complex. Although generation of a K+ diffusion potential results in acidification of the vesicle interior, the magnitude of the induced delta pH (approx. 1 pH unit) is insufficient to account for the dopamine concentration gradient achieved (greater than 200-fold). Further, data presented here suggest that higher uptake levels of dopamine can be achieved when certain anions (ATP and citrate) are entrapped within the LUV system. These anions may complex with the protonated form of dopamine creating a non-equilibrium trapping phenomena resulting in interior concentrations of dopamine in excess of that predicted by a simple Henderson-Hasselbach equilibrium. 相似文献
10.
The passive transbilayer movement-flip-flop-was investigated on planar bilayer lipid membranes (BLMs), containing myristic, stearic, or linoleic long-chain fatty acids (FA). In response to a transbilayer pH gradient, a difference in the surface charges between inner and outer leaflets appeared. Because the BLM was formed from FA and neutral lipid, a surface potential difference was originated solely by a concentration difference of the initially equally distributed ionized FA. As revealed by zeta-potential measurements, the corresponding surface potential difference DeltaPhi(s) was at least twice the value expected from a titration of the FA alone. The additional surface charge was attributed to FA flip-flop induced by the transbilayer pH gradient. DeltaPhi(s) was derived from capacitive current measurements carried out with a direct current (dc) bias and was corrected for changes of membrane dipole potential Phi(d). Dual-wavelength ratiometric fluorescence measurements have shown that Phi(d) values of the pure DPhPC bilayers and BLMs containing 40 mol % FA differ by less than 6%. It is concluded that fast FA flip-flop is not restricted to membranes with high curvature. The role of pH gradient as an effective driving force for the regulation of FA uptake is discussed. 相似文献
11.
The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle. When large vesicles were treated with TNBS, approximately 40% of the total membrane PE was derivatized; in the small vesicles 55% reacted. These values are interpreted as representing the percentage of total membrane PE residing in the outer leaflet of the vesicle bilayer. The large vesicles likely contained approximately 20% of the total membrane lipid as internal membranes. Therefore, in both types of vesicles, PE as a phospholipid class was randomly distributed between the inner and outer leaflets of the bilayer. The proportion of total PE residing in the outer leaflet was unaffected by changes in either the cholesterol or PE content of the vesicles. However, the transbilayer distributions of individual molecular species of PE were not random, and were significantly influenced by radius of curvature, membrane cholesterol content, or both. For example, palmitate- and docosahexaenoate-containing species of PE were preferentially located in the outer leaflet of the bilayer. Membrane cholesterol content affected the transbilayer distributions of stearate-, oleate-, and linoleate-containing species. The transbilayer distributions of palmitate-, docosahexaenoate-, and stearate-containing species were significantly influenced by membrane curvature, but only in the presence of high levels of cholesterol. Thus, differences in membrane curvature and cholesterol content alter the array of PE molecules present on the surfaces of phospholipid bilayers. In cells and organelles, these differences could have profound effects on a number of critical membrane functions and processes. 相似文献
12.
We have measured the transbilayer diffusion at 4 degrees C of spin labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid in the human erythrocyte membrane. Measurements were also carried out in ghosts, released without ATP, and on large unilamellar vesicles made with total lipid extract. As reported previously (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), the amino phospholipids are rapidly transported from the outer to the inner leaflet on fresh erythrocytes, whereas phosphatidylcholine diffuses slowly. We now show that phosphatidic acid behaves like phosphatidylcholine: approximately 10% is internalized in 5 h at 4 degrees C. Under the same experimental conditions, no inward transport of sphingomyelin can be detected. In ghosts resealed without ATP, all glycerophospholipids tested diffuse slowly from the outer to the inner leaflet (approx. 10% in 5 h) while no transport of sphingomyelin is seen. Finally in lipid vesicles, the inward diffusion of all glycerophospholipids is less than 2% in 5 h and a very small transport of sphingomyelin can be measured. These results confirm the existence of a selective inward aminophospholipid transport of fresh erythrocytes and suggest a slow and passive diffusion of all phospholipids on ghosts, resealed without ATP, as well as on lipid vesicles. 相似文献
13.
Contreras FX Villar AV Alonso A Kolesnick RN Goñi FM 《The Journal of biological chemistry》2003,278(39):37169-37174
Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway. 相似文献
14.
15.
Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol 总被引:2,自引:0,他引:2
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The energetic determinants of the distribution of anionic phospholipids across a phosphatidylcholine (PtdCho) bilayer with different packing constraints in the two leaflets were studied, using (13)CH2-ethyl-labeled phosphatidylethanol (PtdEth) as a (13)C NMR membrane probe. PtdEth is unique in exhibiting a split (13)CH2-ethyl resonance in sonicated vesicles, the two components originating from the inner and outer leaflets, thus permitting the determination of the PtdEth concentration in each leaflet. Small and large unilamellar PtdEth-PtdCho vesicles were prepared in solutions of different ionic strengths. A quantitative expression for the transbilayer distribution of PtdEth, based on the balance between steric and electrostatic factors, was derived. The transbilayer difference in packing constraints was obtained from the magnitude of the PtdEth signal splitting. The electrostatic contribution could be satisfactorily described by the transmembrane difference in Gouy-Chapman surface potentials. At low (0.1-0.25%) PtdEth levels and high (up to 500 mM) salt concentrations, PtdEth had a marked fivefold preference for the inner leaflet, presumably because of its small headgroup, which favors tighter packing. At higher PtdEth content (4.8-9.1%) and low salt concentrations, where electrostatic repulsion becomes a dominant factor, the asymmetry was markedly reduced and an almost even distribution across the bilayer was obtained. In less curved, large vesicles, where packing constraints in the two leaflets are approximately the same, the PtdEth distribution was almost symmetrical. This study is the first quantitative analysis of the balance between steric and electrostatic factors that determines the equilibrium transbilayer distribution of charged membrane constituents. 相似文献
16.
It is shown that the equilibrium transmembrane lipid asymmetry strongly depends on the degree of nonideality in the lateral mixing of the lipid components. In two-component bilayers the effect of nonideal lateral mixing is maximal for a given component at mole fractions of this component between 0.35 and 0.4. For asymmetry creating factors about 3 kT correcting for lateral nonidealities typical for lipids can increase as much as three times the transmembrane asymmetry. The relationship between lateral nonideality and transbilayer asymmetry is analysed in detail in the case of electrostatically induced asymmetry by using the Gouy-Chapman theory of electric double layers and the Bragg-Williams (regular solutions) approximation of nonideal lateral mixing. Two representative models are studied: (a) a single flat bilayer with a transmembrane electric potential difference applied on it; (b) two parallel membranes at short separation. In case (a), for transmembrane potentials of about 50-100 mV the introduction of nonideality corrections increases up to 40% the transmembrane asymmetry. In case (b), at physiological electrolyte concentrations the lipid asymmetry and, consequently, the effect of lateral nonideality become significant only at unrealistically small separations between the membranes. The surprisingly great influence of the lateral nonideality on the equilibrium transmembrane asymmetry suggests a significant role for this effect in determining the membrane molecular organization. A restricted lateral lipid miscibility might serve as a peculiar, but rather strong 'amplifier' of the transmembrane asymmetry. The qualitatively different asymmetries found in small unilamellar phosphatidylcholine-phosphatidylethanolamine vesicles of different fatty acid composition (Lentz, B.R. and Litman, B.J. (1978) Biochemistry 17, 5537-5543) can be reasonably well explained as an effect of the lateral nonideality. A hypothesis considering the transmembrane distributions of the major phospholipid species in erythrocytes as evolving from their lateral miscibilities is proposed. 相似文献
17.
The 3-hydroxy group and 4,5-trans double bond of sphingomyelin are essential for modulation of galactosylceramide transmembrane asymmetry
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Malewicz B Valiyaveettil JT Jacob K Byun HS Mattjus P Baumann WJ Bittman R Brown RE 《Biophysical journal》2005,88(4):2670-2680
The structural features of SPM that control the transbilayer distribution of beta-GalCer in POPC vesicles were investigated by (13)C- and (31)P-NMR spectroscopy using lipid analogs that share physical similarities with GalCer or SPM. The SPM analogs included N-palmitoyl-4,5-dihydro-SPM, 3-deoxy-SPM, 1-alkyl-2-amidophosphatidylcholine, and dipalmitoylphosphatidylcholine, a popular model "raft lipid". The transbilayer distributions of the SPM analogs and SPM in POPC vesicles were similar by (31)P-NMR. To observe the dramatic change in GalCer transbilayer distribution that occurs when SPM is included in POPC vesicles, the 3-OH group, 4,5-trans double bond, and amide linkage all were required in SPM. However, inclusion of 2 and 10 mol % dihydroSPM in SPM/POPC (1:1) vesicles mitigated and completely abrogated the effect of SPM on the transbilayer distribution of GalCer. Despite sharing some structural features with GalCer and localizing preferentially to the inner leaflet of POPC vesicles, dimyristoylphosphatidylethanolamine did not undergo a change in transbilayer distribution when SPM was incorporated into the vesicles. The results support the hypothesis that specific interactions may be favored among select sphingolipids in curvature-stressed membranes and emphasize the potential importance of the SPM-dihydroSPM ratio in membrane fission and fusion processes associated with vesicle biogenesis and trafficking. 相似文献
18.
Kinetics and thermodynamics of calcium-induced lateral phase separations in phosphatidic acid containing bilayers 总被引:1,自引:0,他引:1
The effects of calcium on the mixing of synthetic diacylphosphatidylcholines (PC's) and diacylphosphatidylethanolamines (PE's) with the corresponding phosphatidic acids (PA's) have been examined by high-sensitivity differential scanning calorimetry and by measurements of the fluorescence of labeled PA or PC species in PA-PC bilayers. Calorimetrically derived phase diagrams for dimyristoyl- and dielaidoyl-substituted PA-PC and PA-PE mixtures indicate that these species are readily miscible in the absence of calcium but phase-separate very extensively in the presence of high levels of calcium (30 mM). The limiting solubilities of PA (Ca2+) in liquid-crystalline PC or PE bilayers are less than or equal to 10 and approximately 5 mol %, respectively, while approximately 20 mol % of PC or PE can be introduced into the "cochleate" phase of PA (Ca2+) before a distinct PC-rich (or PE-rich) phase appears. The kinetics of calcium-induced lateral phase separations were examined for dioleoyl- and dielaidoyl-substituted PA-PC unilamellar vesicles labeled with fluorescent (C12-NBD-acyl) PA or PC, whose fluorescence becomes partially quenched upon phase separation. Our results indicate that, for the PA-PC system, lateral phase separation is very rapid (approximately less than 1 s) after calcium addition and develops partially (possibly in only one face of the bilayer) when calcium is present only on one side of the bilayer. Moreover, phase separations can develop at a rate faster than that of vesicle diffusion when calcium is added to dilute suspensions of vesicles, suggesting that interbilayer contacts are not essential to promote phase separations. 相似文献
19.
Loss of membrane phospholipid asymmetry during activation of blood platelets and sickled red cells; mechanisms and physiological significance 总被引:3,自引:0,他引:3
Robert F. A. Zwaal Edouard M. Bevers Paul Comfurius Jan Rosing Roland H. J. Tilly Peter F. J. Verhallen 《Molecular and cellular biochemistry》1989,91(1-2):23-31
Membrane phospholipid asymmetry is considered to be a general property of biological membranes. Detailed information is presently available on the non-random orientation of phospholipids in red cell- and platelet membranes. The outer leaflet of the lipid bilayer membrane is rich in choline-phospholipids, whereas amino-phospholipids are abundant in the inner leaflet. Studies with blood platelets have shown that these asymmetries are not maintained when the cells are activated in various ways. Undoing the normal asymmetry of membrane phospholipids in activated blood cells is presumably mediated by increased transbilayer movement of phospholipids. This process, which leads to increased exposure of negatively charged phosphatidylserine at the outer surface, plays an important physiological role in local blood clotting reactions. A similar phenomenon occurs in sickled red cells. Phospholipid vesicles breaking off from reversibly sickled cells contribute similarly to intravascular clotting in the crisis phase of sickle cell disease.The loss of membrane phospholipid asymmetry in activated platelets seems to be strictly correlated with degradation of cytoskeletal proteins by endogenous calpain. It is remarkable that membrane phospholipid asymmetry can be (partly) restored when activated platelets are treated with reducing agents. This leads to disappearance of phosphatidylserine from the outer leaflet where it was previously exposed during cell activation. These observations will be discussed in relation to two mechanisms which have been recognized to play a role in the regulation of membrane phospholipid asymmetry; i.e. the interaction of aminophospholipids to cytoskeletal proteins, and the involvement of a phospholipid-translocase catalyzing outward-inward transbilayer movement of amino-phospholipids. 相似文献
20.
The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%. 相似文献