Transbilayer Distribution Research Articles

Extensive electroporation abolishes experimentally induced shape transformations of erythrocytes: a consequence of phospholipid symmetrization?

As shown in earlier work (M.M. Henszen et al., Mol. Membr. Biol. 14 (1997) 195–204), exposure of erythrocytes to single brief electric field pulses (5–7 kV cm −1) enhances the transbilayer mobility of phospholipids and produces echinocytes which can subsequently be transformed into stomatocytes in an ATP-dependent process. These shape transformations arise from partly reversible changes of the transbilayer disposition of phospholipids, in agreement with the bilayer couple concept. Extensive membrane modification by repetitive (≤20) field pulses followed by 20 h incubation at 37°C is now shown to produce discocytic cells which are resistant to many established shape-transforming treatments, including (A) single electric field pulses, Ca 2+ incorporation and exposure to membrane active amphiphiles, but also (B) metabolic depletion, binding of band 3 ligands, alkaline pH and contact with glass surfaces. The suppression of type A effects can readily be interpreted by a complete symmetrization of the phospholipids in extensively field pulse-modified cells which prevents shape transformations related to the asymmetric disposition of the phospholipids. This symmetrization could be further substantiated by more direct determinations of the transbilayer distribution of phospholipids. Suppression of shape transformations of type B may indicate an involvement of phospholipid asymmetry in these processes on a yet unknown mechanistic basis. Alternatively we discuss field pulse-induced alterations of the disposition of peripheral proteins or of the conformation of integral membrane proteins as mechanisms interfering with shape transformations of type B.

An amphipathic α-helix at a membrane interface: a structural study using a novel X-ray diffraction method

The amphipathic α-helix is a recurrent feature of membrane-active proteins, peptides, and toxins. Despite extensive biophysical studies, the structural details of its affinity for membrane interfaces remain rather vague. We report here the first results of an effort to obtain detailed structural information about α-helices in membranes by means of a novel X-ray diffraction method. Specifically, we determined the transbilayer position and orientation of an archetypal class A amphipathic helical peptide in oriented fluid-state dioleoylphosphatidylcholine (DOPC) bilayers. The peptide, Ac-18A-NH2 (Ac-DWLKAFYDKVAEKLKEAF-NH2), is a model for class A amphipathic helices of apolipoprotein A-I and other exchangeable lipoproteins. The diffraction method relies upon experimental determinations of absolute scattering-length density profiles along the bilayer normal and the transbilayer distribution of the DOPC double bonds by means of specific bromination, and molecular modeling of the perturbed lipid bilayer (derived using the transbilayer distribution of the double bonds) and the peptide. The diffraction results showed that Ac-18A-NH2 was located in the bilayer interface and that its transbilayer distribution could be described by a Gaussian function with a 1/e-halfwidth of 4.5(±0.3) Å located 17.1(±0.3) Å from the bilayer center, close to the glycerol moiety. Molecular modeling suggested that Ac-18A-NH2 is helical and oriented generally parallel with the bilayer plane. The helicity and orientation were confirmed by oriented circular dichroism measurements. The width of the Gaussian distribution, a measure of the diameter of the helix, indicated that the Ac-18A-NH2 helix penetrated the hydrocarbon core to about the level of the DOPC double bonds. Bilayer perturbations caused by Ac-18A-NH2 were surprisingly modest, consisting of a slight decrease in bilayer thickness with a concomitant shift of the double-bond distribution toward the bilayer center, as expected from a small increase in lipid-specific area caused by the peptide.

Detection of Phosphatidylserine Surface Exposure on Human Erythrocytes Using Annexin V-Ferrofluid

The asymmetric transbilayer distribution of phospholipids in the plasma membrane and the regulation of phosphatidylserine (PS) exposure at the cell surface of animal cells are of high physiological significance. It has been shown previously that annexin V is one of the most sensitive tools with which the presence of small amounts of PS on the outer surface of eukaryotic cells can be detected. We present here the covalent coupling of annexin V molecules to magnetic nanoparticles of maghemite. The resulting annexin V-ferrofluid is used in the magnetic separation of PS exposing cells, as illustrated for human erythrocytes modified in their phospholipid transbilayer asymmetry by the use of a calcium ionophore. Results on stored human erythrocytes and comparison with results obtained using iodinated and fluorescein-labeled annexin V are also presented.

Stability of transbilayer phospholipid asymmetry in viable ram sperm cells after cryotreatment.

The transbilayer dynamics of lipids in the plasma membrane of mammalian sperm cells is crucial for the fertilization process. Here, the transbilayer movement and distribution of phospholipids in the plasma membrane of fresh, ejaculated and cryopreserved ram spermatozoa was studied by labeling cells with fluorescent analogues of phosphatidylserine and phosphatidylcholine. By co-labeling cells with the DNA-binding dye propidiumiodide as well as by employing fluorescence microscopy and flow cytometry we were able to determine the transbilayer redistribution of fluorescent phospholipid analogues in intact (propidiumiodide-negative) and in impaired (propidiumiodide-positive) spermatozoa. The transbilayer distribution of the fluorescent phosphatidylserine and phosphatidylcholine analogues was not perturbed in intact sperm cells after cryopreservation. In those cells, the phosphatidylserine analogue became rapidly enriched on the cytoplasmic leaflet by the activity of a putative aminophospholipid translocase similar to intact cells of fresh, ejaculated samples. However, upon cryopreservation the activity of the putative aminophospholipid translocase was significantly reduced in intact cells. Employing annexin V-FITC, we found that even after cryopreservation the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet is maintained in intact cells, but not in impaired cells. The phosphatidylcholine analogue redistributed very slowly remaining essentially confined to the exoplasmic leaflet of the plasma membrane of intact cells from both fresh, ejaculated and cryopreserved samples. The physiological consequences of a perturbed transbilayer asymmetry in sperm plasma membranes is discussed.

Spectrofluorometric and microcalorimetric study of the thermal poration relevant to the mechanism of thermohaemolysis

This study sheds light on the structural changes in erythrocyte membrane during thermally induced poration, an event involved in thermohaemolysis. Two major membrane disturbing events can be induced during transient heating, the denaturation of spectrin and thermoporation. The first one precedes the latter but is not involved in it. Ethanol linearly reduces the onset temperature of both events but with different efficiencies. Thermoporation efficiency exceeds by 3.5 fold that of spectrin denaturation. Thus, at a specific concentration of ethanol (18% v/v), the poration occurs at 39.5 C, which precedes the denaturation of spectrin by 6 C. To induce and study the poration avoiding spectrin denaturation, cells were put in contact with preheated (39 C) isotonic (60mM) NaCl) media containing 18% v/v ethanol and sucrose as an osmotic protectant. After 3 min heating, the porated cells were washed, their membranes isolated and studied. The control cells were processed similarly except that they were incubated at 23 C, thus avoiding thermoporation. Using scanning microcalorimetry, the enthalpy and the temperature of denaturation of spectrin were found to be the same in control as well as in porated membranes which indicates similar spectrin structure in both membranes. While the enthalpy of denaturation of the anion channel was preserved, its denaturation temperature was lowered by 2.5 C after poration. These results confirmed that the heat denaturation of the main membrane proteins was not needed and not involved in thermoporation and, hence, in thermohaemolysis. Analysis of the fluorescence of membrane bound ANS gave an apparent increase in the number of binding sites for ANS in membranes after poration. In relation to the control, the eximerization of pyrene in porated membranes changed, depending on the location of the probe: in the domain of free lipids it decreased by 18% but it increased by 60% in the lipid milieu proximal to membrane proteins. Likewise, the eximerization of N-(3-Pyrene) maleimide bound to membrane proteins increased by 67% after poration, which proves increased intramolecular mobility of proteins following poration. The maximal efficiency for transferring energy from tryptophans to neighbouring pyrene was determined to be 0.93 in control, which is almost the same as in intact membranes, and 0.70 in porated membranes, indicating a strong decrease in the lipid/protein contact zone. This data suggests a mild conformational change, possibly an irreversible perturbance of the transbilayer distribution of membrane proteins in porated membranes in comparison to the control and intact ones.

Determination of the Hydrocarbon Core Structure of Fluid Dioleoylphosphocholine (DOPC) Bilayers by X-Ray Diffraction Using Specific Bromination of the Double-Bonds: Effect of Hydration

Changes in the structure of the hydrocarbon core (HC) of fluid lipid bilayers can reveal how bilayers respond to the partitioning of peptides and other solutes (Jacobs, R. E., and S. H. White. 1989. Biochemistry. 28:3421–3437). The structure of the HC of dioleoylphosphocholine (DOPC) bilayers can be determined from the transbilayer distribution of the double-bonds (Wiener, M. C., and S. H. White. 1992. Biophys. J.61:434–447). This distribution, representing the time-averaged projection of the double-bond positions onto the bilayer normal (z), can be obtained by means of neutron diffraction and double-bond specific deuteration (Wiener, M. C., G. I. King, and S. H. White. 1991. Biophys. J. 60:568–576). For fully resolved bilayer profiles, a close approximation of the distribution could be obtained by x-ray diffraction and isomorphous bromine labeling at the double-bonds of the DOPC sn-2 acyl chain (Wiener, M. C., and S. H. White. 1991. Biochemistry. 30:6997–7008). We have modified the bromine-labeling approach in a manner that permits determination of the distribution in under-resolved bilayer profiles observed at high water contents. We used this new method to determine the transbilayer distribution of the double-bond bromine labels of DOPC over a hydration range of 5.4 to 16 waters per lipid, which reveals how the HC structure changes with hydration. We found that the transbilayer distributions of the bromines can be described by a pair of Gaussians of 1/e half-width ABr located at z=±ZBr relative to the bilayer center. For hydrations from 5.4 waters up to 9.4 waters per lipid, ZBr decreases from 7.97±0.27Å to 6.59±0.15Å, while ABr increased from 4.62±0.62Å to 5.92±0.37Å, consistent with the expected hydration-induced decrease in HC thickness and increase in area per lipid. After the phosphocholine hydration shell was filled at ∼12 waters per lipid, we observed a shift in ZBr to ∼7.3Å, indicative of a distinct structural change upon completion of the hydration shell. For hydrations of 12–16 waters per lipid, the bromine distribution remains constant at ZBr=7.33±0.25Å and ABr=5.35±0.5Å. The absolute-scale structure factors obtained in the experiments provided an opportunity to test the so-called fluid-minus method of structure-factor scaling. We found that the method is quite satisfactory for determining the phases of structure factors, but not their absolute values.

Transbilayer distribution of cholesterol is modified in brain synaptic plasma membranes of knockout mice deficient in the low-density lipoprotein receptor, apolipoprotein E, or both proteins.

Both apolipoprotein E (apoE) and the low-density lipoprotein (LDL) receptor are present in brain; however, little is known regarding the function of these proteins in brain, in particular with respect to brain cholesterol. The role of apoE and the LDL receptor in modulating the transbilayer or asymmetric distribution of cholesterol in the exofacial and cytofacial leaflets of synaptic plasma membranes (SPMs) was examined in mutant mice deficient in apoE, the LDL receptor, or both proteins by using the fluorescent sterol dehydroergosterol and fluorescent quenching procedures. Fluidity of the exofacial and cytofacial leaflets was also measured. Cholesterol asymmetry of SPMs was altered in the mutant mice, with the largest effect observed in the LDL receptor-deficient mice. There was an approximately twofold increase in the percent distribution of cholesterol in the exofacial leaflet of the LDL receptor-deficient mice (32%) compared with C57BL/6J mice (15%). Mice deficient in apoE or both proteins also showed a significantly higher percent distribution of cholesterol (23 and 26%, respectively) in the exofacial leaflet compared with the C57BL/6J mice. Although the percent distribution of cholesterol was highest in the exofacial leaflet of the LDL receptor-deficient mice, fluidity of the exofacial leaflet of that group was significantly lower. However, the cholesterol-to-phospholipid ratio of SPMs of the LDL receptor-deficient mice was significantly lower, and this difference was largely the result of a significant increase in the total amount of SPM phospholipid. This study demonstrates for the first time that SPM lipid structure is altered in mice deficient in apoE or the LDL receptor. Although the mechanism that maintains the asymmetric distribution of cholesterol in plasma membranes is not well understood, data of the present experiments indicate that both apoE and the LDL receptor are involved in maintaining the transbilayer distribution of cholesterol.

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.

Transbilayer motion of spin-labelled phospholipids in the plasma membrane of epididymal and ejaculated ram spermatozoa.

The transbilayer movement and distribution of spin-labelled phospholipid analogues were studied in the plasma membrane of ram sperm cells isolated from functionally different regions of the epididymis (caput, cauda) and from the ejaculate. As already shown for ejaculated cells, (i) a rapid movement of aminophospholipid analogues phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet, and (ii) a slow transbilayer movement of phosphatidylcholine were observed in the plasma membrane of maturating ram sperm cells. This suggests an asymmetric steady state transbilayer distribution of phospholipids, with a preferential enrichment of aminophospholipids on the cytoplasmic leaflet of those cells. The fast inward redistribution of the aminophospholipid analogues is consistent with the presence of an aminophospholipid translocase activity in all three sperm cell preparations. The translocase activity was enhanced slightly during the epididymal transit of spermatozoa. However, compared with epididymal sperm cells, a marked increase in the aminophospholipid translocase activity and a more pronounced phospholipid asymmetry for phosphatidylethanolamine was established for ejaculated spermatozoa. The physiological relevance of the rapid removal of aminophospholipids from the exoplasmic leaflet of the plasma membrane of ram sperm cells is discussed. The quality of sperm cell fractions was characterized by the cell morphology, membrane integrity and the cellular ATP concentration.

Packing constraints and electrostatic surface potentials determine transmembrane asymmetry of phosphatidylethanol

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.

Transbilayer reorientation of phospholipid probes in the human erythrocyte membrane. Lessons from studies on electroporated and resealed cells

In order to characterize in more detail the previously observed (Dressler et al. (1983) Biochim. Biophys. Acta 732, 304–307) increases in transbilayer mobility of phospholipids in the erythrocyte membrane following electroporation at 0°C and subsequent resealing at 37°C of the cells, we have studied rates of flip and flop as well as steady state distributions of the fluorescent N-(NBD)-aminohexanoyl-analogues of the four major membrane phospholipids. Measurements comprised the passive non-mediated components as well as those mediated by specific translocases (flippase and floppase). The major new findings and insights can be summarized as follows. (1) The enhancement of passive transbilayer mobility which increases with the strength, duration, and number of field pulses at 0°C, cannot be fully reversed by subsequent resealing at 37°C. Flip-flop remains considerably elevated relative to the original values.(2) Enhanced mobilities induced by electroporation differ for the probes studied in the sequence SM⋘PS≪PC<PE. Other membrane perturbations going along with enhanced flip-flop share only in part this pattern. (3) Mediated, ATP-dependent components of flip and flop of the probes are suppressed in electroporated/resealed cells, partly due to loss of cellular Mg 2+, partly – in case of flippase – due to competition by externalized endogenous PS. (4) Electroporated/resealed cells provide an elegant means to demonstrate the contribution of various components of flip and flop to the steady state transbilayer distribution of phospholipids, in particular the rôle of passive mobility. The new, detailed information on the displacements of phospholipid between the two leaflets of the membrane bilayer in porated/resealed cells will help to understand erythrocyte shape changes following poration and during resealing (Henszen et al. (1993) Biol. Chem. Hoppe-Seyler 374, 114).

Role of membrane lipid distribution in chlorpromazine-induced shape change of human erythrocytes

This is a study of the morphology and transbilayer lipid distribution of human erythrocytes treated with chlorpromazine (CPZ) over extended time courses. At 0°C, treatment of dilauroylphosphatidyl[1- 14C]choline-labeled erythrocytes with 120 μM CPZ produced an immediate stomatocytic transformation ( t 1/2<5 min) with no concurrent change in transbilayer distribution of radiolabeled lipid, as determined by bovine serum albumin extractability. At 37°C, CPZ treatment of cells produced two sequential morphological effects: immediate stomatocytosis ( t 1/2<1 min) with no concurrent change in radiolabel transbilayer distribution, followed by gradual increase in stomatocytic extent over several hours, with concurrent redistribution of radiolabeled lipid to the inner monolayer. Cells pretreated with vanadate at 37°C exhibited a triphasic morphological response: CPZ produced immediate stomatocytosis, followed by a transient reversion to echinocytes lasting about 2 h, before returning to stomatocytic morphologies over the next several hours. The echinocytic reversion was accompanied by exposure of phosphatidylserine on the cell surface, as indicated by increased activation of exogenous prothrombinase. These findings suggest that while CPZ induces transbilayer lipid redistribution over extended time periods (which may mediate the complex morphological transformations observed), the early stomatocytic response elicited by addition of CPZ is not due to lipid reorganization.

Ultrafast Glycerophospholipid-selective Transbilayer Motion Mediated by a Protein in the Endoplasmic Reticulum Membrane

A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain phospholipids with a new fast translocation assay. Identical results were obtained with both types of probes. Transbilayer motion of glycerophospholipids was found to be much more rapid than previously reported (half-time less than 25 s) and to occur identically for phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Such transport is nonvectorial and leads to a symmetric transbilayer distribution of phospholipids. In contrast, transverse diffusion of sphingomyelin was 1 order of magnitude slower. Phospholipid flip-flop appears to occur by a protein-mediated transport process displaying saturable and competitive behavior. Proteolysis, chemical modification, and competition experiments suggest that this transport process may be related to that previously described in the endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). The relationship between phospholipid flip-flop and nonbilayer structures occurring in the endoplasmic reticulum was also investigated by 31P-NMR. Several conditions were found under which the 31P isotropic NMR signal previously attributed to nonbilayer structures is decreased or abolished, whereas transbilayer diffusion is unaffected, suggesting that the flip-flop process is independent of such structures. It is concluded that flip-flop in the endoplasmic reticulum is mediated by a bidirectional protein transporter with a high efficiency for glycerophospholipids and a low efficiency for sphingomyelin. In vivo, the activity of this transporter would be able to redistribute all changes in phospholipid composition due to biosynthetic processes between the two leaflets of the endoplasmic reticulum membranes within a time scale of seconds.

Temperature-induced changes in the transbilayer distribution of phosphatidylethanolamine in mitoplasts of rainbow trout (Oncorhynchus mykiss) liver

Increased proportions of phosphatidylethanolamine (PE) are a common adaptation of poikilothermic animals to life at cold temperatures. Although amino-phospholipids such as PE are asymmetrically distributed between the two membrane hemilayers, the impact of temperature-induced changes in phospholipid composition on PE asymmetry has not been examined. Accordingly, the distribution of PE between the inner and outer hemilayers of the inner membrane of liver mitochondria was determined in rainbow trout (Oncorhynchus mykiss) acclimated to 20°C and 5°C. Mitochondria were isolated from trout liver by a combination of differential and Nycodenz™ density-gradient centrifugation, resulting in an 11- to 15-fold enrichment in the activity of the mitochondrial enzyme marker succinate cytochrome c reductase. Interestingly, the outer mitochondrial membrane was more stable in 5°C- than 20°C-acclimated trout as indicated by the higher concentration of digitonin (0.6 vs. 0.3 mg digitonin/mg mitochondrial protein) required for the preparation of mitoplasts. Phosphatidylethanolamines in the outer hemilayer of the inner mitochondrial membrane (IMM) were identified as derivatives of 2,4,6-trinitrobenzene sulfonic acid (TNBS), a nonpenetrating reagent that reacts with primary amines. Our results demonstrate 1) a decrease in the phosphatidylcholine:phosphatidylethanolamine ratio in the IMM of 5°C- (1.43 ± 0.09) compared to 20°C- (2.31 ± 0.34) acclimated trout and 2) smaller amounts of PE in the outer hemilayer (6.0 ± 0.51%) of the IMM from 5°C- than 20°C-acclimated trout (8.1 ± 0.55%). These results indicate that the composition of the inner mitochondrial membrane is enriched in PE at cold temperatures and that this enrichment is largely restricted to, and therefore most likely to be of functional significance to, the inner hemilayer. © 1996 Wiley-Liss, Inc.

Sarcolemmal phosphatidylethanolamine reorganization during simulated ischaemia and reperfusion: reversibility and ATP dependency.

Exposure of cultured neonatal rat heart cells to simulated ischaemia results in a cessation of the spontaneous contractile activity and changes at both the level of sarcolemmal phospholipid topology and the ultrastructural level. Reperfusion at a timepoint before irreversible cell damage develops leads to a recovery of contractile activity. Furthermore, the shift in transbilayer distribution of sarcolemmal phosphatidylethanolamine in favour of the outer membrane leaflet, due to the ischaemic period, is reversed during subsequent reperfusion. Also the morphological changes (mitochondrial oedema, reorganization of the mitochondrial cristae and the formation of extrusions at the sarcolemma) are reversible. At the same time total intracellular ATP levels are restored to 80% of control. The role of cellular ATP content on sarcolemmal phospholipid topology was further studied by the use of the calcium antagonist verapamil (10 microM), which preserved cellular ATP content by inhibiting cell contractility before the onset of ischaemia. After 120 min of ischaemia, cell ATP content was still 63% of control in the presence of verapamil, versus 20% of control in untreated cells. Verapamil treatment also prevented the loss of the asymmetrical distribution of phosphatidylethanolamine and sarcolemmal disruption, the latter occurring during 120 min of ischaemia in untreated cells. It is proposed that maintenance of phospholipid asymmetry of the sarcolemma of the myocytes depends on the cellular ATP concentrations, indicating the involvement of an ATP dependent aminophospholipid translocase.

Mechanisms for recognition and phagocytosis of apoptotic lymphocytes by macrophages.

Erythrocytes have an asymmetric distribution of phospholipids across the bilayer of their plasma membranes, maintained by an ATP-dependent aminophospholipid translocase, and dissipated by activation of a non-specific lipid flipsite. Loss of asymmetry provokes recognition by the reticuloendothelial system. In vitro, enhanced phagocytosis of erythrocytes with a symmetric bilayer can be inhibited by artificial lipid vesicles made of phosphatidylserine (PS), indicating that macrophages recognize the PS that appears on the erythrocyte surface upon loss of asymmetry. It is becoming increasingly clear that these same fundamental membrane structure/function relationships established in the erythrocyte paradigm also apply to lymphocytes. All evidence suggests that lymphocytes maintain an asymmetric transbilayer distribution of phospholipids in their plasma membranes, maintained by an aminophospholipid translocase. Asymmetry is lost as part of the program of cell death, by down-regulation of the translocase and activation of the non-specific lipid flipsite, exposing PS on the cell surface. That PS exposure has functional consequences is demonstrated by the ability of artificial lipid vesicles containing PS to inhibit enhanced phagocytosis of apoptotic lymphocytes by macrophages. However, other signals besides PS are also involved in recognition of apoptotic lymphocytes. Studies with other inhibitors indicate that macrophages also utilize integrin-mediated and lectin-like recognition systems, although each is restricted to either unactivated or activated macrophages. These results indicate that although many fundamental features of recognition by the reticuloendothelial system may be analogous in erythrocytes and lymphocytes, the signals for recognition of apoptotic lymphocytes ae more complex and involve multiple recognition systems.

Structure of Mixed Multilayers of Palmitoyloleoylphosphatidylcholine and Oligo(oxyethylene glycol) Monododecyl Ether Determined by X-ray and Neutron Diffraction

The structure of oriented multilayers of the zwitterionic phospholipid palmitoyloleoylphosphatidylcholine containing nonionic surfactants (oligo(oxyethylene glycol) monododecyl ethers, C12En with n = 2, 4, and 6) was studied by X-ray and neutron diffraction. Investigations were done at T = 20 °C in the lamellar liquid-crystalline phase with surfactant/lipid molar ratios RA/L = 0.5 and 1 and hydration levels corresponding to 85 and 97% relative humidity. The transbilayer distribution of the surfactant was determined by neutron diffraction using selectively deuterated C12En. The incorporation of surfactant causes a decrease of the repeat distance and of the thickness of the hydrocarbon core of the membrane. The effect increases with the number of oxyethylene units and with the concentration of the surfactant as well as with the relative humidity. The α-methylene group of the surfactants is anchored near the boundary of the hydrophobic core. The oxyethylene moieties are mainly located in the polar membrane/water interface region. The impact of molecular disorder on the diffraction data is discussed.

A slight asymmetry in the transbilayer distribution of lysophosphatidylcholine alters the surface properties and poly(ethylene glycol)-mediated fusion of dipalmitoylphosphatidylcholine large unilamellar vesicles.

Large, unilamellar vesicles (LUV) composed of dipalmitoylphosphatidylcholine (DPPC) were made asymmetric in L-alpha-lysopalmitoylphosphatidylcholine (LPC) either by adding a small amount (total mole fraction = 0.003) of LPC to their outer leaflets (LUV-LPCout) or by extracting a small amount from outer leaflets which already contained 0.0015 mol fraction LPC (LUV-LPCin). The slow rate of the transbilayer redistribution of LPC allowed the asymmetric vesicles to be characterized with regard both to their physical properties and to their ability to fuse in the presence of poly(ethylene glycol) (PEG). The fraction of LPC extractable with bovine serum albumin was taken as a measure of LPC transbilayer asymmetry. The ratio of LPC available to that unavailable to BSA extraction was 6 for LUV-LPCout and 0.3 for LUV-LPCin. These asymmetries could not be enhanced significantly by increasing the vesicle LPC content. Measurements of the mixing and leakage of vesicle contents showed that LUV-LPCin fused in the presence of 15% (w/w) PEG without loss of contents but that LUV-LPCout did not fuse in the presence of up to 35% PEG. Vesicles prepared from palmitoyloleoylphosphatidylcholine could also be made asymmetric in LPC, but did not fuse even in the presence of 30% PEG. Quasi-elastic light scattering revealed that LUV-LPCin aggregated at 25 degrees C except when swollen by an osmotic gradient while LUV-LPCout were much less likely to aggregate. Trapped volume determinations suggested that neither type of vesicle was perfectly spherical in shape, but no correlation was found between fusogenicity and vesicle shape. Measurements of the fluorescence properties of TMA-DPH and of C6-NBD-PC suggested that the interface region of the outer leaflet of LUV-LPCin was slightly less ordered and less well packed than that of LUV-LPCout. This slight perturbation of the external vesicle surface correlated with the ability of juxtaposed vesicle bilayers to fuse.

Shape Response of Human Erythrocytes to Altered Cell pH

Alteration of red blood cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of phosphatidylcholine and phosphatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH cells. Possible roles of pH-titratable inner monolayer phospholipids were examined by assessing pH-dependent shape in cells in which their levels had been perturbed. In metabolically depleted cells and calcium-treated cells, which have altered levels of phosphatidic acid, phosphatidylinositol-4-phosphate, and/or phosphatidylinositol-4,5-bisphosphate, low cell pH was stomatocytogenic and high cell pH was echinocytogenic, as in control cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for cell pH-mediated shape change.

Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions

The fluorescence of a membrane-bound tryptophan derivative (tryptophan octyl ester, TOE) has been examined as a model for tryptophan fluorescence from proteins in membrane environments. The depth-dependent fluorescence quenching of TOE by brominated lipids was found to proceed via a dynamic mechanism with vertical fluctuations playing a central role in the process. The activation energy for the quenching was estimated to be 1.3 kcal/mole. The data were analyzed using the distribution analysis (DA) method, which extends the conventional parallax method to account more realistically for the transbilayer distributions of both probe and quencher and for possible variations in the probe's accessibility. DA provides a better fit than the parallax method to data collected with TOE in membranes formed of lipids brominated at either the 4,5, the 6,7, the 9,10, or the 11,12 positions of the sn-2 acyl chain. DA yields information on the fluorophore's most probable depth in the membrane, its conformational heterogeneity, and its accessibility to the lipid phase. Previously reported data on cytochrome b5 and melittin were reanalyzed together with data obtained with TOE. This new analysis demonstrates conformational heterogeneity in melittin and provides estimates of the freedom of motion and exposure to the lipid phase of membrane-embedded tryptophans of cytochrome b5.