Mixed-chain phosphatidylcholine bilayers: structure and properties

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.     

Structural transitions in short-chain lipid assemblies studied by (31)P-NMR spectroscopy

The self-assembled supramolecular structures of diacylphosphatidylcholine (diC(n)PC), diacylphosphatidylethanolamine (diC(n)PE), diacylphosphatidyglycerol (diC(n)PG), and diacylphosphatidylserine (diC(n)PS) were investigated by (31)P nuclear magnetic resonance (NMR) spectroscopy as a function of the hydrophobic acyl chain length. Short-chain homologs of these lipids formed micelles, and longer-chain homologs formed bilayers. The shortest acyl chain lengths that supported bilayer structures depended on the headgroup of the lipids. They increased in the order PE (C(6)) < PC (C(9)) < or = PS (C(9) or C(10)) < PG (C(11) or C(12)). This order correlated with the effective headgroup area, which is a function of the physical size, charge, hydration, and hydrogen-bonding capacity of the four headgroups. Electrostatic screening of the headgroup charge with NaCl reduced the effective headgroup area of PS and PG and thereby decreased the micelle-to-bilayer transition of these lipid classes to shorter chain lengths. The experimentally determined supramolecular structures were compared to the assembly states predicted by packing constraints that were calculated from the hydrocarbon-chain volume and effective headgroup area of each lipid. The model accurately predicted the chain-length threshold for bilayer formation if the relative displacement of the acyl chains of the phospholipid were taken into account. The model also predicted cylindrical rather than spherical micelles for all four diacylphospholipid classes and the (31)P-NMR spectra provided evidence for a tubular network that appeared as an intermediate phase at the micelle-to-bilayer transition. The free energy of micellization per methylene group was independent of the structure of the supramolecular assembly, but was -0.95 kJ/mol (-0.23 kcal/mol) for the PGs compared to -2.5 kJ/mol (-0.60 kcal/mol) for the PCs. The integral membrane protein OmpA did not change the bilayer structure of thin (diC(10)PC) bilayers.     

Effect of hydrophobic mismatch and interdigitation on sterol/sphingomyelin interaction in ternary bilayer membranes

Sphingomyelin (SM) is a major phospholipid in most cell membranes. SMs are composed of a long-chain base (often sphingosine, 18:1(Δ4t)), and N-linked acyl chains (often 16:0, 18:0 or 24:1(Δ15c)). Cholesterol interacts with SM in cell membranes, but the acyl chain preference of this interaction is not fully elucidated. In this study we have examined the effects of hydrophobic mismatch and interdigitation on cholesterol/sphingomyelin interaction in complex bilayer membranes. We measured the capacity of cholestatrienol (CTL) and cholesterol to form sterol-enriched ordered domains with saturated SM species having different chain lengths (14 to 24 carbons) in ternary bilayer membranes. We also determined the equilibrium bilayer partitioning coefficient of CTL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes containing 20mol% of saturated SM analogs. Ours results show that while CTL and cholesterol formed sterol-enriched domains with both short and long-chain SM species, the sterols preferred interaction with 16:0-SM over any other saturated chain length SM analog. When CTL membrane partitioning was determined with fluid POPC bilayers containing 20mol% of a saturated chain length SM analog, the highest affinity was seen with 16:0-SM (both at 23 and 37°C). These results indicate that hydrophobic mismatch and/or interdigitation attenuate sterol/SM association and thus affect lateral distribution of sterols in the bilayer membrane.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

X-ray diffraction evidence for fully interdigitated bilayers of 1-stearoyllysophosphatidylcholine

X-ray diffraction experiments have been performed on 1-stearoyllysophosphatidylcholine or C(18):C(0)PC as a function of hydration at temperatures below the order/disorder transition (Tm = 26.2 degrees C). At these temperatures, hydrated C(18):C(0)PC forms lamellae. The bilayer thickness, as determined by the saturation hydration method and electron-density profile, is 35-36 A, and the average area per C(18):C(0)PC molecule at the lipid/water interface is 45.5 A2. The packing geometry of C(18):C(0)PC in the lamella is proposed to adopt a fully interdigitated model in which the long C(18) acyl chain extends across the entire hydrocarbon width of the bilayer. Thus far, three different types of interdigitated bilayers are known for phosphatidylcholines. These various types of chain interdigitation are discussed in terms of the chain length difference between the sn-1 and sn-2 acyl chains.     

Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport

The structural and functional consequences of a mismatch between the hydrophobic thickness d(P) of a transmembrane protein and that d(L) of the supporting lipid bilayer were investigated using melibiose permease (MelB) from Escherichia coli reconstituted in a set of bis saturated and monounsaturated phosphatidylcholine species differing in acyl-chain length. Influence of MelB on the midpoint gel-to-liquid-phase transition temperature, T(m), of the saturated lipids was investigated through fluorescence polarization experiments, with 1,6-diphenyl-1,3,5-hexatriene as the probe, for varying protein/lipid molar ratio. Diagrams in temperature versus MelB concentration showed positive or negative shifts in T(m) with the short-chain lipids DiC12:0-PC and DiC14:0-PC or the long-chain lipids DiC16:0-PC and DiC18:0-PC, respectively. Theoretical analysis of the data yielded a d(L) value of 3.0 +/- 0.1 nm for the protein, similar to the 3.02 nm estimated from hydropathy profiles. Influence of the acyl chain length on the carrier activity of MelB was investigated in the liquid phase, using the monounsaturated PCs. Binding of the sugar to the transporter showed no dependence on the acyl chain length. In contrast, counterflow and Deltapsi-driven experiments revealed strong dependence of melibiose transport on the lipid acyl chain length. Similar bell-shaped transport versus acyl chain length profiles were obtained, optimal activity being supported by diC16:1-PC. On account of a d(P) value of 2.65 nm for the lipid and of various local constraints which would all tend to elongate the acyl chains in contact with the protein, one can conclude that maximal activity was obtained when the hydrophobic thickness of the bilayer matched that of the protein.     

Thermotropic phase behavior of mixed-chain phosphatidylglycerols: implications for acyl chain packing in fully hydrated bilayers

In this communication we report the first systematic investigation of the thermodynamic properties of fully hydrated mixed-chain phosphatidylglycerols (PG) using high-resolution differential scanning calorimetry (DSC). The crystal structure of dimyristoylphosphatidylglycerol shows an acyl chain conformation that is nearly opposite to that of phosphatidylcholine (PC). In PC, the sn-1 chain is straight while the sn-2 chain contains a bend; for PG, the sn-1 contains a bend while the sn-2 chain is in the all-trans conformation (R.H. Pearson, I. Pascher, The molecular structure of lecithin dihydrate, Nature, 281 (1978) 499-501; I. Pascher, S. Sundell, K. Harlos, H. Eibl, Conformational and packing properties of membrane lipids: the crystal structure of sodium dimyristoylphosphatidylglycerol, Biochim. Biophys. Acta, 896 (1987) 77-88). If the structure of PG found in the single crystal can be extrapolated to that in the fully hydrated gel-state bilayer, the observed difference in acyl chain conformations implies that modulation of the acyl chain asymmetry will have an opposite effect on the thermotropic phase behavior of PG and PC. For example, it is expected, based on the crystal structures, that C(15):C(13)PG should have a higher main phase transition temperature (Tm) than C(14):C(14)PG, and C(13):C(15)PG should have a lower Tm than C(14):C(14)PG. However, our DSC studies show clearly that the expectation is not borne out by experimental data. Rather, the Tm values of C(15):C(13)PG, C(14):C(14)PG, and C(13):C(15)PG are 18.2 degrees C, 23.1 degrees C, and 24.4 degrees C, respectively. Several other PGs, each with a unique acyl chain composition, have also been studied in this laboratory using high-resolution DSC. It is shown that the acyl chain conformation of fully hydrated PG in general is nearly opposite to that seen in the PG crystal structure.     

Effects of aromatic residues at the ends of transmembrane alpha-helices on helix interactions with lipid bilayers

We have studied the effects of aromatic residues at the ends of peptides of the type Ac-KKGL(n)()WL(m)()KKA-amide on their interactions with lipid bilayers as a function of lipid fatty acyl chain length, physical phase, and charge. Peptide Ac-KKGFL(6)WL(8)FKKA-amide (F(2)L(14)) incorporated into bilayers of phosphatidylcholines containing monounsaturated fatty acyl chains of lengths C14-C24 at a peptide:lipid molar ratio of 1:100 in contrast to Ac-KKGL(7)WL(9)KKA-amide (L(16)) which did not incorporate at all into dierucoylphosphatidylcholine [di(C24:1)PC]; Ac-KKGYL(6)WL(8)YKKA-amide (Y(2)L(14)) incorporated partly into di(C24:1)PC. Lipid-binding constants relative to that for dioleoylphosphatidylcholine (C18:1)PC were obtained using a fluorescence quenching method. For Y(2)L(14) and F(2)L(14), relative lipid-binding constants increased with increasing fatty acyl chain length from C14 to C24; strongest binding did not occur at the point where the hydrophobic length of the peptide equalled the hydrophobic thickness of the bilayer. For Ac-KKGYL(9)WL(11)YKKA-amide (Y(2)L(20)), increasing chain length from C18 to C24 had little effect on relative binding constants. Anionic phospholipids bound more strongly than zwitterionic phospholipids to Y(2)L(14) and Y(2)L(20) but effects of charge were relatively small. In two phase (gel and liquid crystalline) mixtures, all the peptides partitioned more strongly into liquid crystalline than gel phase; effects were independent of the structure of the peptide or of the lipid (dipalmitoylphosphatidylcholine or bovine brain sphingomyelin). Addition of cholesterol had little effect on incorporation of the peptides into lipid bilayers. It is concluded that the presence of aromatic residues at the ends of transmembrane alpha-helices effectively buffers them against changes in bilayer thickness caused either by an increase in the chain length of the phospholipid or by the presence of cholesterol.     

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T(m)) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T(m) to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T(m) value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.     

Selectivity in lipid binding to the bacterial outer membrane protein OmpF

The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.     

Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis

The effect of the phospholipid acyl chain carbon number on the activity of the branched-chain amino acid transport system of Lactococcus lactis has been investigated. Major fatty acids identified in a total lipid extract of L. lactis membranes are palmitic acid (16:0), oleic acid (18:1) and the cyclopropane-ring containing lactobacillic acid (19 delta). L. lactis membrane vesicles were fused with liposomes prepared from equimolar mixtures of synthetic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) with cis mono-unsaturated acyl chains. The activity of the branched-chain amino acid carrier is determined by the bulk properties of the membrane (Driessen, A.J.M., Zheng, T., In 't Veld, G., Op den Kamp, J.A.F. and Konings, W.N. (1988) Biochemistry 27, 865-872). PE acts as an activator and PC is ineffective. Counterflow and protonmotive-force driven transport of leucine is sensitive to changes in the acyl chain carbon number of both phospholipids and maximal with dioleoyl-PE/dioleoyl-PC. Above the gel to liquid-crystalline phase transition temperature of the lipid species, membrane fluidity decreased with increasing acyl chain carbon number. Our data suggest that the carbon number of the acyl chains of PE and PC determine to a large extent the activity of the transport system. This might be relevant for the interaction of PE with the transport protein. Variations in the acyl chain composition of PC exert a more general effect on transport activity. The acyl chain composition of phospholipids determines the membrane thickness (Lewis, B.A. and Engelman, D.M. (1983) J. Mol. Biol. 166, 211-217). We therefore propose that the degree of matching between the lipid-bilayer and the hydrophobic thickness of the branched-chain amino acid carrier is an important parameter in lipid-protein interactions.     

Membrane thickening by the antimicrobial peptide PGLa

Using x-ray diffraction, solid-state ²H-NMR, differential scanning calorimetry, and dilatometry, we have observed a perturbation of saturated acyl chain phosphatidylglycerol bilayers by the antimicrobial peptide peptidyl-glycylleucine-carboxyamide (PGLa) that is dependent on the length of the hydrocarbon chain. In the gel phase, PGLa induces a quasi-interdigitated phase, previously reported also for other peptides, which is most pronounced for C18 phosphatidylglycerol. In the fluid phase, we found an increase of the membrane thickness and NMR order parameter for C14 and C16 phosphatidylglycerol bilayers, though not for C18. The data is best understood in terms of a close hydrophobic match between the C18 bilayer core and the peptide length when PGLa is inserted with its helical axis normal to the bilayer surface. The C16 acyl chains appear to stretch to accommodate PGLa, whereas tilting within the bilayer seems to be energetically favorable for the peptide when inserted into bilayers of C14 phosphatidylglycerol. In contrast to the commonly accepted membrane thinning effect of antimicrobial peptides, the data demonstrate that pore formation does not necessarily relate to changes in the overall bilayer structure.     

Effect of fatty acyl domain of phospholipids on the membrane-channel formation of Staphylococcus aureus alpha-toxin in liposome membrane.

By use of carboxyfluorescein-loaded multilamellar liposomes prepared from synthetic phosphatidylcholine (PC) or sphingomyelin and cholesterol in a molar ratio of 1:1, we studied whether or not fatty acyl domain of the phospholipids affects the membrane-damaging action (or channel formation) of Staphylococcus aureus alpha-toxin on the phospholipid-cholesterol membranes. Our data indicated: (1) that toxin-induced carboxyfluorescein-leakage from the liposomes composed of saturated fatty acyl residue-carrying PC and cholesterol was decreased with increasing chain length of the acyl residues between 12 and 18 carbon atoms, although toxin-binding to the liposomes was not significantly affected by the length of fatty acyl residue; (2) that unsaturated fatty acyl residue in PC or sphingomyelin molecule conferred higher sensitivity to alpha-toxin on the phospholipid-cholesterol liposomes, compared with saturated fatty acyl residues; and (3) that hexamerization of alpha-toxin, estimated by SDS-polyacrylamide gel electrophoresis, occurred more efficiently on the liposomes composed of PC with shorter fatty acyl chain or unsaturated fatty acyl chain. Thus, hydrophobic domain of the phospholipids influences membrane-channel formation of alpha-toxin in the phospholipid-cholesterol membrane, perhaps by modulating packing of phospholipid, cholesterol and the toxin in membrane.     

Hydrocarbon chain packing and the effect of ethanol on the thermotropic phase behavior of mixed-chain phosphatidylglycerols

Previous studies in this laboratory have delineated the relationship between the acyl chain asymmetry of mixed-chain phosphatidylcholines and the effect of ethanol concentration ([EtOH]) on their melting behavior (Li et al., Biophys J., 70 (1996) 2784-2794). This present investigation extends these findings to another phospholipid family by using high-resolution differential scanning calorimetry (DSC) to characterize the effect of ethanol concentration on the main phase transition temperature (Tm) of five molecular species of mixed-chain phosphatidylglycerol (PG). For C(14):C(18)PG, C(15):C(17)PG, C(16):C(16)PG, and C(17):C(15)PG, a biphasic profile in the Tm versus [EtOH] plot was observed, and the minimum in the plot for each PG occurred at 33, 15, 19, and 36 mg/ml, respectively. This biphasic behavior is typical of phospholipids whose acyl chain asymmetry is fairly small. For C(18):C(14)PG, only a linear decrease in the Tm was observed as a function of ethanol concentration; this effect is characteristic of highly asymmetric phospholipids. Our DSC results obtained with mixed-chain PG in the presence of ethanol demonstrate that the acyl chain asymmetry of the five lipids studied can be ranked as follows: C(15):C(17)PG相似文献   

Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics

The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of monounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transmembrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be important for the conformational mobility of the Na,K-ATPase changing the distribution of conformations. Lipid fluidity was important for several parameters of reconstitution, e.g., the amount of protein inserted and the orientation in the liposomes. The temperature dependence of the Na,K-ATPase as well of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATPase activity of Na,K-ATPase reconstituted with monounsaturated phospholipids. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnover. In the case of ATPase reconstituted without cholesterol, the minimum free energy of activation and the maximum turnover both shifted to longer acyl chain lengths of about 22.     

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Molecular mechanisms for the induction of peroxidase activity of the cytochrome c-cardiolipin complex

Induction of the peroxidase activity of cytochrome c (cyt c) by cardiolipin (CL) and H(2)O(2) in mitochondria is suggested to be a key event in early apoptosis. Although electrostatic interaction between the positively charged cyt c and negatively charged CL is a predominant force behind the formation of a specific cyt c-CL complex and sequential induction of the peroxidase activity, molecular mechanisms of hydrophobic interactions involving the fatty acyl chains of CL remain to be investigated. To elucidate the function of the acyl chains, particularly the role of the double bond, we synthesized a variety of CL analogues and examined their peroxidase inducing activity. Irrespective of the number of double bonds in the acyl chains, the peroxidase activity of cyt c induced by liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) and a different CL (9:1 molar ratio) was similar, except for that of 1,1',2,2'-tetrastearoylcardiolipin (TSCL, C18:0)-containing liposomes. The peroxidase inducing activity of TSCL-containing liposomes was 3-4-fold greater than that of other CL-containing liposomes. The peroxidase activity induced by all CL-containing liposomes was much lower at high ionic strengths than that at low ionic strengths because of diminution of the electrostatic interaction. The peroxidase inducing effects of various CL-containing liposomes were related well to their ability to associate with cyt c. Thus, our results revealed that at low CL levels, the saturated acyl chain of CL is favorable for the activation of peroxidase activity of CL-bound cyt c and the proposed critical role of the double bond is not a general feature of the cyt c-CL interaction. The polarity of the membrane surface of TSCL-containing liposomes was slightly, but significantly, lower than that of other CL-containing liposomes, suggesting that the higher activating ability of TSCL-containing liposomes may be due to a reduced level of hydration of the polar head region reflecting tighter packing of the fully saturated acyl chains. Moreover, using CL analogues in which a central glycerol head moiety was modified, we revealed that the natural structure of the head moiety is not critical for the formation of the active cyt c-CL complex. The effects of the CL content of the liposomal membrane on the cyt c-CL interaction are discussed.     

Acyl and alkyl chain length of GPI-anchors is critical for raft association in vitro

We determined the acyl and alkyl chain composition of GPI-anchors isolated from MDCK and Fischer rat thyroid (FRT) cells. Both cell lines synthesize GPI-anchors containing C16/C18 or C18/C18 saturated acyl and alkyl chains. The GPI-anchored placental alkaline phosphatase (PLAP) expressed in both cells is raft-associated and PLAP purified from FRT cells is raft-associated in vitro when reconstituted into liposomes containing raft lipids. In contrast, the GPI-anchored variant surface glycoprotein from Trypanosoma brucei which contains C14 acyl and alkyl chains shows no significant raft association after reconstitution in vitro. These data indicate that the acyl and alkyl chain composition of GPI-anchors determines raft association.