Dispersions Of Dimyristoylphosphatidylcholine Research Articles

Effects of polypeptide-phospholipid interactions on bilayer reorganizations Raman spectroscopic study of the binding of polymyxin B to dimyristoylphosphatidic acid and dimyristoylphosphatidylcholine dispersions

The interactions of the antibiotic polymixin B, a polycationic cyclic polypeptide containing a branched acyl side chain, with dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidic acid (DMPA) bilayers were investigated by Raman spectroscopy for a wide range of lipid/polypeptide mole fractions. Temperature profiles, constructed from peak height intensity ratios derived from the lipid methylene C-H stretching and acyl chain C-C stretching mode regions, reflected changes originating from lateral chain packing effects and intrachain trans / gauche rotamer formation, respectively. For DMPC/polymyxin B bilayers the temperature dependent curves indicate a broadening of the gel-liquid crystalline phase transition accompanied by an approx. 3 C deg. increase in the phase transition temperature from 22.8°C for the pure bilayer to 26°C for the polypeptide complex. For a 10:1 lipid/polypeptide mole ratio the temperature profile derived from the C-C mode spectral parameters displays a second order/disorder transition, at approx. 35.5°C, associated with the melting behavior of approximately three bilayer lipids immobilized by the antibiotic's charged cyclic headgroup and hydrophobic side chain. For the 10:1 mole ratio DMPA/polypeptide liposomes, the temperature profiles indicate three order/disorder transitions at 46, 36 and 24°C. Pure DMPA bilayers display a sharp lamellar-micellar phase transition at 51°C.

Interaction of the serum amyloid A proteins with phospholipid.

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.

The effect of cholesterol and gramicidin A′ on the carbonyl groups of dimyristoylphosphatidylcholine dispersions

Proton-enhanced carbon-13 magnetic resonance measurements have been made of the natural abundance carbon-13 carbons in hydrated L α phase dispersions of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) codispersed with cholesterol or with the polypeptide gramicidin A′. The carbonyl group spectrum consists of a superposition of two peaks derived from the two carbonyl sites within the lipid. In the L α phase of DMPC both carbonyl sites contribute axially symmetric spectra, one with a chemical shift anisotropy of –29 ppm and the other with a chemical shift anisotropy of less than –5 ppm. The chemical shift anisotropy of the broader carbonyl resonance was found to increase with increasing cholesterol content. However, in DMPC dispersions with gramicidin A′, the chemical shift anisotropy of the broader carbonyl signal initially increased slightly from that of pure DMPC and then decreased with increasing concentrations of gramicidin A′. The width of the narrower spectral component was essentially unaltered by cholesterol or gramicidin A′. The presence of a narrow component at all concentrations of cholesterol or gramicidin A′ suggests that it is unlikely that any significant conformational changes have occurred at the carbonyl level of the bilayer. We propose that the major effect of cholesterol or gramicidin A′ is to alter the molecular order parameter, S mol, which reflects the range of angles through which the local molecular long axis of the phospholipid is tumbling.

Studies on the soap films stabilized by phospholipids: I. Effects of metal ions on the free energy of black soap films

In order to study the interaction of some phosphorylcholine-containing lipids with mono-, di-, and trivalent cations over a wide concentration range, the free energy (Δ F) of formation of black soap film has been obtained by direct measurement of film tension. The black films were prepared from sonicated aqueous dispersions of dimyristoylphosphatidylcholine as well as from solutions of palmitoyllysophosphatidylcholine and hexadecylphosphorylcholine at concentrations above the CMC. Monovalent cations give little effect, whereas di- and trivalent cations show remarkable effects on Δ F in the three lipids. The effects of ions on the reduction of | ΔF| at the lower concentrations of electrolytes were compared with the affinity of the ions for the film molecules derived from the DLVO-type theory. The effects of ions at higher concentrations must be explained by the specific interaction of calcium and magnesium chlorides with phospholipids, in addition to the shielding effect. It was observed that two types of black films coexisted in the same film near the electrolyte concentrations, at which | ΔF| gives the minimal value. This was taken to be the transition from Newton black film to common one. The fact that the black soap films of the phospholipids resemble the multilamellar phase in their physical properties, indicates that soap films stabilized by phosphatidylcholine are suitable for the study of model membranes, and particularly the interaction between membranes.

Electron-electron double resonance and saturation-recovery studies of nitroxide electron and nuclear spin-lattice relaxation times and Heisenberg exchange rates: lateral diffusion in dimyristoyl phosphatidylcholine.

Lateral diffusion constants of the stearic acid nitroxide radical spin label 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolindinyl oxide in dispersions of dimyristoyl phosphatidylcholine have been measured. Electron-electron double resonance methods were used to determine the product of the bimolecular collision frequency and T1e, the electron spin-lattice relaxation time. T1e in turn was measured by the technique of saturation recovery. The theoretical model of Träuble and Sackmann was then used to relate the bimolecular collision frequencies to the diffusion constants. Results are in agreement with other methods. Lower spin-label concentrations than were used in previous electron paramagnetic resonance studies are needed (label-to-lipid ratio less than 0.5 mol%). Analysis of the data also yields values of the nitrogen nuclear spin-lattice relaxation time of the nitroxide moiety. These values are indicative of membrane fluidity.

Mobilization of cholesterol from cholesterol ester-enriched tissue culture cells by phospholipid dispersions

The accumulation of cholesterol esters in foam cells of the arterial intima is an important characteristic of fatty streak lesions of atherosclerosis. We wished to know if cholesterol ester accumulations in cells could be mobilized by altering their external milieu. Thus, phospholipid dispersions were used to remove cholesterol from a cholesterol ester-enriched cell line. Rat hepatoma cells, Fu5AH, were loaded with cholesterol esters by incubation in medium supplemented with hyperlipemic rabbit serum. After removing the loading medium, we incubated the cells in serum-free medium containing egg phosphatidylcholine dispersions. Unesterified cellular cholesterol level decreased in the first 4 h and then remained at a constant level. The cholesterol esters decreased after a lag time of about 2 h and the triacylglycerol level increased after 3 h. The decrease in cellular cholesterol ester depended on the amount of phospholipid in the medium. Cellular cholesterol ester decreased with increasing concentration of medium phospholipid to 2 μmol/ml and then plateaued. The removed cellular sterols appeared in the medium as free cholesterol. Since there was no measurable cholesterol esterase activity in the medium, the cholesterol ester in the cells was hydrolyzed before it appeared in the medium. The fatty acyl composition of the cellular cholesterol esters remained unchanged after significant reduction, suggesting that the hydrolysis of cholesterol esters was not specific for the acyl chain. Sphingomyelin and dimyristoyl phosphatidylcholine dispersions, though cytotoxic, were also effective in reducing cellular cholesterol esters. These experiments demonstrate that cholesterol ester accumulations in these cells can be reduced when phospholipid dispersions are used as cholesterol acceptors in the extracellular medium.

Effects of oxygen on EPR spectra of nitroxide spin-label probes of model membranes

The use of a methylpentene polymer, TPX, for construction of sample containers that allow easy equilibration of electron paramagnetic resonance samples with nitrogen is described. The effects of oxygen-dependent shortening of the electron spin relaxation times of nitroxide spin labels were studied in dispersions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylchohne (DPPC). First-harmonic, in-phase, absorption spectra of deoxygenated samples of 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16SASL) in DMPC display decreased linewidths and increased peak-to-peak heigths and resolution of 13C splittings. Continuous-wave (cw) saturation studies of 16SASL/DMPC and both lipid- and aqueous-phase components of 2,2,6,6-tetramethyl-piperidinooxyl (Tempo) partitioned into DPPC show that the rf field at which the signal intensity is maximized decreases when aerated samples are equilibrated with nitrogen. Second-harmonic, out-of-phase, absorption (saturation transfer) spectra of deoxygenated samples of 16SASUDMPC at −22°C and 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (5SASL) in DPPC at 35°C display increased signal intensity and lineshape changes. Electron-electron double resonance (ELDOR) spectra display much greater ELDOR reduction in signal intensity when a deoxygenated sample of 16SASL/DMPC is used. Our results indicate that the routine use of deoxygenated samples in biologically relevant studies using spin-label probes should be considered.

Exchange and aggregation in dispersions of dimyristoyl phosphatidylcholine vesicles containing myristic acid.

Processes occurring in dispersions of dimyristoyl phosphatidylcholine containing myristic acid have been studied by light scattering of dilute dispersions (concn. less than or equal to 1 mg/ml) at temperatures above and below the phase transition temperatures of these dispersions. The transition temperatures increase with increasing mol fraction of myristic acid. Above these temperatures, vesicles with different mol fractions of myristic acid exchange lipid molecules. The exchange process leads to vesicles having phase transition temperatures and radii, which ar both intermediate between the initial transitions and radii, respectively. In contrast with the observations above the phase transitions, it was found that when dimyristoyl phosphatidylcholine/myristic acid vesicles were cooled to a few degrees below the phase transition, larger particles were formed. These observations are consistent with a mechanism consisting of vesicle aggregation followed by fustion of the aggregated vesicles. The aggregation process is of second order in the vesicle concentration, and its rate increases with increasing mol fraction of myristic acid.

Cooperativity of the phase transition in single- and multibilayer lipid vesicles

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersions of dimyristoyl phosphatidylcholine, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6- tetramethylpiperdine-N- oxyl , phosphatidylcholine and stearic acid, and also 90° light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526–535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/✓σ, is a measure of the mean number of molecules, per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.