Equilibrium studies of lecithin-cholesterol interactions I. Stoichiometry of lecithin-cholesterol complexes in bulk systems

The maximum molar ratio of lecithin:cholesterol in aqueous dispersions has been reported to be 2:1, 1:1, or 1:2. The source of the desparate results has been examined in this study by analyzing (a) the phase relations in anhydrous mixtures (from which most dispersions are prepared) and (b) various methods of preparing aqueous dispersions, with the purpose of avoiding the formation of metastable states that may be responsible for the variability of the lecithin-cholesterol stoichiometry. Temperature-composition phase diagrams for anhydrous mixtures of cholesterol (CHOL) with dimyristoyl (DML) and with dipalmitoyl (DPL) lecithin were obtained by differential scanning calorimetry (DSC). Complexes form with molar ratios for lecithin:CHOL of 2:1 and 1:2; they are stable up to 70°C. When x(CHOL) < 0.33, two phases coexist: complex (2:1) plus pure lecithin; when 0.33 < x(CHOL) < 0.67 complexes (2:1) and (1:2) coexist as separate phases. The corresponding phase diagram in water for these mixtures was determined by DSC and isopycnic centrifugation in D₂O-H₂O gradients. Aqueous dispersions were prepared by various methods (vortexing, dialysis, sonication) yielding identical results except as noted below. The data presented supports the following phase relations. When x(CHOL) < 0.33, two lipid phases coexist: pure lecithin plus complex (2:1) where the properties of the lecithin phase are determined by whether the temperature is below or above T_c, the gel-liquid crystal transition temperature. Therefore, complex (2:1) will coexist with gel state below T_c and with liquid crystal above T_c. The densities follow in the order gel > complex (2:1) > liquid crystal. The density of complex (2:1) is less sensitive to temperature in the range 5°-45°C compared to the temperature dependence for DML and DPL where large changes in density occur at T_c. When x(CHOL) > 0.33, CHOL phase coexists with complex (2:1); anhydrous complex (1:2) is apparently not stable in H₂O. The results are independent of the method and temperature used for preparing the lipid dispersions. However, when dispersions are prepared by sonication or with solvents at T > T_c, an apparent 1:1 complex is formed. Evidence suggests the 1:1 complex is metastable.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

The effect of sonication on the hydrocarbon chain conformation in model membrane systems: A Raman spectroscopic study

Raman spectra are presented for egg lecithin above and below the gel-liquid crystal phase transition, and several regions of the Raman spectrum are shown to be sensitive to conformational changes in the hydrocarbon chains. These regions are used to investigate the effect of sonication on the structure of egg lecithin and dipalmitoyl lecithin vesicles.Sonication of both egg lecithin above T_m, and dipalmitoyl lecithin above and below T_m produces no change in the relative population of trans and gauche isomers in any of the systems studied. Sonication does however appear to effect interchain interactions, a possible consequence of imperfect packing towards the center of the bilayers in vesicle systems.     

Probing water compartments and membrane permeability in plant cells by 1H NMR relaxation measurements

¹H NMR relaxation times (T₁ and T₂) in parenchyma tissue of apple can identify three populations of water with different relaxation characteristics. By following the uptake of Mn²⁺ ions in the tissue it is shown that the observed relaxation times originate from particular water compartments: the vacuole, the cytoplasm, and the cell wall/extracellular space.

Proton exchange between these compartments is controlled by the plasmalemma and tonoplast membranes. During the Mn²⁺ penetration experiment, conditions occur that cause the relaxation times of protons of cytoplasmic water to be much shorter than their residence time in the cytoplasm. Then the tonoplast permeability coefficient P_d for water can be calculated from the vacuolar T₁ and T₂ values to be 2.44 10^-5 m·s^-1.

     

Oxygen diffusion in phospholipid artificial membranes studied by Fourier transform nuclear magnetic resonance

Spin-lattice (T_i) relaxation mesurements can provide information about the presence of oxygen in the environment of a nucleus, since oxygen, by virtue of its paramagnetic properties, increases T_i relaxation rates. Spin-lattice relaxation times were measured for the choline, fatty acid methylene, and fatty acid methyl protons of sonicated dimyristoyl phosphatidyl choline vesicles in D₂O at several oxygen pressures. The increase in relaxation rate due to oxygen was found to be greater for the fatty acid resonances than for the choline resonance. This was interpreted to indicate the presence of oxygen in the hydrocarbon core of the bilayer. In addition, the T_i relaxation data permitted calculation of the oxygen diffusion coefficient in the water and lipid phases.     

Magnetic resonance studies on membrane and model membrane systems: III. Fatty acid motions in aqueous lecithin dispersions

Magnetic resonance spectra and relaxation rates of sonicated and unsonicated vesicles of egg yolk lecithin are reviewed and compared. The NMR relaxation rates differ by about two orders of magnitude while the ESR order parameters show no such variation. The apparent contradiction may be removed by proposing that the ESR data reflect the order of segments of the fatty acids while the NMR relaxation rates reflect positional fluctuations. Macroscopic vesicular tumbling contributes insignificantly to the relaxation rates. Resonance and non-resonance data converge on a dynamic model in which the fatty acid molecules are configurationally mobile yet relatively ordered.     

1H-NMR Relaxation Investigation of Water Bound to Bovine Rod Outer Segment Disk Membranes

Spin-lattice relaxation times T₁ in deuterated aqueous dispersions of lecithin and rod outer segment disk membranes were measured at various concentrations and temperatures. Fast chemical exchange between two loosely defined phases of water molecules was shown to fit the data, allowing the dynamic features of “bound” water and the hydration of the biological membrane to be evaluated. The state of the water was shown to be also involved in vision physiology.     

The nature of bile salt micelles as studied by deuterium NMR

Deuterium NMR of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid was studied. Molecular sizes obtained from deuterium spin-lattice relaxation time (T₁) data of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in methanol and in water are in accordance with monomeric and tetrameric structures in the two media, respectively. The deuterium T₁ and intensity of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in aqueous solution at pH 8.0–8.8 were studied as functions of NaCl and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaCl, and that 3α, 12α-dihydroxy-7,7-dideutero-5β-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

Preparation and characterization of large dioctadecyldimethylammonium chloride liposomes and comparison with small sonicated vesicles

Dioctadecyldimethylammonium chloride (DODAC) unilamellar liposomes with a mean external diameter of 0.5 μm and sharp gel-to-liquid-crystalline phase transition temperatures (T_c) were obtained by chloroform vaporization and compared with small sonicated DODAC vesicles. Sucrose, impermeant through large DODAC liposomes and sonicated vesicles, was used for internal volume determinations. The internal volumes for large DODAC liposomes and sonicated DODAC vesicles were 9.0 ± 1.3 and 0.13 ± 0.2 l/mol, respectively. Ideal osmometer behaviour, towards KCl (0–50 mM) and sucrose, was observed only for large DODAC liposomes. Sonicated DODAC vesicles were osmotically non-responsive towards sucrose and flocculated upon addition of KCl. At temperatures near the T_c, a steep increase in the initial shrinkage rate and a minimum for the total extent of shrinkage were observed for large DODAC liposomes. Large DODAC liposomes are proposed as an adequate synthetic membrane model.     

Carbon-13 nuclear magnetic resonance studies of cerebroside derivatives and their properties in lecithin bilayers (1)

The ¹³C NMR chemical shifts and spin-lattice relaxation times of D-galactosylsphingosine derivatives in CDCl₃-CD₃OD and in egg-yolk lecithin vesicles in D₂O, and of N-acetylpsychosine micelles, are reported. Results with sonicated, unilamellar vesicles containing cerebroside and EYL^a show that (1) cerebrosides decrease the fluidity of the lecithin bilayer membrane and have the greatest effect on the glycerol backbone and choline methyl carbons. (2) N-acetylpsychosine experiences a greater freedom of motion in the galactose region than does cerebroside and does not reduce the fluidity of the lecithin as much as cerebroside. (3) Ac-Psy/EYL vesicles formed are permeable to Yb³⁺ but cerebroside/lecithin vesicles are not. (4) The choline groups on the inner bilayer surface are less mobile than those on the outer surface according to preliminary T₁ measurements of the Yb³⁺-separated resonances. (5) Yb³⁺-induced chemical shifts of choline methyl and choline $\text{C}\text{H}{}_2$OP peaks in mixed cerebroside-lecithin vesicle systems indicate a small preference for cerebroside in the outside monolayer. The data show that these molecules have significant effects on bilayer conformational mobilities, particularly near the surface, and thus demonstrate one mechanism for modulation of cell surface properties by glycosphingolipids.     

HeteroTOCSY-based experiments for measuring heteronuclear relaxation in nucleic acids and proteins

Summary While both ³¹P and ¹¹³Cd are present at locations of interest in many different macromolecular systems, heteronuclear-detected relaxation measurements on these nuclei have been restrained by limitations in either resolution or signal-to-noise ratio. We have developed hetero TOCSY-based methods to overcome both of these problems. Two-dimensional versions of these experiments were utilized to measure ³¹P T₁ and T₂ values in DNA oligonucleotides; the additional resolution offered by a second dimension allowed determination of these values for most of the ³¹P resonances in a DNA dodecamer. The results from the experiments indicated that there was little significant variation in T₁ values for the different phosphates in the DNA dodecamer; however, the T₂ values showed a clear pattern, with lower values in the interior of the sequence than at the ends of the helix. Furthermore, a significant correlation between ³¹P chemical shifts and T₂ values was observed. One-dimensional, frequency-selective versions of these experiments were also developed for use on systems containing a smaller number of heteronuclear spins. These methods were applied to investigate the heteronuclear relaxation properties of ¹¹³Cd in ¹¹³Cd₂LAC9(61), a Cys₆Zn₂ DNA-binding domain. Data from the experiments confirm biochemical evidence that more significant differences occur in the metal-protein interactions between the two metal-binding sites than has been previously identified for proteins containing this motif.     

Anisotropic and restricted molecular motion in lecithin bilayers

A treatment has been developed which accounts for the prominent features of the PMR spectra and proton relaxation data of unsonicated lecithin bilayers. The spin-lattice relaxation time T₁ and the spin-spin relaxation time T₂ of the protons of a methyl top undergoing anisotropic and restricted reorientation have been calculated employing the theory of Woessner, but modified to include the effects of orientational anisotropies on long time scales. Analysis of the nmr data in terms of this theory permitted determination of the mobility of the lecithin hydrocarbon chains in the bilayer phase. These results indicate that the hydrophobic core of an unsonicated bilayer is more appropriately described as a soft solid rather than as a highly mobile fluid, contrary to the suggestions of recent esr spin label studies.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

Single bilayer vesicles prepared without sonication. Physico-chemical properties.

Single shelled lecithin vesicles of uniform size (diameter = 300 A) are prepared without sonication by solubilizing unsonicated lecithin dispersions with sodium cholate and removing the detergent from the mixed lecithin - cholate micelles by gel filtration on Sephadex G-50. A homogeneous population of pure lecithin single-bilayer vesicles free of multilamellar structures is obtained. The vesicle diameter is somewhat larger than the average diameter of sonicated vesicles. The curvature of the bilayer seems to be sufficiently large to allow for similar packing densities (areas/molecule) on the outer and inner layer of the bilayer. The morphology and some physico-chemical properties of these vesicles are described and compared with those of sonicated vesicles.     

Lipid requirements for axenic cultivation ofEntamoeba histolytica

Fatty acids, cholesterol and glucose present in axenic medium are utilized by growingEntamoeba histolytica but the amoeba is unable to synthesize cholesterol from [U^-14 C^- ] glucose although the label is incorporated into the fatty acids and non-saponifiable fractions of the organism. Exogenously-added sonicated dispersions of cholesterol, Β-sitosterol, lanosterol, lecithin and lauric, palmitic, linoleic and stearic acids are ingested by the amoebae with subsequent loss in amoeboid movement. After a few hours the movement is regained. Cholesterol, lecithin and the fatty acids stimulate amoebic multiplication but are unable to replace serum in the medium either singly or in combination. CDRI Communication No. 2516.     

NMR observation of the interaction of small oligopeptides with phospholipid vesicles

Using proton spin-lattice relaxation times, the interaction of small oligopeptides with sonicated vesicles of synthetic β-γ-dimyristoyl L-α-lecithin has been monitored at 29°C in D₂O. The measured relaxation times for the lecithin choline methyl, alkyl chain, and terminal methyl protons were observed to shorten markedly with increasing concentration of peptide, the relaxation remaining exponential. Noticeable resonance broadening was observed at the highest peptide concentration studied. The data reported are for the effect of the pharmacologically active pentapeptide methionine-enkephalin. Similar results have been observed for the effect of tetraglycine. The relaxation of the observable resonances of the added peptide appear to be unaffected. The results are discussed in terms of peptide-vesicle interactions.     

P relaxation responses associated with n(2)/o(2) diffusion in soybean nodule cortical cells and excised cortical tissue

N₂-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O₂ followed by N₂ or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (δ 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O₂ perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T₁), and spin-spin relaxation time (T₂), under perfusion with N₂ or Ar as opposed to O₂, indicated that loss of signal was due to T₁ saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T₁, values derived from biexponential relaxation processes under perfusing O₂ were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N₂ these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T₁ relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T₂ values for cortical tissue were also biexponential but were essentially the same under O₂ (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N₂ (39% 0.07 ± 0.01 s and 61% 0.37 ± 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T₁ decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T₁ experiments demonstrate that O₂ does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O₂ perfusion) is responsible for the observed relaxation changes.     

Influence of membrane fluidity on transport mediated by ubiquinones through phospholipid vesicles

Coenzymes Q₁₀ and Q₃ are incorporated into dipalmitoylphosphatidylcholine and egg yolk lecithin liposomes. Dithionite reduction of ferricyanide trapped inside these phospholipid vesicles is taken as a measure of ubiquinone-mediated transport of reducing equivalents. The reaction shows complex pattern with a high order for CoQ. The initial transport rates are very sensitive to the membrane physical state, being considerably reduced at temperatures below the phase transition of the pure dipalmitoylphosphatidylcholine, both for CoQ₁₀ and CoQ₃ reconstituted with this phospholipid. It is suggested that a different reaction mechanism operates in fluid and rigid membranes. This suggestion is related to the possible organization of CoQs in phospholipid membranes.     

Multi-conformational peptide dynamics derived from NMR data: A new search algorithm and its application to antamanide

Summary A search algorithm, called MEDUSA, is presented which allows the determination of multiple conformations of biomolecules in solution with exchange rate constants typically between 10³ and 10⁷ s^–1 on the basis of experimental high-resolution NMR data. Multiples of structures are generated which are consistent as ensembles with NMR cross-relaxation rates (NOESY, ROESY), scalar J-coupling constants, and T_1p measurements. The algorithm is applied to the cyclic decapeptide antamanide dissolved in chloroform. The characteristic radio-frequency field dependence of the T_1p relaxation rates found for the NH protons of Val¹ and Phe⁶ can be explained by a dynamical exchange between two structures.     

Carbon-13 spin-lattice relaxation studies of intramolecular motion in lysine and a series of oligolysines

¹³C Spin-lattice relaxation times T₁ for individual carbon nuclei have been measured in a series of oligo-l-lysines, as well as lysine and glycine monomers. Anomalous behavior of profiles of T₁ versus pD occurs for lysine and glycine; the T₁ values of the C_α and CO groups are maximal at pH values corresponding to zwitterionic structures. This is interpreted in terms of the hindered intramolecular rotation around the carbonyl-C_α bond at acidic and basic pD values. Lysine monomer manifests a much less pronounced increase in T₁ values from the α- to the ?-carbon than does lysine in an oligomer or polymer. The rate of reorientation of the C_α and C_β carbons of N-terminal groups are faster than those of the central and C-terminal residues, especially at pD greater than 10 for tri-l-lysine hydrochloride and penta-l-lysine acetate. This is interpreted in terms of interaction between the ?-amino groups and the negatively charged carboxyl groups at pD < 10. Segmental motion is shown to make a significant contribution to relaxation of side-chain carbons, making them less sensitive to molecular size than the carbonyl carbons.