High-resolution proton and carbon-13 NMR of membranes: why sonicate?

We have obtained high-field (11.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance (NMR) spectra of egg lecithin and egg lecithin-cholesterol (1:1) multibilayers, using "magic-angle" sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:1) vesicles, using conventional FT NMR methods. Resolution of the proton and carbon-13 MASS NMR spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS NMR spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13C NMR, we demonstrate that most 13C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, 13C MASS NMR, spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon-13 NMR spectra.     

Comparison of membrane organization in mitochondria from yeast and rat liver by nuclear magnetic resonance spectroscopy

Summary Proton magnetic resonance (PMR) and carbon-13 magnetic resonance (CMR) spectra of intact, unsonicated yeast and rat liver motochondria show differences which may be correlated with the composition of the membranes. High resolution PMR and CMR signals in intact yeast mitochondria have been assigned to regions of fluid lipid-lipid interaction on the basis of spectra of extracted lipid and protein, and the temperature dependence of NMR signals from the intact membrane. PMR spectra suggest that about 20% of total yeast phospholipid is in regions where both intramolecular fatty acid chain mobility and lateral diffusion of entire phospholipid molecules are possible. No such regions appear to exist in rat liver mitochondria. For both yeast and rat liver mitochondria, comparison of PMR and CMR spectra suggests that about 50% of phospholipid appears to be in regions where intramolecular fatty acid chain motion is considerable, but lateral diffusion is restricted. The remaining phospholipid appears to have little inter- or intramolecular mobility. Since NMR observation of lipid extracts from membranes indicates that phospholipid-sterol interactions do not account for the spectra of intact mitochondria, these effects are interpreted in terms of extensive lipid-protein interactions.     

Freeze-etch electron microscopy of erythrocytes,Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B

Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150–250Åin diameter, in the membranes of rat erythrocytes, in cholesterol-containing membranes ofAcholeplasma laidlawii cells and in egg lecithin-cholesterol liposomes. No change in fracture faces was observed when cholesterol was absent in the membranes ofA. laidlawii, and lecithin liposomes.Amphotericin B does not visibly affect the freeze-etch morphology of erythrocytes, cholesterol-containingA. laidlawii cells and lecithin-cholesterol liposomes.     

Molecular order in acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids

The first application of deuterium magnetic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37°C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.     

Interaction of the fluorescent probe diS-C3-(5) with lecithin-cholesterol membranes

Behaviour of fluorescent carbocyanine probe disS-C3(5) in the egg lecithin-cholesterol membrane suspension was studied in relation to the lecithin/cholesterol ratio. The partition coefficient of the probe between aqueous and lipid phases decreases unlinearly with increase of cholesterol molar part in a bilayer. This parameter over molar part units was estimated to be (2.4 +/- 0.1) X 10(6) for egg lecithin membranes and (1.8 +/- 0.2) X 10(6) for 10 mol% cholesterol, (1.2 +/- 0.1) X 10(6) for 20, (0.8 +/- 0.1) X 10(6) for 30, and (0.48 +/- 0.02) X 10(6) for 50 mol% cholesterol. It is suggested that the probe partition coefficient value consists of two components: one caused by pure lecithin bilayer regions and another by local lecithin concentration fluctuations in the mixed lecithin-cholesterol regions.     

A 31P NMR study of the thermal transition of dipalmitoyl lecithin vesicles.

Phosphorous-31 nuclear magnetic resonance (31P NMR) was used to study the liquid crystalline transition of sonicated dipalmitoyl lecithin vesicles in D2O. Linewidths were dependent upon temperature and changed dramatically through the transition region. The potential usefulness of 31P NMR spectroscopy for probing the thermal behavior of phospholipid membranes is evaluated and discussed.     

The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

Capacitance and resistance of the bilayer lipid membrane formed of phosphatidylcholine and cholesterol

Capacity and electric resistance of lipid membranes composed of lecithin and cholesterol were determined. The components were chosen for the study because they were present in biological membranes. Capacitance of the lecithin and cholesterol membranes amounts to 0.38 and 0.61 microF/cm(2), and resistance to 1.44(10(4)and 2.12(10(6)Omega cm(2), respectively. A 1:1 complex appears as a result of lecithin-cholesterol membrane formation. Parameters of the membrane formed of the lecithin-cholesterol complex were determined: surface concentration (Gamma(3)), capacitance (C(3)), and conductance (R;(3)(-1), as well as the stability constant (K) of the complex. The mean values of those magnitudes are as follows: 4.265(10(-6)mol/m(2), 0.54 microF/cm(2), 1.381(10(-6)Omega(-1)cm(-2)and 3.748(10(7), respectively.     

The interaction of 1-anilino-8-naphthalenesulfonate with thyroid lipids and membranes: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) spectra of thyroid cell membranes and their total lipid extracts, in the presence of 1-anilino-8-naphthalenesulfonate (ANS), have been studied. The addition of ANS causes a shifting of the head group PMR signal, a splitting of the signal into two components and an increase in total spectral intensity. The data suggest that ANS interacts with phospholipid in the membrane as it does in total lipid vesicles. Evidence is also presented for the removal of lipids from the membrane, by ANS, and the subsequent formation of micelles. The membrane results are compatred with our earlier work on the interaction of ANS with egg phosphatidycholine (P.C.) vesicles and the results are used in explaining the inhibition of iodide transport in isolated thyroid slices.     

Interactions between biliary lipid micelles and intestinal brush border membranes investigated by 1H and 31P nuclear magnetic resonance

The effect of taurocholate and lecithincholesterol-taurocholate mixed micelles on the structure of isolated intestinal brush border membranes was investigated by nuclear magnetic resonance (NMR). Rabbit brush border membranes isolated by a Mg²⁺ precipitation step were chosen for this study because of their stability and integrity as revealed by ³¹P NMR. Incubation of taurocholate with the brush border membranes does not induce significant solubilization of these membranes even when the taurocholate/phospholipid ratio reaches 3.0 ¹H NMR studies indicate that taurocholate is included in the membrane bilayer at low concentration (3 mM). However this biliary salt produces a size diminution of the vesicles when its concentration increases. Incorporation of lecithin or lecithin-cholesterol in micelles of taurocholate and subsequent incubation with brush border membranes lead simultaneously to a decrease in the ³¹P NMR isotropic/bilayer line ratio, and to an increase in . These results indicate a protective effect of these compounds against lytic damage of taurocholate. Futhermore the equilibrium distribution of lecithin between mixed micelles and the membrane bilayer is strongly in favour of complete integration of micellar components in the bilayer. These data suggest that uptake of lipids from the micellar phase by isolated brush border membranes involves an interaction of the micelles with membranes followed by a fusion process.     

The interaction of 2,4-dinitrophenol with phospholipids at phospholipid-water interfaces

The effect of 2, 4-dinitrophenol, DNP, on monolayers of egg lecithin, hydrogenated egg lecithin, dipalmitoyl lecithin and mitochondrial lipids has been examined. Both the undissociated and dissociated forms of DNP bind to the phospholipid polar groups. Binding of the acid form leads to a decrease in monolayer surface potential and an expansion of the monolayer. The amount of penetration of the acid form into lecithin monolayers appears to depend on the London-Van der Waals attractions between the lecithin hydrocarbon chains. Binding of the 2,4-dinitro-phenolate anion is reflected in a decrease in surface potential for lecithin monolayers, and an increase in surface potential for mitochondrial lipid monolayers. The adsorption of dinitro-phenolate to egg lecithin has been further investigated by micro-electrophoresis of lecithin liposomes. It is suggested that binding of DNP to phospholipid-water interfaces is important in determining its action as an uncoupler of oxidative phosphorylation, and as a compound that increases the electrical conductance of artificial lipid membranes.     

Adamantyl nitroxide: A spin label for probing membrane surfaces

In an effort to understand more about the perturbing properties of adamantane-like molecules on biological membranes, the spin probe adamantyl nitroxide (2,2′-dimethyl-5-adamantyl oxazolidine-N-oxyl) was synthesized, purified and characterized. Electron paramagnetic resonance (EPR) spectra were then obtained from 1:50 and 1:200 mixtures of adamantyl nitroxide with dipalmitoyl and dipalmityl phosphatidylcholine multibilayers. Above the phase transition temperature of these lipids (41°C for dipalmitoyl phosphatidylcholine and 43°C for dipalmityl pholphatidylcholine) the spectra of adamantyl nitroxide are similar to control spectra obtained in liquid oleic acid. Below the phase transition temperatures, however, spectral differences were observed depending on: (1) the concentration of the spin probe in the lipid; (2) the linkage between the polar head group and the hydrocarbon tails of the phospholipid; (3) the temperature of the sample. Partitioning of adamantyl nitroxide between the aqueous and hydrocarbon phases of the sample is most prominent at probe-to-lipid ratios of 1:200 and at temperatures below the pre-transition temperature of the lipid (around 33°C). Computer simulations of the above results, as well as additional experiments performed at 35 GHz, show that the results arise from true partitioning and not from asymmetric probe motion.Two conclusive results of these experiments are that spectra of adamantyl nitroxide in phospholipid multibilayers are sensitive to probe concentration and to the physical characteristics of the phospholipid which they probe. The spectral differences which arise when adamantyl nitroxide is used with ether- and ester-linked phospholipids indicate that it is a sensitive probe of membrane surfaces. Employment of this molecule in membrane research should prove to be useful in obtaining additional information about membrane surface events.     

The interaction of mequitazine with phospholipid model membranes

The effect of increasing concentrations of mequitazine, a quinuclidinylmethyl-phenothiazine, on the phase transition temperature (T_c), the broadening of the transition peak, the enthalpy and entropy of transition of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholine (DPPC) liposomes was studied. Pest critical micelle concentrations of mequitazine (CMC = 5.23 X 10^?2M), caused broadening of the transition peak and lowering of the T_c of pure liposomes. The ratio of peak heights from the nuclear magnetic resonance (NMR) spectra of egg phosphatidycholine liposomes was used as a criterion for assessing the interaction of the drug with phospholipid membranes. Mequitazine interacts with both the polar head groups and hydrophobic membrane interior.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

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.     

Interaction of phospholipid vesicles with cells. Endocytosis and fusion as alternate mechanisms for the uptake of lipid-soluble and water-soluble molecules

Depending on their phospholipid composition, liposomes are endocytosed by, or fuse with, the plasma membrane, of Acanthamoeba castellanii. Unilamellar egg lecithin vesicles are endocytosed by amoeba at 28 degrees C with equal uptake of the phospholipid bilayer and the contents of the internal aqueous space of the vesicles. Uptake is inhibited almost completely by incubation at 4 degrees C or in the presence of dinitrophenol. After uptake at 28 degrees C, the vesicle phospholipid can be visualized by electron microscope autoradiography within cytoplasmic vacuoles. In contrast, uptake of unilamellar dipalmitoyl lecithin vesicles and multilamellar dipalmitoyl lecithin liposomes is only partially inhibited at 4 degrees C, by dinitrophenol and by prior fixation of the amoebae with glutaraldehyde, each of which inhibits pinocytosis. Vesicle contents are taken up only about 40% as well as the phospholipid bilayer. Electron micrographs are compatible with the interpretation that dipalmitoyl lecithin vesicles fuse with the amoeba plasma membrane, adding their phospholipid to the cell surface, while their contents enter the cell cytoplasm. Dimyristoyl lecithin vesicles behave like egg lecithin vesicles while distearoyl lecithin vesicles behave like dipalmitoyl lecithin vesicles.     

Differential mobility of the N-terminal headpiece in the lac-repressor protein.

It is shown by resolution enhancement and relaxation studies of the 360 MHz ¹H nuclear magnetic resonance spectra of the lac-repressor of Escherichia coli and the two fragments derived from it by limited tryptic digestion (the N-terminal headpiece and remaining T-core) that the majority of the relatively mobile residues in the intact lac-repressor are located in the headpiece. Although nuclear magnetic resonance data clearly indicate that the headpiece is a highly structured entity, even when isolated, it is a more mobile part of the repressor than the T-core.     

Haptenic activity of galactosyl ceramide and its topographical distribution on liposomal membranes. I. Effect of cholesterol incorporation

The relation between the immune-reaction of phosphatidylcholine liposomes containing spin-labeled galactosyl ceramide with or without cholesterol and the topographical distribution of the glycolipid in membranes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and antibody binding occurred, irrespectively of the presence of cholesterol, though the motion of the fatty acyl chain of spin-labeled galactosyl ceramide was restricted by cholesterol. In dipalmitoyl phosphatidylcholine liposomes, unlike in egg yolk phosphatidylcholine liposomes, the immune-reaction depended on the cholesterol content. The electron spin resonance (ESR) spectra of spin-labeled galactosyl ceramide in dipalmitoyl phosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled galactosyl ceramide in the liposomes. Without cholesterol, most of the spin-labeled galactosyl ceramide was clustered on the dipalmitoyl phosphatidylcholine membrane, but with increase of cholesterol, random distribution of hapten on the membrane increased. The cholesterol-dependent change in the topographical distribution of hapten on the membranes was parallel with that of immune reactivity. 'Aggregates' composed solely of galactosyl ceramide did not show any binding activity with antibody. The findings suggest that the recognition of galactosyl ceramide by antibody depended on the topographical distribution of hapten molecules. Phosphatidylcholine and/or cholesterol may play roles as 'spacers' for the proper distribution of 'active' haptens on the membranes. The optimum density of haptens properly distributed on liposomal membranes is discussed.     

The interaction of 1-anilino-8-naphthalenesulfonate with lipids: A nuclear magnetic resonance study

The proton magnetic resonance (PMR) and phosphorus magnetic resonance (PhMR) spectra of egg phosphatidylcholine in the presence of 1-anilino-8-naphthalenesulfonate (ANS) have been studied. At low ratios of ANS to phospholipid, the spectra indicate that ANS molecules are in the lipid interface region where they interact with the head-group protons. ANS also penetrates into the hydrocarbon region to some extent. As the ANS/phospholipid ratio approaches one, a significant splitting of the head-group signal occurs. This splitting is associated with head-group signals from inner and outer molecules of the phospholipid vesicles. As the ANS/phospholipid ratio is further increased, a gel phase often occurs. The spectra for this gel phase suggest a highly mobile head-group. Further ANS addition results in a PMR spectrum suggestive of ANS—phospholipid micelle formation. The results for a phospholipid—cholesterol complex and for the total lipid extract from a cell membrane show that the ANS effect is more complicated in these cases.