Phosphocholine Headgroup Research Articles

Interaction of melittin with phosphatidylcholine membranes. Binding isotherm and lipid head-group conformation.

The binding of melittin to nonsonicated bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was studied with an ultracentrifugation assay and with 2H and 31P nuclear magnetic resonance. Melittin binding could best be described by a partition equilibrium with Kp = (2.1 +/- 0.2) X 10(3) M-1, measuring the binding isotherm in the concentration range of 0-100 microM melittin and taking into account electrostatic effects by means of the Gouy-Chapman theory. This partition coefficient is smaller than that deduced for small sonicated vesicles and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium magnetic resonance revealed a conformational change of the phosphocholine head group upon melittin binding. The quadrupole splittings of the alpha and beta segments of the choline head group varied linearly with the amount of bound melittin but in opposite directions; i.e., the alpha splitting decreased, and the beta splitting increased. This conformational change is not specific to melittin but is a response of the phosphocholine head group to positive membrane surface charges in general. Quantitatively, melittin is one of the most efficient head-group modulators, the efficiency per unit charge comparable to that of charged local anesthetics or hydrophobic ions.

31P and 2H NMR studies of structure and motion in bilayers of phosphatidylcholine and phosphatidylethanolamine.

The structural and motional properties of mixed bilayers of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) have been examined by using wide-line 31P, 14N, and 2H NMR. 2H and 14N NMR data showed that in mixed bilayers containing both PC and PE the conformations of the head-group moieties are essentially identical with those observed for bilayers containing a single phospholipid species. Equimolar amounts of cholesterol induce also only a small change in head-group conformation. 31P T1 relaxation measurements (at 300 MHz) at various temperatures of bilayers containing phospholipids with a mixture of phosphocholine and phosphoethanolamine head-groups and unsaturated fatty acid residues revealed in all cases a clearly defined minimum corresponding to the condition omega O tau C-1 approximately 1. For all phospholipid mixtures studied, the 31P T1 relaxation was homogeneous over the whole powder spectrum and could be fitted to a single-exponential decay. The 31P vs temperature profiles were analyzed by a simple correlation model following the analysis of Seelig et al. (1981) [Seelig, J., Tamm, L., Hymel, L., & Fleischer, S. (1981) Biochemistry 20, 3922-3932]. Rotational diffusion of the phosphate moiety in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was slower than that of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the activation energy was increased by a factor of 1.7 to 31.4 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Partitioning of local anesthetics into membranes: surface charge effects monitored by the phospholipid head-group

The binding of the charged form of two local anesthetics, dibucaine and etidocaine, to bilayers composed of 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC) was measured simultaneously with ultraviolet spectroscopy and deuterium magnetic resonance. Because of their amphiphilic molecular structure, both drugs intercalate between the lipid molecules, increasing the surface area and imparting a positive electric charge onto the membrane. The ultraviolet (UV) binding isotherms were therefore analyzed in terms of a model which specifically took into account the bilayer expansion as well as the charge-induced concentration variations near the membrane surface. By formulating a quantitative expression for the change in surface area upon drug intercalation and combining it with the Gouy-Chapman theory, the binding of charged dibucaine and etidocaine to the lipid membrane was best described by a partition equilibrium, with surface partition coefficients of 660 ± 80 M −1 and 11 ± 2 M −1 for dibucaine and etidocaine, respectively (pH 5.5, 0.1 M NaCl/50 mM buffer). Deuterium magnetic resonance demonstrated further that the binding of drug changed the head-group conformation of the lipid molecules. Invoking the intercalation model, a linear variation of the deuterium quadrupole splittings of the choline segments with the surface charge density was observed, suggesting that the phosphocholine head-group may act as a ‘molecular electrometer’ with respect to surface charges.

Structure and dynamics of the phosphatidylcholine and the phosphatidylethanolamine head group in L-M fibroblasts as studied by deuterium nuclear magnetic resonance.

Mouse fibroblast L-M cells were grown in tissue culture medium containing selectively deuterated choline or ethanolamine. Both compounds were incorporated into the corresponding phospholipids at levels greater than 50% thus leading to a selective deuteration of these phospholipid head groups. Choline and ethanolamine were labeled at either the alpha- or the beta-carbon atom and well-resolved deuterium and phosphorus n.m.r. spectra were obtained from intact cells, crude plasma membranes and lipid extracts, leading to the following conclusions. (i) A large fraction, if not all, of the phospholipids in the intact L-M cell membranes were organized in a liquid crystalline bilayer. (ii) The phosphoethanolamine and the phosphocholine head group conformation were found to be remarkably similar in pure lipid bilayers and in intact L-M cell membranes with the head group dipoles being oriented parallel to the membrane surface. (iii) The deuterium T1 spin lattice relaxation times fell in the range of 7-25 ms and were similar in intact L-M cells and in pure lipid model membranes, suggesting that the two head groups are not involved in strong interactions with membrane proteins. The rotational diffusion rate of the two head groups was reduced by at least a factor of 10 compared to molecules of the same size in aqueous solution. (iv) The phosphocholine head group was sensitive to the size and sign of membrane surface charges as verified in mixing experiments with charged lipids. In L-M cell membranes the phosphocholine appeared to sense an electrically neutral environment in spite of the fact that L-M cell membranes contain 10-20% negatively charged lipids.

The inhibition of diacylglycerol-stimulated intracellular phospholipases by phospholipids with a phosphocholine-containing polar group. A possible physiological role for sphingomyelin.

Phosphatidylinositol phosphodiesterase activated by diacylglycerol is substantially inhibited by all phospholipids containing a phosphocholine head group, including phosphatidylcholine, hydrogenated phosphatidylcholine, choline plasmalogen, lysophosphatidylcholine, lysocholine plasmalogen, sphingomyelin and sphingosylphosphocholine. The sphingosine-containing phospholipids are the most inhibitory. Phosphatidic acid does not inhibit, and phosphatidylethanolamine activates the hydrolysis still further. Sphingomyelin is highly inhibitory to a diacylglycerol-stimulated intestinal mucosal phospholipase A2, or a liver lysosomal phospholipase A1 + A2, both hydrolysing a phosphatidylcholine substrate. Sphingomyelin [20% molar (20 mol of sphingomyelin/80 mol of phosphatidylethanolamine)] activates phosphatidylethanolamine hydrolysis by intestinal mucosal phospholipase A2, and then at higher concentrations (40% molar) substantially inhibits the activity. The results are discussed in relation to possible molecular reorganizations brought about in the hydrated phospholipid substrate complex, and in particular the possible stabilizing role of sphingomyelin in the maintenance of membrane structure, and hence in the modulation of phospholipase activity.

Hydration Dependence of the Head Group Mobility in Phospholipid (DMPC) Membranes

AbstractThe dielectric dispersion in the frequency range 2–800 MHz of the zwitterionic dipolar phosphocholine head group has been measured from −20 to +60°C for DMPC depending on the amount of bound water. It is found that the head group rotation shows a sudden change at the gel‐fluid phase transition, 1. the rotation frequency becomes much higher and 2. the related dielectric response becomes much more pronounced in the fluid phase. Below 15 H2O molecules bound per lipid molecule the head group rotation frequency decreases with decreasing number of bound water. This observation reveals for the first time that there is a correlation between hydration dependence of the chain melting temperature and head group rotation. The thermal activation of the head group rotation in the fluid phase made us conclude that three hydrogen bonds have to be broken in order to rotate the head group.

14N NMR of lipid bilayers: effects of ions and anesthetics.

The interaction of divalent and trivalent metal cations, ferricyanide, a lipophilic ion (tetraphenylborate), and a local anesthetic (tetracaine) with the phosphocholine head group of egg lecithin was investigated by using wide-line 14N and 31P NMR. Measurements of the 14N quadrupolar splittings in the presence of a variety of perturbing agents demonstrated that the 14N NMR technique can be used to directly monitor ion or anesthetic binding. The 14N quadrupolar splitting (delta nu Q) is a measure of the order parameter of the C beta-N bond segment, and changes in delta nu Q as large as 3.5 kHz were observed. Moreover, a comparison of the changes in the quadrupolar splittings induced by the binding of ions or anesthetics provided a sensitive method of discriminating between these perturbing agents in their ability to alter the orientational order of the C beta-N bond segment of the phosphocholine moiety. Without exception, addition of metal ions or anesthetics always resulted in a decrease of the 14N delta nu Q. This reduction reflects a change in the average orientation or degree of motional averaging at the C beta-N bond segment position. In the case of metal ion binding, the strength of the interaction increased with the charge of the metal ion in the order Ca2+ less than Ln3+, in agreement with a previous 2H NMR study [Akutsu, H., & Seelig, J. (1981) Biochemistry 20, 7366-7373].(ABSTRACT TRUNCATED AT 250 WORDS)

The interactions between monovalent ions and phosphatidyl cholines in aqueous bilayers.

The interactions of lithium and sodium ions and water with phosphatidylcholine bilayers have been studied by means of 7Li, 23Na and 2H NMR quadrupole splittings. The experimental results are interpreted in terms of a simple three-site model with two anisotropic sites ('binding' sites) and one isotropic site ('free' ions and water molecules). The findings obtained for the zwitterionic model membrane are compared with previous investigations of lamellar phases composed of ionic and nonionic amphiphiles. It is shown that the data obtained are compatible with our previous suggestion [Lindblom, G., Persson, N.-O., and Arvidson G. (1976) Adv. Chem. Ser. 152, 121] that an increase in the salt content in the water layer induces a conformational change in the polar head group of phosphatidylcholine. Thus at high salt concentration the phosphocholine head group tends to be oriented perpendicular to the lipid bilayer surface. The study also shows that increasing the amount of salt leads to a squeezing out of water between the bilayers. This is interpreted in terms of a reduction of the repulsion forces between the bilayers.

2H- and 31P-NMR studies of bilayers composed of 1-acyllysophosphatidylcholine and fatty acids

1-Palmitoyllysophosphatidylcholine has been mixed in equimolar amounts with specifically deuterated palmitic acid and the structural properties of the lipid/water phase have been studied by 2H- and 31P-nuclear magnetic resonance. The order profile of the free palmitic acid is very similar to that of the parent compound 1,2- dipalmitoyl-sn- glycero-3-phosphocholine at temperatures above the gel-to-liquid crystal phase transition. The bending of the sn-2 chain which is typical for diacyl lipids is not observed for the free palmitic acid. The mixture of lysolipid and palmitic acid exhibits well-defined quadrupole splittings even at temperatures below the gel-to-liquid crystal phase transition. Hence it is possible for the first time to establish an order profile in the gel-state of the lipid bilayer phase. Between carbon atoms 5 to 12 the palmitic acid chain is found to assume the extended all- trans conformation with a very small contribution from gauche defects. Towards the methyl terminal a distinct increase in the gauche probability can be noted. The motion of the phosphocholine headgroup was also studied by 2H- and 31P-NMR using selectively deuterated 1-palmitoyllysophosphatidylcholine. The headgroup has a considerably larger motional freedom in the mixture of lysolipid and palmitic acid than in 1,2- dipalmitoyl-sn- glycero-3-phosphocholine . In addition, the average headgroup conformations are also different in the two systems.

Lipid solvation of cytochrome c oxidase. Deuterium, nitrogen-14, and phosphorus-31 nuclear magnetic resonance studies on the phosphocholine head group and on cis-unsaturated fatty acyl chains.

Lipid solvation of cytochrome c oxidase. Deuterium, nitrogen-14, and phosphorus-31 nuclear magnetic resonance studies on the phosphocholine head group and on cis-unsaturated fatty acyl chains.

A comparison of the headgroup conformation and dynamics in synthetic analogs of dipalmitoylphosphatidylcholine

14N-NMR spectra and relaxation times for dipalmitoylphosphatidylcholine and three analogs were obtained in both the liquid crystal and gel phases. The analogs either changed the PO 4 − to N + (CH 3) 3 distance (P-N) within the headgroup by increasing the number of CH 2 groups from two in the phosphocholine headgroup (PN-2) to six in the phospho-( N′, N′, N′-trimethyl)hexanolamine headgroup (PN-6), or replaced the ester linkages to the hydrocarbon chains with ether linkages. 31P-NMR spectra were obtained for the four samples in the liquid-crystal phase. (1) The 14N- and 31P-NMR spectra and 14N relaxation times all indicate that increasing the P-N distance within the headgroup causes changes in both the average orientation of the C-N bond and its dynamics. (2) The 14N-NMR spectra provide evidence for a change in orientational order of the headgroup as a result of changing the linkage to the acyl chains. On the other hand, the relaxation time measurements indicate that the molecular motion for the headgroup is independent of the type of linkage. (3) The thermal behaviour of the four samples is clearly reflected in the 14N-NMR spectra. The second moments of the spectra show distinct changes at each of the phase tansitions. (4) The 14N-NMR spectra show that the average conformation of the headgroups is not significantly altered by the main phase transition. For the PN-2 samples, T 2e, the decay of the quadrupolar echo, decreases discontinuously in the P β, phase, which is evidence for a possible exchange process between two molecular states within this phase.

Phase Transition of the Bilayer Membrane of Synthetic Dialkyl Amphiphiles as Studied by Differential Scanning Calorimetry

AbstractThe phase transition behavior of dilute aqueous solutions of the bilayer membrane of dialkyl (C12 to C18 in most cases) amphiphiles was investigated by differential scanning calorimetry (DSC). The extent of the sample dispersion affected the DSC behavior for dialkyl amphiphiles with ammonium, phosphate and sulfonate head groups. The well‐sonicated aqueous sample of the ammonium amphiphiles showed broader transition peaks at lower temperatures than the corresponding frozen sample did. The discrepancy became less pronounced when the interacting group was introduced at or near the head group. The wel DSC behavior was not observed for amphiphiles with nonionic (polyoxyethylene) and zwitterionic (phosphocholine) head groups. The phase transition temperature (0 – 80°C) was enhanced progressively with increasing alkyl chain lengths (C12 to C18), but the transition enthalpy (2–12 kcal/mol) did not obey simple relations.

31P NMR study of head group behavior in sonicated phosphatidylcholine liposomes in the gel and in the liquid state

We measured the 31P[ 1H] Nuclear Overhauser Effect (NOE) as a function of temperature and of 1H irradiation frequency, the linewidth Δν 1 2 as a function of temperature and the relaxation time T 1 above and below the thermal transition temperature, of the 31P-NMR signal in sonicated liposomes of 1,2-dimiristoyl-3- sn-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-3- sn-phosphatidylcholine (DPPC) and 1,2-dimiristoyl-3- sn-phosphatidylcholine (DSPC). The same measurements were repeated in the presence of high molecular weight dextrans. They strongly reduce the NOE and produce longer relaxation times T 1. According to the current models, we were able to evaluate, in the different situations, the correlation time of the internal motion τ G and the distance r between interacting groups in the region of the polar head groups. While the first parameter changes abruptly through the phase transition and under the effect of dextrans, the latter does not appear modified in any case. These results are discussed in terms of a conformational change of the phosphocholine head groups.

Bilayers of phosphatidylglycerol. A deuterium and phosphorus nuclear magnetic resonance study of the head-group region

The structural properties of the phosphoglycerol polar head group in bilayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol have been studied with deuterium and phosphorus-31 nuclear magnetic resonance. For this purpose, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol was deuterated chemically or biosynthetically at all three segments of the glycerol head group. Both the D and the L configurations of the glycerol head group have been synthesized, and the correct stereochemical configuration of the polar group was verified by an enzymtic assay, as direct measurement of the optical rotation was insufficiently sensitive to distinguish between these diastereomers. The phosphatidylglycerol sodium salts were dispersed in excess buffer (0.1 M NaCl, pH 7.0), and the bilayer properties were characterized by small-angle X-ray diffraction and differential scanning calorimetry. The deuterium quadrupole splittings, delta vQ, and the phosphorus-31 chemical shielding anisotropy, delta sigma, were measured as a function of temperature in the range 20-60 degrees C, yielding the following results. (1) Well-resolved deuterium signals were obtained for all head-group segments and could be assigned unambiguously. (2) Almost identical spectra were obtained for 1,2-dipalmitoyl-sn-glycero-3-phospho-1'-glycerol (natural L,D configuration) and 1,2-dipalmitoyl-sn-glycero-3-phospho-3'-glycerol (L,L configuration), suggesting very similar head-group motions and orientations for both diastereomers. (3) The spatial anisotropy of motion and the segmental fluctuations of the negatively charged phosphoglycerol are similar to those of the zwitterionic phosphocholine and phosphoethanolamine head groups but differ distinctly from those of phosphoserine which also carries a net negative charge. (4) The motional inequivalence of geminal deuterons in 1,2-dipalmitoyl-sn-glycero-3-phospho-3'-glycerol was demonstrated by synthesis of a stereospecifically monolabeled analogue.

The influence of cholesterol on head group mobility in phospholipid membranes

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0–70°C for various mixtures of 1,2- dipalmitoyl-sn- glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f 2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42°C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42°C no sudden changes in f 2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42°C, however, a decrease in f 2 at cholesterol contents up to 20–30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.

Phase behavior of synthetic phosphatidylglycerols and binary mixtures with phosphatidylcholines in the presence and absence of calcium ions

Using differential thermal analysis, scanning calorimetry and light scattering, transition temperatures and enthalpy data for the gel to liquid crystalline phase transitions of five synthetic phosphatidylglycerol sodium salts (PG-Na+) were measured. The values obtained were almost identical with literature values for the corresponding phosphatidylcholines (PC). However, transition temperatures for the fully protonated forms of the saturated phosphatidylglycerols (PG-H+) were approximately 20 degrees C higher. For binary mixtures of PG-Na+ and PC in which the acyl chains of the two species were identical, the width of the thermal transition for the phase change was not appreciably greater than that observed with either of the two components alone. In contrast, mixing of PG-Na+ and PC with different chain lengths increases the transition width. In the presence of Ca2+, narrow transitions were also observed with mixtures of PG and PC when the chain length of the PG-Ca2+ was equal to or two carbons shorter than the PC but the transition width was clearly increased when the chain length of the PG-Ca2+ was two carbons longer than the PC. Mixing lipids with greater differences in chain length or mixing saturated lipids with unsaturated lipids in the presence of Ca2+ produced two minima in the thermograms, clearly indicative of phase separation. In sum, these results provide evidence for a high degree of miscibility of the phosphoglycerol and phosphocholine head groups, either in the presence or absence of Ca2+, such that the characteristic phase behavior of each mixture is determined primarily by differences in the hydrocarbon chain structure.