Dextran

ABSTRACT The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca2+ bridges results in a perturbation of lipid packing at physiologically relevant Ca?+ concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by 2H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca2+ results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCI and Ca2+ concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of AA = 2.7 *2 per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca2+/DS-complexes. Lipid area compression after addition of DS and Ca2+ to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca2+ bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca2+ concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.

INTRODUCTION

A key molecular process in the pathogenesis of arteriosclerosis is the association of low-density lipoproteins (LDL) with glycosaminoglycans (GAG) of the arterial connective tissue. The GAG molecules can be exposed to the bloodstream at defects of the endothelium of the arterial walls (Rudel et al., 1986; Camejo et al., 1985; Camejo, 1982). Attractive forces arise from electrostatic interactions between clusters of positively charged amino acids of the protein component of LDL and negatively charged sulfate groups of the GAG molecules (Arnold et al., 1989; Cardin and Weintraub, 1989; Weisgraber and Rall, Jr. 1987; Iverius, 1972). It has been shown that the zwitterionic phospholipids of LDL also contribute to the association by formation of Ca2+ bridges to the GAGs (Kim and Nishida, 1979; Srinivasan et al., 1975; Nishida and Cogan, 1970). The Ca2+-mediated interaction between zwitterionic phospholipids and sulfated polyelectrolytes must be a rather strong contribution since pure phosphatidylcholine (PC) liposomes and micelles form large aggregates with GAG at physiological Ca2+ concentrations, as found in the extracellular space (2-3 mM) (Steffan et al., 1994; Krumbiegel and Arnold, 1990; Arnold et al., 1990; Kim and Nishida, 1977).

Although the biophysical properties of LDL/Ca2+/GAG and phospholipid/Ca2+/GAG complexes are studied rather well (Arnold, 1995; Krumbiegel and Arnold, 1990; Arnold et al., 1990; Kim and Nishida, 1977, 1979; Gigli et al., 1992; Cardin et al., 1989), little is known about consequences of this association on the lipid packing. Fluorescence measurements revealed lipid surface dehydration upon Ca2+-mediated GAG association (Steffan et al., 1994). Lateral diffusion of phospholipid molecules in the LDL monolayer is restricted by GAG binding (Fenske and Cushley, 1990). In a recent study we described the response of the PC headgroup to Ca2+-mediated adsorption of dextran sulfate (DS) (Huster and Arnold, 1998). 2H-NMR detected a small reorientation of the phospholipid headgroup toward the membrane surface as a consequence of DS binding. Ca2+-mediated DS adsorption to PC surfaces was described as a complex equilibrium between attractive forces, caused by Ca2+ bridge formation between lipid headgroups and DS molecules, and repulsive forces between adsorbed DS strands due to electrostatic interactions. These electrostatic forces are screened by higher NaCl concentrations, which results in weaker binding of the polyelectrolyte through fewer Ca2+ bridges. However, because the lateral repulsive forces between adsorbed DS strands are also screened, the amount of adsorbed DS to the PC surface is larger at higher NaCl concentration.

Although there is agreement that GAG binding involves the phospholipid component of LDL, it is unknown how this interaction influences the lipid packing in the phospholipid monolayer as well as in the core of LDL particles. In a DSC study it was established that the core lipids of LDL (cholesterylesters, triglycerides) form a fluid isotropic phase at body temperature. However, after binding to GAG via Ca2+ these lipids form a liquid ordered state (Bihari-Varga et al., 1981). The mechanism that leads to this phase transition is not clear at the moment.

In this paper we investigated the influence of Ca2+mediated GAG binding on phospholipid packing in a model system. We studied the influence of Ca2+-mediated DS adsorption on PC chain packing in both monolayers and bilayers. We suggest that changes in lipid packing could be responsible for the phase transition in the LDL core lipids. Indeed, it was demonstrated that lipid-lipid interaction is sensitive to binding of peptides and proteins in both monolayers and bilayers (Diederich et al., 1996; Maksymiw et al., 1987; Gawrisch et al., 1995; Roux et al., 1989). ZH-NMR on deuterated lipids has been shown to be a very sensitive method for detection of changes in lipid chain packing (Davis, 1983; Seelig, 1977). Chain order parameters can be reproduced with a precision of 0.2% (Holte et al., 1996). From order parameter changes, differences in average area per lipid molecule in the bilayer are estimated with similar precision (Koenig et al., 1997).

Lipid monolayers represent a well-suited system to study the interaction of polyelectrolytes with phospholipid molecules at the lipid/water-interface, because lipid density as well as subphase properties can be easily modified. Penetration of polyelectrolyte into the monolayer is indicated by an increase of the surface tension, while interaction with the surface often reduces the surface tension. Interface and lattice properties, as well as lateral packing of anionic phospholipid monolayers, are strongly influenced by interaction with cationic polyelectrolytes (de Meijere et al., 1997) or model peptides (Johnson et al., 1991).

Here, we report quantitative investigations of area compression in DMPC monolayers and bilayers induced by Ca2+-mediated polyelectrolyte binding. DS was used as a model compound for GAG that is available in a variety of rather well-defined chain lengths. Area condensation was investigated as a function of mono- and divalent cation concentration that modify electrostatic interactions at the membrane surface.

MATERIALS AND METHODS

Materials

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine (DMPC-d54) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Dextran sulfate 500 kDa (DS 500) was purchased from Serva, Feinbiochemica, Heidelberg, Germany; dextran sulfate 40 kDa (DS 40) from ICN Biochemicals (Cleveland, OH), dextran sulfate 8 kDa (DS 8) from Sigma Chemical Co. (St. Louis, MO), and dextran sulfate I kDa (DS 1) from Pfeifer and Langen, Dormagen, Germany.

NMR and x-ray sample preparation

For each lipid sample, 10 mg lipid powder was weighed into plastic containers and hydrated in I ml buffer (10 or 100 mM NaCl, 10 mM Hepes, pH 7.4, buffer prepared in deuterium-depleted water) containing aliquots of Caz+ added from a 0.1 M CaC12 stock solution resulting in multilamellar lipid vesicles. Preparations were vortexed, followed by five freeze-thaw cycles to ensure homogeneous ion distribution between the bilayers. DS was added to the suspensions from 100 mg/ml stock solutions. Mixtures were subjected to intense vortexing and 10 additional freeze-thaw cycles to equilibrate phospholipid/DS preparations. The suspensions were centrifuged at 20,000 x g, and the pellet was transferred into 5-mm glass tubes and sealed for NMR measurement or into glass capillaries (1 mm diameter) and sealed for x-ray diffraction.

RESULTS

Characterization of DMPC/Ca2+/DS 500 complex structure

According to the anisotropy of chemical shift of the 31p_ NMR powder spectra, all preparations investigated at 37degC were in a lamellar liquid-crystalline phase (data not shown).

Fig. 1 shows x-ray repeat spacings for DMPC in the presence of 1 mg/ml DS 500 as a function of the Ca2+ concentration. Due to the Ca2+-mediated DS binding to DMPC, a drastic decrease in the x-ray repeat spacing is observed yielding similar repeat spacings for all investigated Ca2+ concentrations.

Ca2+ concentration dependence

In Fig. 2, typical 2H-NMR powder spectra of DMPC-d54/DS 500 mixtures are shown. In the absence of Ca2+, no influence of DS 500 on lipid chain order is detected, as indicated by identical average order parameters of (S) = 0.167. With increasing Ca2+ concentration an increase in the lipid chain quadrupolar splittings is observed, indicating higher lipid chain order as a result of Ca2+-mediated DS binding to DMPC. No changes in the average order parameters (within experimental error (0.002)) were observed in the absence of DS 500 at Ca2+ concentrations up to 15 mM. Smoothed chain order parameter profiles of the spectra in Fig. 2 are shown in Fig. 3 A. To evaluate the order changes along the chain, difference order parameter profiles were plotted (Nezil and Bloom, 1992). Fig. 3 B shows the difference between the chain order parameters of the DMPC/DS 500 mixtures in the presence and absence of different Ca2+ concentrations. As seen from the plot, there is a rather uniform order increase over the entire chain due to Ca2+-mediated binding of DS to DMPC.

In Fig. 4, lipid area reduction in DMPC/DS 500 mixtures and average order parameters are recorded as a function of Ca2+ concentration at 10 and 100 mM NaCl. Ca2+-mediated binding of DS 500 to DMPC surfaces increases lipid chain order, which is analogous to a reduction in the average lipid area per molecule (see Eq. 3). Lipid area reduction upon Ca2+-induced DS 500 binding is of larger magnitude at 10 mM NaCl compared to 100 mM NaCl. A rather steep decrease in area per molecule is observed at Ca2+ concentrations from 0 to 5 mM, while only small changes are detected at higher Ca2+ concentrations.

DS molecular weight dependence

The interaction of DMPC with DS of varying molecular weight was investigated at 3 mM Ca2+, the approximate extracellular concentration of Ca2+. Again, experiments were carried out at two different electrostatic screening lengths that were adjusted by 10 or 100 mM NaCl. Average order parameters and reduction in lipid area per molecule are shown in Fig. 5. Within experimental error, the short chain DS 1, consisting of only four sulfated glucose subunits, does not induce an area change, suggesting that no interaction with the DMPC surface occurs. DS of longer chain lengths, however, induce a significant reduction in lipid area per molecule starting at molecular weights >=8000. Almost identical area reduction is observed at 10 mM NaCl for all DS with longer chains. At 100 mM NaCl a moderate area decrease due to DS 8 and approximately uniform area compression of DMPC due to DS 40 and DS 500 is detected.

Surface pressure in DMPC monolayers

Within experimental error, no changes of the surface pressure of pure buffer solution due to DS in the presence or absence of Ca2+ were detected suggesting that DS alone is not surface-active at the concentration used in this study. However, substantial changes of DMPC monolayer surface pressure as a result of Ca2+-mediated interaction with DS 500 were investigated. The initial surface pressure was adjusted to 32 mN/m in all monolayer experiments. In the presence of Ca2+, addition of DS to the subphase leads to a decrease of monolayer surface pressure of several mN/m within a few minutes. Fig. 6 shows the reduction of DMPC surface pressure upon Ca2+-induced DS 500 association as a function of Ca2+ concentration. At 10 mM NaCl, a steep decrease of surface pressure is observed, reaching a plateau value at Ca2+ concentrations of -4 mM. A smaller surface pressure decrease is observed at 100 mM NaCl.

Finally, surface pressure changes were recorded for the four DS chain lengths investigated in this study at 3 mM Ca2+ and salt concentrations of 10 and 100 mM NaCl. Results are shown in Fig. 7. Within experimental error, no surface pressure reduction is observed for the short DS 1. At 10 mM NaCl, the monolayer is clearly compressed by higher molecular weight DS with increasing magnitude from DS 8 to DS 500. At 100 mM NaCl no change in surface tension is recorded for DS 1 and DS 8, while at higher molecular weight a small reduction in surface pressure is found.

DMPC phase transition

The influence of Ca2+-mediated DS 500 association on the DMPC phase transition was investigated by ZH-NMR. Spectral first moments of 2H-NMR powder spectra of DMPC-d54 liposomes in the presence of 15 mM Ca2+ (10 mM NaCl) are plotted in Fig. 8. In the absence of DS 500, a rather sharp phase transition of DMPC-d54 at -20.SoC is indicated by a sudden decrease of the first spectral moment. In the presence of 1 mg/ml DS 500, the phase transition is much broader, with a midpoint of 26.7C. At 5 mg/ml DS 500 the phase transition is narrow again and occurs at -28.6C. At these ion and DS 500 concentrations, a large increase of the chain order parameters in the fluid phase is observed ((S) = 0.197) that corresponds to an area compression of AA = 2.74 k2 per lipid molecule.

DISCUSSION

Lipid chain order

The interaction of anionic GAGs with zwitterionic phospholipids is the result of formation of Ca2+ bridges between sulfate groups of DS and phosphate groups of the zwitterionic phospholipids that only involves the phospholipid headgroup (Fenske and Cushley, 1990; Steffan et al., 1994; Iverius, 1972; Kim and Nishida, 1977). In a previous paper we investigated DS binding to DMPC in the presence of Ca2+ (Huster and Arnold, 1998). At 1 mg/ml DS, as in the bilayer experiments of this study, the molar ratio of anionic charge to DMPC is 1 1 to 2.3, assuming two sulfate groups per DS monomer. At 10 mM NaCl and 3 mM Ca2+, the molar DMPC/Ca2+/DS monomer ratio is 10:1.7:2.2, and at 15 mM 10:3.3:2.2. Such molar ratios translate into local Ca2+ concentrations in the lipid headgroup region of the order of 1 M.

The binding of DS to zwitterionic phospholipid surfaces via Ca2+ bridges increases phospholipid chain order (Fig. 3). The magnitude of the ordering increases with Ca2+ concentrations (Fig. 4). Clearly, membrane perturbation is a function of the number of Ca2+ bridges formed. At 100 mM NaCl, compared to 10 mM NaCl, electrostatic screening of the charges and binding competition between Na+ and Ca2+ ions result in formation of fewer Ca2+ bridges. The increase in chain order is the result of the presence of Ca2+ ions and DS. Ca2+ ions alone have no measurable effect on lipid chain order up to concentrations of 15 mM. Measurable membrane packing perturbations require Ca2+ concentrations higher than 25 mM (Zidovetzki et al., 1989).

DS induced lipid ordering occurs over the entire length of the lipid chains (Fig. 3 B). In contrast, a partial penetration of the bilayer by DS would have disordered mostly chain segments in the center of the hydrophobic core. Therefore, a penetration of DS into the upper hydrophobic region near the lipid/water interface is unlikely (Barry and Gawrisch, 1995). The influence of DS binding on lipid packing is similar to previously published data on the influence of binding of cationic polypeptide chains to acidic bilayers (Laroche et al., 1990). For example, binding of polylysine to phosphatidic acid bilayers increased lipid chain order and raised the phospholipid phase transition temperature. From that perspective, the more complicated mechanism of binding the polyelectrolyte via Ca2+ bridges is comparable to the purely electrostatic attraction between oppositely charged membrane and polyelectrolyte.

DS of lower molecular weight interact only weakly with PC membranes. This is attributed to binding energy provided by the relatively few Ca2+ bridges, which is insufficient to counterbalance repulsion by thermal fluctuation and configurational entropy losses of an aligned polyelectrolyte chain. Therefore, no effect of DS 1 on the order parameters is seen (Fig. 5). Sufficient adsorption energies accumulate, however, for longer chain DS, resulting in strong binding as reflected by increased lipid chain order parameters.

Reduction of lipid area per molecule

Ca2+-mediated binding of DS to DMPC membranes resulted in a decrease of average area per lipid molecule in the bilayer (Figs. 4 and 5). The lipid area is determined by an equilibrium of attractive and repulsive forces between lipid molecules (Marsh, 1996; Evans and Skalak, 1980; Israelachvili et al., 1980). Attractive forces between lipids arise from the hydrophobic effect and the van der Waals interactions between the atoms. Repulsive forces are due to the electric charges of the lipid headgroups, headgroup hydration, and steric repulsion between atoms. Furthermore, at close approach the hydrocarbon chain motional degrees of freedom are severely restricted, which is entropically unfavorable. In equilibrium, the repulsive forces are counterbalanced by attractive interactions and the hydrophobic effect resulting in a tension-free state of the lipid bilayer. The decrease in average area per lipid molecule in response to DS binding via Ca2+ could be the result of a reduction in magnitude of the repulsive forces, an increase of the attractive forces, or, most likely, a combination of both.

We have evidence that the presence of DS raises the Ca2+ concentration between the lipid bilayers by orders of magnitude (Huster and Arnold, 1998). The increase of Ca2+ concentration in the lipid headgroup region due to the presence of the polyelectrolyte can be explained by the Manning condensation theory (Manning, 1978). Binding of Ca2+ ions to the lipid phosphate groups occurs, and Ca2+ ions may also form bridges between two neighboring phospholipid molecules, which establishes an attractive electrostatic interaction. This attractive force may reduce the area per lipid molecule. A similar effect on the packing of PC bilayers has been observed at high Ca2+ concentrations without the presence of DS (Huster et al., 1997; Zidovetzki et al., 1989). Furthermore, the binding of DS to the lipid via Ca2+-induced electrostatic interactions may result in an energetically favorable structural arrangement of phospholipids, calcium, and polyelectrolyte with fast ion exchange between multiple binding sites and formation of Ca2+ bridges. Because DS itself has no surface activity and does not penetrate into the hydrophobic region of the bilayer, an influence of DS on the hydrophobic effect is unlikely but cannot entirely be ruled out.

The attractive forces between the lipid molecules, induced by Ca2+-mediated DS binding, stabilize the more tightly packed lipid gel phase, as seen by an increase of the lipid phase transition temperature. As the lateral tension increases, the chain melting is shifted toward higher temperatures. Similar shifts in the phase transition in PC membranes at high Ca2+ concentration have been reported (Blatt and Vaz, 1986). Increased lipid phase transition temperatures, due to divalent cation mediated GAG binding, have also been measured by DSC (Voszka et al., 1989; Steffan et al., 1994).

DS bridging between lipid surfaces

In the presence of DS and Ca2+, the x-ray repeat spacing of DMPC bilayers is reduced (Fig. 1). In contrast, the presence of Ca2+ alone results in electrostatic repulsion between lipid bilayers that increases the repeat spacing (Lis et al., 1981). The reduction in spacing can be explained by the binding of DS strands to both bilayer surfaces via Coulomb interactions, a process that has been called "bridging of the polyelectrolyte" (Akesson et al., 1989; Podgornik, 1991, 1992) (see Fig. 9). The bridging mechanism provides an attractive force that causes the lipid surfaces to approach each other. The attractive bridging forces counterbalance repulsive hydration, electrostatic, and steric forces.

The x-ray repeat spacing is the sum of bilayer and water layer thickness. The determination of the bilayer thickness from the repeat spacings was shown to be model-dependent (Petrache et al., 1998; Lemmich et al., 1999; Janiak et al., 1976). Because lipid chains are extended as a result of DS binding (as revealed by an increase of the lipid chain order parameter, see Eq. 3) the observed reduction in the x-ray repeat spacing must be the result of a changed thickness of the water/DS layer between the lipid bilayers. For example, at 5 mM Ca2+ and 1 mg/ml DS 500, the thickness of the water layer is reduced by 6.8 *. Assuming an average area per DMPC molecule of 59.5 AZ (Koenig et al., 1997), the reduction in x-ray repeat spacing translates into a loss of -7 water molecules per lipid.

According to this rough estimate, DS bridging results in a close approach between lipid surfaces squeezing out -30% of the water molecules from the space between bilayers (assuming that the number of waters per lipid molecule at full hydration is 23 (Arnold and Gawrisch, 1993)). Fluorescence measurements by Steffan et al. (1994) on GAG binding to bilayer surfaces via Ca2+ also suggested bilayer dehydration. This raises the question whether lipid dehydration is a primary effect of DS interaction or a secondary result of the presence of strong DS-mediated attractive forces between bilayers. Membrane dehydration also results in increased lipid chain order and decreased area per lipid molecule (Gawrisch and Holte, 1996; Holte and Gawrisch, 1996). We consider the observed dehydration a secondary effect of Ca2+/DS binding to the lipid. Because both DS and Ca2+ alone are likely to attract water to the membrane interface because of their high charge density, the gain in free energy from bridging must be large enough to overcompensate the repulsive forces. An even closer approach is prevented by a steep increase in hydration repulsion, but also by a loss in configurational entropy of the polyelectrolyte. This model is supported by preliminary magic angle spinning NMR results that suggest a strong reduction in mobility of lipid-bound DS (unpublished results).

Monolayer surface pressure reduction

The equilibrium of attractive and repulsive forces in a lipid bilayer results in a tension-free state. Lipid monolayers require application of an outside surface pressure for stability. The pressure equivalent to a bilayer was reported to be -30 to 35 mN/m (Marsh, 1996). Therefore, the monolayer experiments in this study were carried out at a surface pressure of 32 mN/m.

Ca2+-mediated binding of DS to DMPC monolayers reduced the surface tension at constant area (Figs. 6 and 7). This confirms that DS molecules do not penetrate deep into the lipid monolayer; otherwise, an increased monolayer surface pressure would have been observed (de Meijere et al., 1997; Demel et al., 1989). The decrease of surface tension is consistent with the reduction in lipid area per molecule that was calculated from the lipid chain order parameters. Very recent experiments on DS interactions with DPPE monolayers via Ca2+ also observed denser lipid packing (de Meijere et al., 1998). D.H. is grateful for a grant by the Studienstiftung des deutschen Volkes. The work was supported by the Deutsche Forschungsgemeinschaft (SFB 197, A10).

REFERENCES

Akesson, T., C. E. Woodward, and B. Jonsson. 1989. Electric double layer forces in the presence of polyelectrolytes. J. Chem. Phys. 91:2461-2469.

Arnold, K. 1995. Cation-induced vesicle fusion modulated by polymers and proteins. In Structure and Dynamics of Membranes. R. Lipowsky and E. Sackmann, editors. Elsevier Science B. V., Amsterdam. 903-957.

Arnold, K., J. Amhold, O. Zschornig, D. Wiegel, and M. Krumbiegel. 1989. Characterization of chemical modifications of surface properties of low density lipoproteins. Biomed. Biochim. Acta. 48:735-742.

Arnold, K., and K. Gawrisch. 1993. Effects of fusogenetic agents on membrane hydration: a deuterium nuclear magnetic resonance approach. Methods EnzymoL 220:143-157.

Arnold, K., S. Ohki, and M. Krumbiegel. 1990. Interaction of dextran sulfate with phospholipid surfaces and liposome aggregation and fusion. Chem. Phys. Lipids. 55:301-307.

Barry, J. A., and K. Gawrisch. 1995. Effects of ethanol on lipid bilayers containing cholesterol, gangliosides, and sphingomyelin. Biochemistry. 34:8852-8860.

Bihari-Varga, M., J. Sztatisz, and S. Gal. 1981. Changes in the physical behavior of low density lipoprotein in the presence of glycosaminoglycans and high density lipoprotein. Atherosclerosis. 39:19-23.

Blatt, E., and L. C. Vaz. 1986. The effect of Ca2+ on lipid diffusion. Chem. Phys. Lipids. 41:183-194.

Bloom, M., E. Evans, and 0. G. Mouritsen. 1991. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q. Rev. Biophys. 24:293-397.

Bunn, C. W. 1939. The crystal structure of long-chain paraffin hydrocarbons. Trans. Faraday Soc. 35:482-491.

Camejo, G. 1982. The interaction of lipids and lipoproteins with the intercellular matrix of arterial tissue: its possible role in atherogenesis. Adv. Lipid. Res. 19:1-53.

Camejo, G., A. Lopez, F. Lopez, and J. Quinones. 1985. Interaction of low density lipoproteins with arterial proteoglycans. The role of charge and sialic acid content. Atherosclerosis. 55:93-105.

Cardin, A. D., R. L. Jackson, B. Elledge, and D. Feldhake. 1989. Dependence on heparin chain-length of the interaction of heparin with human plasma low density lipoproteins. Int. J. Biol. Macromol. 11:59-62.

Cardin, A. D., and H. J. R. Weintraub. 1989. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis. 9:21-32.

Davis, J. H. 1983. The description of membrane lipid conformation, order and dynamics by 2H-NMR. Biochim. Biophys. Acta. 737:117-lil.

Davis, J. H., K. R. Jeffrey, M. Bloom, M. I. Valic, and T. P. Higgs. 1976. Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem. Phys. Lett. 42:390-394.

de Meijere, K., G. Brezesinski, and H. Mohwald. 1997. Polyelectrolyte coupling to a charged lipid monolayer. Macromolecules. 30:2337-2342.

de Meijere, K., G. Brezesinski, O. Zschornig, K. Arnold, and H. Mohwald. 1998. Structure studies of a phospholipid monolayer coupled to dextran sulfate. Physica B. 248:269-273.

Demel, R. A., W. Jordi, H. Lambrechts, H. van Damme, R. Hovius, and B. de Kruijff. 1989. Differential interactions of apo and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria. J. Biol. Chem. 264:3988-3997.

Diederich, A., C. Sponer, D. Pum, U. B. Sleytr, and M. Losche. 1996. Reciprocal influence between the protein and lipid components of a lipid-protein membrane model. Colloids Surf, B. 6:335-346.

Evans, E., and R. Skalak. 1980. Mechanics and Thermodynamics of Biomembranes. CRC Press, Boca Raton, FL.

Fenske, D. B., and R. J. Cushley. 1990. Insoluble complex formation between low density lipoprotein and heparin. A 3'P-NMR study. Chem. Phys. Lipids. 54:9-16.

Gawrisch, K., J. A. Barry, L. L. Holte, T. M. Sinnwell, L. D. Bergelson, and J. A. Ferretti. 1995. Role of interactions at the lipid-water interface for domain formation. Mol. Membr. Biol. 12:83-88.

Gawrisch, K., and L. L. Holte. 1996. NMR investigations of non-lamellar phase promoters in the lamellar phase state. Chem. Phys. Lipids. 81: 105-116.

Gigli, M., A. Consonni, G. Ghiselli, V. Rizzo, A. Naggi, and G. Tom. 1992. Heparin binding to human plasma low-density lipoproteins: dependence on heparin sulfation degree and chain length. Biochemistry. 31:5996-6003.

Holte, L. L., and K. Gawrisch. 1996. Influence of lipid hydration on hydrocarbon chain order: a 2H-NMR study. Biophys. J. 70:418a. (Abstr.).

Holte, L. L., F. Separovic, and K. Gawrisch. 1996. Nuclear magnetic resonance investigations of hydrocarbon chain packing in bilayers of polyunsaturated phospholipids. Lipids. 31:S199S203.

Huster, D., and K. Arnold. 1998. Ca2+-mediated interaction between dextran sulfate and dimyristoyl-sn-glycero-3-phosphocholine surfaces studied by 2H-NMR. Biophys. J. 75:909-916.

Huster, D., A. J. Jin, K. Arnold, and K. Gawrisch. 1997. Water permeability of polyunsaturated lipid membranes measured by 17O-NMR. Biophys. J. 73:855-864.

Israelachvili, J., S. Marcelja, and R. G. Horn. 1980. Physical principles of membrane organization. Q. Rev. Biophys. 13:121-200.

Iverius, P. H. 1972. The interaction between human plasma lipoproteins and connective tissue glycosaminoglycans. J. Biol. Chem. 247: 2607-2613.

Janiak, M. J., D. M. Small, and G. G. Shipley. 1976. Nature of the thermal pretransition of synthetic phospholipids: dimyristolyl- and dipalmitoyllecithin. Biochemistry. 15:4575-4580.

Johnson, S. J., T. M. Bayerl, W. Weihan, H. Noack, J. Penfold, R. K. Thomas, D. Kanellas, A. R. Rennie, and E. Sackmann. 1991. Coupling of spectrin and polylysine to phospholipid monolayers studied by specular reflection of neutrons. Biophys. J. 60:1017-1025.

Kim, Y. C., and T. Nishida. 1977. Nature of interaction of dextran sulfate with lecithin dispersions and lysolecithin micelles. J. Biol. Chem. 252: 1243-1249.

Kim, Y. C., and T. Nishida. 1979. Nature of the interaction of dextran sulfate with high and low density lipoproteins in the presence of Ca2+. J. Biol. Chem. 254:9621-9626.

Koenig, B. W., H. H. Strey, and K. Gawrisch. 1997. Membrane lateral compressibility measured by NMR and x-ray diffraction of acyl chain polyunsaturation. Biophys. J. 73:1954-1966.

Krumbiegel, M., and K. Arnold. 1990. Microelectrophoresis studies of the binding of glycosaminoglycans to phosphatidylcholine liposomes. Chem. Phys. Lipids. 54:1-7.

Lafleur, M., B. Fine, E. Sternin, P. R. Cullis, and M. Bloom. 1989. Smoothed orientational order profile of lipid bilayers by 2H-nuclear magnetic resonance. Biophys. J. 56:1037-1041.

Laroche, G., E. J. Dufourc, Me Pezolet, and J. Dufourcq. 1990. Coupled changes between lipid order and polypeptide conformation at the membrane surface. A 2H-NMR and Raman study of polylysine-phosphatidic acid systems. Biochemistry. 29:6460-6465.

Lemmich, J., K. Mortensen, J. He Ipsen, T. Honger, R. Bauer, and O. G. Mouritsen. 1999. Small-angle neutron scattering from multilamellar lipid bilayers: theory, model, and experiment. Phys. Rev. E. 53: 5169-5180.

Lis, L. J., V. A. Parsegian, and R. P. Rand. 1981. Binding of divalent cations to dipalmitoylphosphatidylcholine bilayers and its effect on bilayer interaction. Biochemistry. 20:1761-1770.

Maksymiw, R., S. F. Sui, H. Gaub, and E. Sackmann. 1987. Electrostatic coupling of spectrin dimers to phosphatidylserine containing lipid lamellae. Biochemistry. 26:2983-2990.

Manning, G. S. 1978. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q. Rev. Biophys. 11:179-246.

Marsh, D. 1992. Handbook of Lipid Bilayers. CRC Press, Boca Raton, FL.

Marsh, D. 1996. Lateral pressure in membranes. Biochim. Biophys. Acta. 1286:183-223.

McCabe, M. A., and S. R. Wassall. 1995. Fast-Fourier-transform DePaking. J. Magn. Reson. B. 106:80-82.

Nagle, J. F. 1993. Area/lipid of bilayers from NMR. Biophys. J. 64: 1476-1481.

Nezil, F. A., and M. Bloom. 1992. Combined influence of cholesterol and synthetic amphiphilic peptides upon bilayer thickness in model membranes. Biophys. J. 61:1176-1183.

Nishida, T., and U. Cogan. 1970. Nature of the interaction of dextran sulfate with low density lipoproteins of plasma. J. Biol. Chem. 245: 4689-4697.

Petrache, H. I., N. Gouliaev, S. Tristram-Nagle, R. Zhang, R. M. Suter, and J. F. Nagle. 1998. Interbilayer interactions from high resolution x-ray scattering. Phys. Rev. E. 57:7014-7024.

Podgornik, R. 1991. Electrostatic forces between charged surfaces in the presence of a polyelectrolyte chain. J. Phys. Chem. 95:5249-5255.

Podgornik, R. 1992. Self-consistent-field theory for confined polyelectrolyte chains. J. Phys. Chem. 96:884-896.

Roux, M., J.-M. Neumann, R. S. Hodges, P. F. Devaux, and M. Bloom. 1989. Conformational changes of phospholipid headgroups induced by a cationic integral membrane peptide as seen by deuterium magnetic resonance. Biochemistry. 28:2313-2321.

Rudel, L. L., J. S. Parks, F. L. Johnson, and J. Babiak. 1986. Low density lipoproteins in atherosclerosis. J. Lipid Res. 27:465-474.

Seelig, J. 1977. Deuterium magnetic resonance: theory and application to lipid membranes. Q. Rev. Biophys. 10:353-418.

Seelig, A., and J. Seelig. 1974. The dynamic structure of fatty acyl chains in a phospholipid bilayer measured by deuterium magnetic resonance. Biochemistry. 13:4839-4845.

Srinivasan, S. R., B. Radhakrishnamurthy, and G. S. Berenson. 1975. Studies on the interaction of heparin with serum lipoproteins in the presence of Ca"+, Mg2+, and Mn2+. Arch. Biochem. Biophys. 170: 334-340.

Steffan, G., S. Wulff, and H. J. Galla. 1994. Divalent cation-dependent interaction of sulfated polysaccharides with phosphatidylcholine and mixed phosphatidylcholine/phosphatidylglycerol liposomes. Chem. Phys. Lipids. 74:141-150.

Sternin, E., M. Bloom, and L. MacKay. 1983. De-Pake-ing of NMR spectra. J. Magn. Reson. 55:274-282.

Voszka, I., S. Gyorgyi, and M. Bihari-Varga. 1989. Effect of glycosaminoglycans and divalent cations on the thermal properties of dipalmitoylphosphatidylcholine. Chem. Phys. Lipids. 51:67-71.

Weisgraber, K. H., and S. C. Rall, Jr. 1987. Human apolipoprotein B-100 heparin-binding sites. J. Biol. Chem. 262:11097-11103.

Zidovetzki, R., A. W. Atiya, and H. de Boeck. 1989. Effect of divalent cations on the structure of dipalmitoylphosphatidylcholine and phosphatidylcholine/phosphatidylglycerol bilayers: A 2H-NMR study. Membr. Biochem. 8:177-186.

Daniel Huster,*# Gerrit Paasche,* Undine Dietrich, Olaf Zschornig,* Thomas Gutberlet, Klaus Gawrisch,# and Klaus Arnold*

*Institute of Medical Physics and Biophysics, University of Leipzig, 04103 Leipzig, Germany; "Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland 20852 USA; and Institute for Experimental Physics I, Department of Physics of Biomembranes, University of Leipzig, 04103 Leipzig, Germany

Received for publication 1 October 1998 and in final form 7 May 1999.

Address reprint requests to Dr. Klaus Arnold, Institute of Medical Physics and Biophysics, University of Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany. Tel.: +49-(0)-341-97-15701; Fax: +49-(0)-341-97-15709; E-mail: arnold@server .medizin.uni-leipzig.de.

Copyright Biophysical Society Aug 1999
Provided by ProQuest Information and Learning Company. All rights Reserved