Temperature dependence of the repulsive pressure between phosphatidylcholine bilayers.

Bilayer structure and interbilayer repulsive pressure were measured from 5 to 50 degrees C by the osmotic stress/x-ray diffraction method for both gel and liquid crystalline phase lipid bilayers. For gel phase dibehenoylphosphatidylcholine (DBPC) the bilayer thickness and pressure-distance relations were nearly temperature-independent, and at full hydration the equilibrium fluid spacing increased approximately 1 A, from 10 A at 5 degrees C to 11 A at 50 degrees C. In contrast, for liquid crystalline phase egg phosphatidylcholine (EPC), the bilayer thickness, equilibrium fluid spacing, and pressure-distance relation were all markedly temperature-dependent. As the temperature was increased from 5 to 50 degrees C the EPC bilayer thickness decreased approximately 4 A, and the equilibrium fluid spacing increased from 14 to 21 A. Over this temperature range there was little change in the pressure-distance relation for fluid spacings less than approximately 10 A, but a substantial increase in the total pressure for fluid spacings greater than 10 A. These data show that for both gel and liquid crystalline bilayers there is a short-range repulsive pressure that is nearly temperature-independent, whereas for liquid crystalline bilayers there is also a longer-range pressure that increases with temperature. From analysis of the energetics of dehydration we argue that the temperature-independent short-range pressure is consistent with a hydration pressure due to polarization or electrostriction of water molecules by the phosphorylcholine moiety. For the liquid crystalline phase, the 7 A increase in equilibrium fluid spacing with increasing temperature can be predicted by an increase in the undulation pressure as a consequence of a temperature-dependent decrease in bilayer bending modulus.     

Cholesterol interactions with phospholipids in membranes.

Mammalian cell membranes are composed of a complex array of glycerophospholipids and sphingolipids that vary in head-group and acyl-chain composition. In a given cell type, membrane phospholipids may amount to more than a thousand molecular species. The complexity of phospholipid and sphingolipid structures is most likely a consequence of their diverse roles in membrane dynamics, protein regulation, signal transduction and secretion. This review is mainly focused on two of the major classes of membrane phospholipids in eukaryotic organisms, sphingomyelins and phosphatidylcholines. These phospholipid classes constitute more than 50% of membrane phospholipids. Cholesterol is most likely to associate with these lipids in the membranes of the cells. We discuss the synthesis and distribution in the cell of these lipids, how they are believed to interact with each other, and what cellular consequences such interactions may have. We also include a discussion about findings in the recent literature regarding cholesterol/phospholipid interactions in model membrane systems. Finally, we look at the recent trends in computer and molecular dynamics simulations regarding phospholipid and cholesterol/phospholipid behavior in bilayer membranes.     

Cholesterol movement between human skin fibroblasts and phosphatidylcholine vesicles

Cholesterol readily exchanges between human skin fibroblasts and unilamellar phospholipid vesicles. Only a fraction of the exchangeable cholesterol and only 10–15% of the total cellular free cholesterol is available for net movement or depletion to cholesterol-free phosphatidylcholine vesicles. [¹⁴C]Cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles does not readily equilibrate with fibroblast cholesterol labelled endogenously from [³H]mevalonic acid. While endogenously-synthesized [³H]cholesterol readily becomes incorporated into a pool of esterified cholesterol, little, if any, of the [¹⁴C]cholesterol introduced into the fibroblast plasma membrane by exchange from lipid vesicles becomes available for esterification. We interpret these findings as suggesting that: (1) net cholesterol movement from fibroblasts to an acceptor membrane is limited to a small percentage of the plasma membrane cholesterol, and (2) separate pools of cholesterol exist in human skin fibroblasts, one associated with the plasma membrane and the second associated with intracellular membranes, and equilibration of cholesterol between the two pools is a very limited process.     

An electron spin resonance study of interactions between phosphatidylcholine and phosphatidylserine in oriented membranes.

A detailed electron spin resonance (ESR) study of mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and phosphatidylserine (POPS) in oriented multilayers in the liquid crystalline phase is reported with the purpose of characterizing the effects of headgroup mixing on the structural and dynamical properties of the acyl chains. These studies were performed over a range of blends of POPC and POPS and temperatures, utilizing the spin-labeled lipids 16-phosphatidylcholine and 5-phosphatidylcholine as well as cholestane (CSL). The ESR spectra were analyzed by nonlinear least-squares fitting using detailed spectral simulations. Whereas CSL shows almost no variation in ordering and rotational dynamics versus mole fraction POPS, (i.e. XPS), and 5-PC shows small effects, the weakly ordered end-chain labeled 16-PC shows large relative effects, such that the orientational order parameter, S is at a minimum for XPS = 0.5 where it is about one-third the value observed for XPS = 0 and 1. This is directly reflected in the ESR spectrum as a substantial variation in the hyperfine splitting with XPS. The least-squares analysis also shows a reduction in rotational diffusion coefficient, R perpendicular by a fractor of 2 for XPS = 0.5 and permits the estimation of S2, the ordering parameter representing deviations from cylindrically symmetric alignment. These results are contrasted with 2H NMR studies which were insensitive to effects of mixing headgroups on the acyl chains. The ESR results are consistent with a somewhat increased disorder in the end-chain region as well as a small amount of chain tilting upon mixing POPC and POPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a biomimetic chromatographic stationary phase for study of the interactions occurring between inorganic anions and phosphatidylcholine membranes

A liquid chromatographic method for the study of ion-membrane interactions is reported. A phosphatidylcholine biomimetic stationary phase was established by loading dimyristoylphosphatidylcholine (DMPC) onto a reversed-phase octadecylsilica packed column. This column was then used to study the interaction of some inorganic anions with the stationary phase by UV and conductivity detection. Ten inorganic anions were selected as model ions and were analyzed with the proposed chromatographic system. Anion-DMPC interactions of differing magnitudes were observed for all of the model anions. Perchlorate-DMPC interactions were strongest, followed by thiocyanate-DMPC, iodide-DMPC, chlorate-DMPC, nitrate-DMPC, bromide-DMPC, chloride-DMPC, fluoride-DMPC, and then sulfate-DMPC. Cations in the eluent, especially H(+) ions and divalent cations such as Ca(2+), showed strong effects on anion-DMPC interactions. The chromatographic data suggest that DMPC interacts with both the anions and the cations. Anion-DMPC interactions were dependent on the surface potential of the stationary phase: at low surface potentials anion-DMPC interactions were predominantly solvation dependent in nature whereas at more positive surface potentials anion-DMPC interactions were predominantly electrostatic in nature. Cation-DMPC interactions served to raise the surface potential, causing the anion-DMPC interactions to vary from solvation dependent to electrostatic. The chromatographic data were used to provide quantitative estimates of the enthalpies of the anion-DMPC interactions.     

Fluorescence and thermotropic studies of the interactions of PEI-cholesterol based PEI-chol lipopolymers with dipalmitoyl phosphatidylcholine membranes

Numerous PEI derived polymers have been explored for their use in gene delivery. Nine PEI-chol lipopolymers based on cholesterol grafting on three polyethyleneimines (PEI) of different molecular weights have been synthesized. Firstly their aggregation behavior has been studied using transmission electron microscopy and then their interactions with l-α-dipalmitoyl phosphatidylcholine (DPPC) membranes have been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These PEI-chol lipopolymers are found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes, depending upon the cholesterol grafting on PEI. These PEI-chol lipopolymers act as filler molecules in membranes. Electron microscopy shows the different aggregation behavior of these new PEI-chol lipopolymers depending upon the molecular weight of PEI and percentage of cholesterol grafting. Detailed analysis of the fluorescence anisotropy and DSC data indicate that the nature of perturbation induced by PEI-chol lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI. In general, PEI-chol lipopolymers rigidify the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase unlike natural cholesterol, which is known to fluidize the gel phase of the membranes.     

Monosaccharide composition, chain length and linkage type influence the interactions of oligosaccharides with dry phosphatidylcholine membranes

Sugars play an important role in the desiccation tolerance of most anhydrobiotic organisms and disaccharides have been extensively investigated for their ability to stabilize model membranes in the dry state. Much less is known about the ability of oligosaccharides to protect dry membranes. However, it has been shown that different structural families of oligosaccharides have different efficacies to interact with and protect membranes during drying. Here, we have compared three families of linear oligosaccharides (fructans, malto-oligosaccharides, manno-oligosaccharides) for their chain-length dependent lyoprotective effect on egg phosphatidylcholine liposomes. We found increased protection with chain length for the fructans, a moderate decrease in protection with chain length for malto-oligosaccharides, and a strong decrease for manno-oligosaccharides. Using Fourier-transform infrared spectroscopy and differential scanning calorimetry, we show that the degree of lyoprotection of the different sugars is closely related to their influence on the gel to liquid-crystalline phase behavior of the dry membranes and to the extent of H-bonding to different groups (C=O, P=O, choline) in the lipids. Possible structural characteristics of the different oligosaccharides that may determine the extent to which they are able to interact with and protect membranes are discussed.     

Monosaccharide composition, chain length and linkage type influence the interactions of oligosaccharides with dry phosphatidylcholine membranes

Sugars play an important role in the desiccation tolerance of most anhydrobiotic organisms and disaccharides have been extensively investigated for their ability to stabilize model membranes in the dry state. Much less is known about the ability of oligosaccharides to protect dry membranes. However, it has been shown that different structural families of oligosaccharides have different efficacies to interact with and protect membranes during drying. Here, we have compared three families of linear oligosaccharides (fructans, malto-oligosaccharides, manno-oligosaccharides) for their chain-length dependent lyoprotective effect on egg phosphatidylcholine liposomes. We found increased protection with chain length for the fructans, a moderate decrease in protection with chain length for malto-oligosaccharides, and a strong decrease for manno-oligosaccharides. Using Fourier-transform infrared spectroscopy and differential scanning calorimetry, we show that the degree of lyoprotection of the different sugars is closely related to their influence on the gel to liquid-crystalline phase behavior of the dry membranes and to the extent of H-bonding to different groups (CO, PO, choline) in the lipids. Possible structural characteristics of the different oligosaccharides that may determine the extent to which they are able to interact with and protect membranes are discussed.     

Cholesterol interactions with tetracosenoic acid phospholipids in model cell membranes: role of the double-bond position

The synthesis and thermotropic properties of 1,2-di-(9Z)-9-tetracosenoylphosphatidylcholine [delta 9-PC(24:1,24:1), 1], 1,2-di-(5Z)-5-tetracosenoylphosphatidylcholine [delta 5-PC(24:1,24:1), 2], and 1,2-di-(15Z)-15- tetracosenoylphosphatidylcholine [delta 15-PC(24:1,24:1), 3] are reported. Liposomes prepared from these phospholipids differ from those of the natural sponge phospholipids, 1,2-di-(5Z,9Z)-5,9-hexacosadienoylphosphatidylcholine (4a) and the corresponding ethanolamine (4b), both of which virtually exclude cholesterol from their bilayers. The behavior of 1 and 2 is similar to that of 1,2-di-(6Z,9Z)-6,9-hexacosadienoylphosphatidylcholine (5), which exhibits a partial molecular interaction with cholesterol. In the case of 3, cholesterol appears to interact with the saturated acyl chain regions of this phospholipid in a manner similar to that of its interaction with DPPC acyl chains. This study delineates the effect of the double-bond location in long fatty acyl chains of phospholipids on their interactions with cholesterol.     

Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance

Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Theory of hapten binding to IgM: the question of repulsive interactions between binding sites.

Various workers in their studies of the binding of haptens to IgM have observed that at low hapten concentration IgM has an apparent valence of five or near five, while at high hapten concentration IgM has a valence of ten. A possible explanation for this is that there is an interaction between binding sites on the same F(ab')2 region of the IgM molecule. In this paper the theory for such an interaction is presented and an expression for the apparent valence is derived. It is shown that the apparent valence depends on both the interaction between binding sites on the IgM molecule and on the width of the affinity distribution which characterizes the antiserum. A broad affinity distribution can give an apparent valence of five even when there is no interaction between sites, i.e., even when the ten binding sites on the IgM molecule are identical and independent. The general properties of a Scatchard plot are also discussed. When there is no interaction between sites it is shown that the average affinity and the variance of the affinity distribution can be obtained from a Scatchard plot. To illustrate the theory, an antiserum with affinities characterized by a normal distribution is considered and a simple method is presented for determining alpha, the parameter which measures the width of the normal distribution.     

Ionophorous properties of the 20,000-dalton fragment of (Ca2++Mg2+)-ATPase in phosphatidylcholine: Cholesterol membranes

Summary The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca²⁺+Mg²⁺)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem.251:4147) to have Ca²⁺-selective ionophoric activity. The Ca²⁺-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca²⁺-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same,P _Ba>P _Ca>P _Sr>P _Mg>P _Mn. This selectivity sequence is the same as that for the intact (Ca²⁺ +Mg²⁺)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except thatP _Mn>P _Mg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca²⁺-ionophoric activity of the (Ca²⁺+Mg²⁺)-ATPase.     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.