Interaction of basic polypeptides with phospholipid monolayers

Adsorption of the polylysine and of the copolypeptides: L-lysine/L-serine and L-lysine/L-phenylalanine on phospholipid monolayers has been investigated. The charge density of the monolayers was varied by using the negatively charged phosphatidyl serine and the neutral phosphatidyl choline at different ratios. The surface concentrations of the adsorbed polypeptides was determined by measuring the surface radiation of their radioactive label.The adsorbing capacity of the monolayer surfaces increases with their negative charge, however with respect to polypeptides the surface activity sequence is pL < pLS < pLφ. From the dependence of adsorption on the ionic strength it was concluded that it is controlled by three types of interaction: (1) electrostatic attraction to the negatively charged surface; (2) electrostatic repulsion between adsorbed polybases; (3) hydrophobic interactions involving specific structural arrangements. This is true even of the apparently neutral PC monolayer where the fixed phosphate groups form an electrical double layer with the more mobile choline groups which can be interpenetrated by the charged groups of the basic polypeptides.     

Influence of dicarboxylic phosphatidylcholines on the stability and phase transition of phosphatidylcholine liposomes

Utilizing reflectance spectrophotometry, hemoperfusion, rate of oxygen consumption and redox level of mitochondrial cytochrome c (+c1) in livers in situ of anesthetized rats were measured. The transition to the anoxic state was induced by raising the pressure on the liver surface to more than the hepatic blood pressure by pressing with the tip of the optical guide of the reflectance spectrophotometer. During this transition, the average oxygen saturation of hemoglobin in the liver in situ decreased linearly with time until it became 10--20% of the total. This was followed by reduction of mitochondrial cytochrome c (+c1), which reached completion in 10--20 s. The measured O2 consumption rate remained constant until the percentage of oxyhemoglobin in situ decreased to a critical level. There was then a decrease in the rate of O2 consumption which was accompanied by a progressive reduction of cytochrome c (+c1). It was shown that amounts of hemoglobin and mitochondrial respiratory chain cytochromes in the liver in situ could be measured non-invasively and could provide important signals for vital cellular functions. The changes in hemoperfusion and rate of O2 consumption of the liver in situ following ethanol ingestion were also shown in rats, and are briefly discussed with respect to possible application of this method to study the pathophysiology of tissues.     

Anisodamine induces the hexagonal phase in phospholipid liposomes

The effects of anisodamine on the polymorphic phase behaviour of cardiolipin and dioleoylphosphatidylcholine liposomes have been investigated by freeze-fracture electron microscopy. Anisodamine induces the formation of lipidic particles in cardiolipin liposomes at pH 7.0 and hexagonal HII tubes at pH 8.8. When the molar ratio of anisodamine and dioleoylphosphatidylcholine is 4 to 1, lipidic particles can be observed in the fracture faces.     

Solution effects on the thermotropic phase transition of unilamellar liposomes

We have investigated the effect of two monosaccharides, glucose and fructose, and two disaccharides, sucrose and trehalose, on the thermotropic phase transition of unilamellar extruded vesicles of DPPC. All the sugars investigated raise the main transition temperature (Tm) of some fraction of the lipid, but there are differences between the effect of glucose and the other three sugars. At low concentrations of glucose, Tm is lowered. At high concentrations of glucose there are two transitions, one with a low Tm and one with a high Tm. The data suggest that at low concentrations, all of the glucose present may bind to the bilayer and increase headgroup spacing by physical intercalation or increased hydration. The appearance of a Tm above that of pure hydrated DPPC suggests the possibility of the dehydration of some other population of phospholipid molecules. The other three sugars increase Tm, but at high concentrations of trehalose, sucrose, and fructose a second peak occurs at a low Tm. The other sugars appear to dehydrate the bilayer at low concentrations, but may show some binding or increased hydration of some portion of the lipid at very high concentrations. The sugar effects on unilamellar vesicles are strikingly different from the effects of these sugars on multilamellar vesicles.     

Influence of the phospholipid structure on the stability of liposomes in serum

The effect of serum on the structural integrity of liposomes consisting of ether and/or carbamyl analogs of 1,2-diester phosphatidylcholine (PC) has been evaluated by measuring both the efflux of the entrapped 6-carboxyfluorescein and the lipid transfer to serum proteins, and the results have been compared with the egg PC liposomes. Replacement of the C-1 ester bond in PC by an ether linkage did not significantly enhance the liposome stability, but it was markedly increased upon introducing further structural changes in the C-2 ester region of the resulting 1-ether-2-ester PC. However, the stability was not influenced by altering the steric configuration of the latter phospholipid. These results strongly suggest that lysis of liposomes in serum can be prevented by structurally modifying the ester bond(s) in the phospholipid component of liposomes.     

The effect of hashish compounds on phospholipid phase transition

The interaction of hashish compounds, Δ¹-tetrahydrocannabinol and cannabidiol, with dipalmitoyl phosphatidylcholine was investigated using differential scanning calorimetry. Both drugs affect the transition of dipalmitoyl phosphatidylcholine from the gel to liquid crystalline state, decreasing both the melting temperature and the enthalpy of melting. At a drug to dipalmitoyl phosphatidylcholine ratio of approx. 1:5, two peaks appear in the transition profile, suggesting a phase separation in the drug dipalmitoyl phosphatidylcholine mixture.     

Fluorometric detection of the bilayer-to-hexagonal phase transition in liposomes

We have used the fluorescent probe N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) to detect the bilayer-to-hexagonal phase transition. The fluorescence intensity of the probe was found to increase during the bilayer-to-hexagonal transition. The bilayer-to-hexagonal transitions of various types of phosphatidylethanolamine or cardiolipin measured by this method are consistent with results obtained by differential scanning calorimetry. To establish this method for wider use, agents known to alter the bilayer-to-hexagonal transition were examined, and the results are comparable with the published data. The added advantage of this fluorometric method over other currently available techniques is that it is applicable not only for aggregated lipid samples but also for dilute liposome suspensions. This is especially important when one of the components of the system under study can partition between lipid and aqueous phase. Since NBD is located near the headgroup region of the bilayer, it most likely detects the change of the environment surrounding that region. On the basis of our present study, it appears that NBD-PE is sufficiently sensitive to detect bilayer-to-hexagonal phase transition.     

Influence of carbamoylation on some analytical properties of basic polypeptides

The effect of carbamoylation on the assay or identification of histones and polylysine was investigated. Incubation with sodium cyanate decreased the positive charge on these polypeptides as judged by changes in the binding of methyl orange or the electrophoretic mobility. Histones in chromatin appeared less accessible to carbamoylation than isolated histones. Carbamoylation of proteins under conditions in which there was little or no effect on the Lowry procedure could affect their assay by methods utilizing metachromasia with Coomassie Blue G. The Bradford assay has low sensitivity for Hl histone and polylysine but this can be increased by preincubation with sodium cyanate. More extensive carbamoylation of polylysine caused decreased sensitivity which was the only response seen with core nucleosomal histones and bovine serum albumin when preincubated with sodium cyanate. It was concluded that the sensitivity for Hl histone and polylysine in assays dependent on metachromasia with Coomassie Blue G may be changed by factors which decrease the positive charge on these polypeptides.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.     

Disc formation in cholesterol-free liposomes during phase transition

Cryogenic transmission electron microscopy (cryo-TEM) images of lysolipid-containing thermosensitive liposomes (LTSL) revealed that open liposomes and bilayer discs appeared when liposomes were cycled through the gel (L_β′) to liquid-crystalline (L_α) phase transition. The amount of bilayer discs generated was dependent on the combined presence of PEG-lipid and lysolipid in the membrane. We hypothesize that micelle-forming membrane components stabilize the rim of bilayer openings and membrane discs that form when liposomes are cycled through T_C.     

Disc formation in cholesterol-free liposomes during phase transition

Cryogenic transmission electron microscopy (cryo-TEM) images of lysolipid-containing thermosensitive liposomes (LTSL) revealed that open liposomes and bilayer discs appeared when liposomes were cycled through the gel (Lbeta') to liquid-crystalline (Lalpha) phase transition. The amount of bilayer discs generated was dependent on the combined presence of PEG-lipid and lysolipid in the membrane. We hypothesize that micelle-forming membrane components stabilize the rim of bilayer openings and membrane discs that form when liposomes are cycled through TC.     

A theory of the electric field-induced phase transition of phospholipid bilayers

Improving the statistical mechanical model of Jacobs et al. (Jacobs, R.E., Hudson, B. and Andersen, H.C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3993–3997) we have constructed a model which describes not only the temperature but also the external field dependence of the membrane structure of phospholipid bilayers. In addition to the interactions between head groups, between hydrocarbon chains, and the internal conformational energy of the chains (which were considered in Jacobs' model), our model includes the energy of deformation and the field energy as well.By the aid of this model we can explain the phenomenon of dielectric breakdown, the non-linearity of current-voltage characteristics, and the mechanism of membrane elasticity.The free energy of the membrane, the average number of the gauche conformations in the hydrocarbon interior and at the membrane surface, gauche distribution along the chain, the membrane thickness, area and volume are calculated at different temperatures and voltages. The calculation also gives the temperature dependence of Young's modulus and that of the linear thermal expansion coefficient.     

A theory of the electric field-induced phase transition of phospholipid bilayers.

Improving the statistical mechanical model of Jacobs et al. (Jacobs, R.E., Hudson, B. and Andersen, H.C. (1975) Proc. Natl. Acad. Sci. U.S. 72, 3993--3997) we have constructed a model which describes not only the temperature but also the external field dependence of the membrane structure of phospholipid bilayers. In addition to the interactions between head groups, between hydrocarbon chains, and the internal conformational energy of the chains (which were considered in Jacobs' model), our model includes the energy of deformation and the field energy as well. By the aid of this model we can explain the phenomenon of dielectric breakdown, the non-linearity of current-voltage characteristics, and the mechanism of membrane elasticity. The free energy of the membrane, the average number of the gauche conformations in the hydrocarbon interior and at the membrane surface, gauche distribution along the chain, the membrane thickness, area and volume are calculated at different temperatures and voltages. The calculation also gives the temperature dependence of Young's modulus and that of the linear thermal expansion coefficient.     

Effect of 1-anilinonaphthalene-8-sulphonate on phase transition temperature of dipalmitoylphosphatidylcholine liposomes

The effect of 1-anilinonaphthalene-8-sulfonate (ANS) on the thermotropic phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by differential scanning calorimetry. The main phase transition temperature was found to be shifted to lower values in the presence of the probe. The shift strongly depends on pH and the presence of salts. This indicates that the penetration of the probe of the hydrocarbon moiety of the bilayer is influenced by coulombic interactions. Pretransition phenomena are also affected. The implications for the interpretation of experimental data of biomembrane studies are discussed.     

Substrate-induced deformation and adhesion of phospholipid vesicles at the main phase transition

The physiochemical properties of phospholipid vesicle, e.g. permeability, elasticity, etc., are directly modulated by the chain-melting transition of the lipid bilayer. Currently, there is a lack of understanding in the relationship between thermotropic transition, mechanical deformation and adhesion strength for an adherent vesicle at temperature close to main phase transition temperature T(m). In this study, the contact mechanics of dimyristoyl-phosphatidylcholine (DMPC) vesicle at the main phase transition are probed by confocal reflectance interference contrast microscopy in combination with phase contrast microscopy. It is shown that DMPC vesicles strongly adhere on pure fused silica substrate at T(m) and the degree of deformation as well as the adhesion energy is a decreasing function against the mid-plane diameter of the vesicles. Furthermore, an increase of osmotic pressure at the gel/liquid crystalline phase co-existence imposes insignificant changes in both the degree of deformation and adhesion energy of adherent vesicles when the lipid bilayer permeability is maximized. With the reverse of substrate charge, the mechanical deformation and adhesion strength for larger vesicles (mid-plane diameter >18 microm) are significantly reduced. By monitoring the parametric response of substrate-induced vesicle adhesion during main phase transition, it is shown that the degree of deformation and adhesion energy of adhering vesicle is increased and unchanged, respectively, against the increase of temperature.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.