Contributions of lipids and proteins to the surface charge of membranes. An electron microscopy study with cationized and anionized ferritin

The surface charge of cultured neurons was investigated with the electron microscope markers anionized ferritin (AF) and cationized ferritin (CF). To determine which membrane components could react with the markers, model reactions were used. Both protein-coated Sepharose beads and lipid vesicles were reacted at physiological pH. Results with these model reactions indicate that the following groups may contribute to the surface charge: acidic groups--the sialic acid of both glycoproteins and gangliosides, the carboxyl group of proteins, and the phosphates of phospholipids; basic groups--the amines of proteins. The effect of chemical fixation on the surface charge was investigated. Glutaraldehyde fixation was shown to increase the charge of neutral proteins but not by a mechanism involving unbound aldehydes. Glutaraldehyde fixation of phospholipid vesicles in the presence of CF showed that amine-containing phospholipids were cross-linked to CF. This cross-linkage was seen with the electron microscope as the clumping of CF and the burying of CF in the membrane. Paraformaldehyde fixation had a lesser effect on the charge of proteins but did react with phospholipids as did glutaraldehyde. It is concluded that at physiological pH: (a) most of the charged proteins and lipids on cell surface can contribute to the membrane surface charge, and (b) the membrane surface charge of cells can be greatly changed by chemical fixation.     

Changes in physico-chemical properties of human large intestine tumour cells membrane

Tumour cells produce and excrete to blood many substances which are present in the cell itself in trace amounts only. Our work has been aimed at the determination of changes in electric charge and in phospholipid composition of large intestine normal mucosa and colorectal cancer cells.Surface charge density of tumour unaffected mucosa and of tissue sections from tumours, was measured by electrophoresis. The measurements were carried out at various pH of solution. Membrane isoelectric point was determined by measuring its electric charge in function of pH as well as total positive charge at low pH and total negative charge at high pH. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC. Four phospholipid classes were identified: PI, PS, PE and PC and their surface concentrations were determined.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.Tumour changes provoke an increase in surface charge density of large intestine membrane, whereas the content of individual phospholipids increased or decreased depending on a patient.     

Electrophoretic mobility of UV-irradiated bacterial cells

Electrophoretic mobility of Escherichia coli cells exposed to various doses of UV-radiation was investigated. The method of free flow electrophoresis was used to study a correlation between membrane protein charge and cell surface electric charge. The change in the cell surface charge and electrophoretic motility was associated with the damage to membrane proteins and the survival of UV-irradiated bacteria.     

Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

Effect of pH on the surface charge density of plant membranes. Comparison of microsomes and liposomes

The surface potential of microsomes of horse bean roots was compared to the one of liposomes prepared from the whole phospholipid extracts. The surface potential was determined from the affinity of the membranes for the anilinonaphthalene sulphonate dye. The effect of pH was studied at two KCl concentrations. It appeared from this comparison that the surface charge density was nearly the same on both materials in the neutral pH range. The isoelectric point was pH 1.7 for the liposomes and pH 4.0 for the microsomes. The implication of these observations is that the surface charge density of microsomes is nearly the same above the lipid and protein components of the membrane. This hypothesis was checked by measuring the activity of a microsomal enzyme with an anionic substrate, while modifying the net surface charge of the membrane. The biological significance of the results is discussed.     

Interaction of enkephalin peptides with anionic model membranes.

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen(2),D-Pen(5)]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

Interaction of the nuclear proteins protamine and histone with charged bilayer membranes]

The interaction of nuclear proteins of protamine and histone with neutral and charged BLM was studied. Anion and cation detergents were used to create the surface charge. The surface density of charges in BLM was comparable with that in biomembranes. Protamine and histone increased the electroconductivity of negatively charged BLM for anions and cations correspondingly. It is suggested that the surface charge of the membrane may influence the ion transport directly and indirectly due to the interaction of the membrane structures with charged proteins present in the surrounding medium.     

Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts

The influence of membrane surface charge on cation uptake was investigated in protoplasts prepared from roots of mung bean (Vigna radiata L.). Confocal laser scanning microscopy showed that a fluorescent trivalent cation accumulated to very high concentrations at the surface of the protoplasts when they were incubated in medium containing low concentrations of Ca or other cations, but that this accumulation could be completely reversed by suppression of membrane surface negativity by high cation concentrations. Influx of 63Ni was strongly reduced by a range of divalent cations. Increasing the Ca concentration in the medium from 25 microM to 10 mM inhibited 63Ni influx by more than 85%. 63Ni influx was also inhibited by 85% by reducing the pH from 7 to 4. Computation of the activity of Ni at the membrane surface under the various treatment conditions showed that Ni uptake was closely correlated with its activity at the membrane surface but not with its concentration in the bulk medium. It was concluded that the effects on Ni uptake of addition of monovalent, divalent and trivalent cations, and of variations in pH are all consistent with the proposition that the activity of Ni at the membrane surface is the major determinant of the rate of Ni influx into mung bean protoplasts. It is proposed that the surface charge on the plasma membrane will influence the membrane transport of most charged molecules into cells.     

Adsorption equilibria between liposome membrane formed of phosphatidylcholine and aqueous sodium chloride solution as a function of pH

The effect has been studied of the adsorption of ions (H(+), Na(+), OH(-), Cl(-)) which are present in solution upon the electric charge of the liposome membrane formed of phosphatidylcholine (PC). The surface charge density of the membrane was determined as a function of pH and electrolyte concentration from electrophoretic mobility measurements. The measurements were carried out by the laser-Doppler microelectrophoresis method. A four-equilibria model has been proposed to describe the phenomena occurring on the membrane surface. The equilibria in which the adsorption of other ions on the liposome membrane surface was involved were assumed to exist beside the equilibria in which the H(+) and OH(-) ions were engaged. The idea was confirmed by mathematical calculations. Association constants of the liposome membrane surface with ions of solution (K(AH), K(ANa), K(BOH), K(BCl)) were determined. The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the lecithin membrane with the experimental data.     

Energization-induced redistribution of charge carriers near membranes

The electric field arising from proton pumping across a topologically closed biological membrane causes accumulation close to the membrane of ionic charges equivalent to the charge of the pumped protons, positive on the side towards which protons are pumped, negative on the other side. We shall call this the 'active surface charge'. We here use the Poisson-Boltzmann equation to evaluate the effects of zwitterionic buffer molecules and uncharged proteins in the aqueous phase bordering the membrane on the magnitude and ionic composition of the active surface charge. For the positive side of the membrane, the main results are: (1) If the membrane is freely accessible to bulk phase ions, pumped protons exchange with these ions, such that the active surface charge consists of salt cations. (2) If a significant fraction of the ions in bulk solution consists of buffer molecules, then some of the pumped protons will remain close to the membrane and constitute a major fraction of the active surface charge. (3) If a protein layer borders the membrane, a significant part of the transmembrane electric potential difference exists within that protein layer and protons inside this layer dominate the active surface charge. (4) On the negative side of the membrane the corresponding phenomena would occur. (5) All these effects are strictly dependent on the transmembrane electric potential difference arising from proton pumping and would come in addition to the well known effects of buffers and electrically charged proteins on the retention of scalar protons. (6) No additional proton diffusion barrier may be required to account for a deficit in number of protons observed in the aqueous bulk phase upon aeration-induced proton pumping.     

Protective effect of green tea on erythrocyte membrane of different age rats intoxicated with ethanol

It is known that aging is characterized by changes in cell metabolism resulting in modification of the structure and function of cell membrane components which is mainly the consequence of reactive oxygen species action. These disturbances are also enhanced by different xenobiotics, e.g. ethanol. Therefore, the aim of this paper is to examine green tea influence on total antioxidant status (TAS) and on composition and electric charge of erythrocyte membrane phospholipids in ethanol intoxicated rats of various ages. Antioxidant abilities of erythrocytes were estimated by measuring TAS. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC, while the extent of erythrocytes lipid peroxidation was estimated by HPLC measurement of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels. Electrophoresis was used to determine the surface charge density of the rat erythrocyte membrane. It was shown that the process of aging was accompanied by a decrease in TAS and in the total amount of phospholipids as well as by enhancement of lipid peroxidation and increase in surface charge density of erythrocyte membrane. Ethanol administration caused, in term, decrease in TAS and increase in the level of all phospholipids and lipid peroxidation products. Ethanol as well significantly enhanced changes in surface charge density of erythrocyte membrane. The ingestion of green tea partially prevented decrease in erythrocyte antioxidant abilities observed during aging and ethanol intoxication. Moreover, long-term drinking of green tea protects the structure of the erythrocytes membrane disturbed during aging process and/or chronic ethanol intoxication.     

Purification and characterization of mitochondrial thioesterase from rabbit myocardium and its inhibition by palmitoyl carnitine

The inhibitory effects of saturated fatty acids with 4 to 18 carbon atoms on ADP-induced aggregation of bovine platelets were investigated. The inhibitory effects of the acids increased with increase of their alkyl chain length up to C14. On the other hand, from C16 the inhibitory effects tended to decrease with increase of chain length, and stearic acid (C18) was not inhibitory. There was a linear relationship between the inhibitory effects and alkyl chain lengths up to C12. This linear relation and the slope of the linear regression line suggested that the inhibitory effects of the acids depended on their partition into the membrane. The fatty acids decreased the fluorescence of the surface charge probe 2-p-toluidinylnaphthalene-6-sulfonate, indicating that they increased the negative charge on the membrane surface. The relative effects of the acids on the fluorescence were consistent with their relative inhibitory effects on aggregation. These results suggest that the inhibition of platelet aggregation by saturated fatty acids is due to a change in the membrane surface charge of the platelet plasma membrane.     

Interaction of enkephalin peptides with anionic model membranes

According to the model for passive transport across the membranes, the total flow of permeant molecules is related to the product of the water-membrane partition coefficient and the diffusion coefficient, and to the water-membrane interfacial barrier. The effect of membrane surface charge on the permeability and interaction of analgesic peptide ligands with model membranes was investigated. A mixture of zwitterionic phospholipids with cholesterol was used as a model membrane. The lipid membrane charge density was controlled by the addition of anionic 1-palmitoyl-2-oleoylphosphatidylserine. Two classes of highly potent analgesic peptides were studied, c[D-Pen²,D-Pen⁵]enkephalin (DPDPE) and biphalin, a dimeric analog of enkephalin. The effect of increased surface charge on the permeability of the zwitterionic DPDPE is a relatively modest decrease, that appears to be due to a diminished partition coefficient. On the other hand the binding of the dicationic biphalin ligands to membranes increases proportionally with increased negative surface charge. This effect translates into a significant reduction of biphalin permeability by reducing the diffusion of the peptide across the bilayer. These experiments show the importance of electrostatic effects on the peptide-membrane interactions and suggest that the negative charge naturally present in cell membranes may hamper the membrane transport of some peptide drugs, especially cationic ones, unless there are cationic transporters present.     

A test of discreteness-of-charge effects in phospholipid vesicles: measurements using paramagnetic amphiphiles

A new series of negatively charged, paramagnetic alkylsulfonate probes was synthesized and can be used to measure both the internal and the external surface potentials of model membrane systems. We tested for discreteness-of-charge effects in lipid membranes by comparing the surface potentials, estimated by use of these negatively charged amphiphiles, with that of a series of positively charged alkylammonium nitroxides in charged membranes. From the partitioning of these probes, the membrane surface potential was estimated in phosphatidylcholine membranes containing either phosphatidylserine or didodecyldimethylammonium bromide. The surface potentials, estimated with either positive or negative probes, were identical, within experimental error, in either positive or negative membranes, and they were well accounted for by a simple Gouy-Chapman-Stern theory. This symmetry, with respect to the sign of the charge, indicates that discreteness-of-charge effects are not significant in determining the potential-sensitive phase partitioning of these probes in model membranes. Thus, despite the fact that charge on membranes is discrete, models that assume a uniform density of charge in the plane of the membrane adequately account for the potentials measured by these amphiphilic probes.     

Lipid and protein contributions to the membrane surface potential of vesicular stomatitis virus probed by a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin

The surface potential of membranes of vesicular stomatitis virus and liposomes was determined by shift of ionization over a wide pH range of the membrane-inserted fluorophore, 4-heptadecyl-7-hydroxycoumarin. Incorporation into sonicated vesicles of negatively charged phosphatidylserine markedly increased the surface potential of uncharged phosphatidylcholine, but no significant effect on surface potential was produced by polar but uncharged glucocerebroside incorporated in phosphatidylcholine vesicles. The membrane of vesicular stomatitis virus was found to have a moderately high surface potential. Contributing to this viral membrane surface potential were glycoprotein spikes and phospholipid headgroups as determined by lowered charge after treatment of intact virions with thermolysin to remove glycoprotein or phospholipase C to remove phospholipid headgroups. The role of viral glycoprotein was confirmed by demonstrating increased surface charge of vesicles reconstituted with both viral glycoprotein and lipids compared with vesicles reconstituted with viral lipids alone. An unexpected finding was the large contribution to surface potential of cholesterol present in viral membrane. Increasing cholesterol concentration in virions by interaction with cholesterol-complexed serum lipoproteins resulted in a marked decrease in surface potential, whereas 75% depletion of virion cholesterol by interaction with sphingomyelin-complexed serum lipoproteins resulted in a significant increase in virion membrane surface potential. Although removal of glycoprotein spikes or depletion of cholesterol causes reduction in infectivity of vesicular stomatitis virus, no direct correlation could be found between alteration in surface charge and infectivity.     

Do plasma proteins distinguish between liposomes of varying charge density?

Cationic liposomes (CLs) are one of the most employed nonviral nanovector systems in gene therapy. However, their transfection efficiency is strongly affected by interactions with plasma components, that lead to the formation of a “protein corona” onto CL surface. The interactions between nanoparticles entering the body and biomolecules have an essential role for their biodistribution. Because the knowledge of proteins adsorbed onto vector surface could be useful in the screening of new, more efficient and more biocompatible liposomal formulations, the behavior of three CLs with different membrane charge densities was investigated. The proteins of the three coronas were identified by nano-liquid chromatography-tandem mass spectrometry, and quantified with label-free spectral counting strategy. Fibrinogen displayed higher association with CLs with high membrane charge density, while apolipoproteins and C4b-binding protein with CLs with low membrane charge density. These results are discussed in terms of the different lipid compositions of CLs and may have a deep biological impact for in vivo applications. Surface charge of nanoparticles is emerging as a relevant factor determining the corona composition after interaction with plasma proteins. Remarkably, it is also shown that the charge of the protein corona formed around CLs is strongly related to their membrane charge density.     

Conceptualisation of articular cartilage as a giant reverse micelle: a hypothetical mechanism for joint biocushioning and lubrication

Phospholipid (PL) molecules form the main structure of the membrane that prevents the direct contact of opposing articular cartilage layers. In this paper we conceptualise articular cartilage as a giant reverse micelle (GRM) in which the highly hydrated three-dimensional network of phospholipids is electrically charged and able to resist compressive forces during joint movement, and hence loading. Using this hypothetical base, we describe a hydrophilic-hydrophilic (HL-HL) biopair model of joint lubrication by contacting cartilages, whose mechanism is reliant on lamellar cushioning. To demonstrate the viability of our concept, the electrokinetic properties of the membranous layer on the articular surface were determined by measuring via microelectrophoresis, the adsorption of ions H, OH, Na and Cl on phospholipid membrane of liposomes, leading to the calculation of the effective surface charge density. The surface charge density was found to be -0.08+/-0.002cm(-2) (mean+/-S.D.) for phospholipid membranes, in 0.155M NaCl solution and physiological pH. This value was approximately five times less than that measured in 0.01M NaCl. The addition of synovial fluid (SF) to the 0.155M NaCl solution reduced the surface charge density by 30% which was attributed to the binding of synovial fluid macromolecules to the phospholipid membrane. Our experiments show that particles charge and interact strongly with the polar core of RM. We demonstrate that particles can have strong electrostatic interactions when ions and macromolecules are solubilized by reverse micelle (RM). Since ions are solubilized by reverse micelle, the surface entropy influences the change in the charge density of the phospholipid membrane on cartilage surfaces. Reverse micelles stabilize ions maintaining equilibrium, their surface charges contribute to the stability of particles, while providing additional screening for electrostatic processes.     

The antibacterial properties of secreted phospholipases A2: a major physiological role for the group IIA enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall.

The antibacterial properties of human group IIA secreted phospholipase A(2) against Gram-positive bacteria as a result of membrane hydrolysis have been reported. Using Micrococcus luteus as a model system, we demonstrate the very high specificity of this human enzyme for such hydrolysis compared with the group IB, IIE, IIF, V, and X human secreted phospholipase A(2)s. A unique feature of the group IIA enzyme is its very high pI due to a large excess of cationic residues on the enzyme surface. The importance of this global positive charge in bacterial cell membrane hydrolysis and bacterial killing has been examined using charge reversal mutagenesis. The global positive charge on the enzyme surface allows penetration through the bacterial cell wall, thus allowing access of this enzyme to the cell membrane. Reduced bacterial killing was associated with the loss of positive charge and reduced cell membrane hydrolysis. All mutants were highly effective in hydrolyzing the bacterial membrane of cells in which the cell wall was permeabilized with lysozyme. These same overall characteristics were also seen with suspensions of Staphylococcus aureus and Listeria innocua, where cell membrane hydrolysis and antibacterial activity of human group IIA enzyme was also lost as a result of charge reversal mutagenesis.     

Anatomical distribution of voltage-dependent membrane capacitance in frog skeletal muscle fibers

Components of nonlinear capacitance, or charge movement, were localized in the membranes of frog skeletal muscle fibers by studying the effect of 'detubulation' resulting from sudden withdrawal of glycerol from a glycerol-hypertonic solution in which the muscles had been immersed. Linear capacitance was evaluated from the integral of the transient current elicited by imposed voltage clamp steps near the holding potential using bathing solutions that minimized tubular voltage attenuation. The dependence of linear membrane capacitance on fiber diameter in intact fibers was consistent with surface and tubular capacitances and a term attributable to the capacitance of the fiber end. A reduction in this dependence in detubulated fibers suggested that sudden glycerol withdrawal isolated between 75 and 100% of the transverse tubules from the fiber surface. Glycerol withdrawal in two stages did not cause appreciable detubulation. Such glycerol-treated but not detubulated fibers were used as controls. Detubulation reduced delayed (q gamma) charging currents to an extent not explicable simply in terms of tubular conduction delays. Nonlinear membrane capacitance measured at different voltages was expressed normalized to accessible linear fiber membrane capacitance. In control fibers it was strongly voltage dependent. Both the magnitude and steepness of the function were markedly reduced by adding tetracaine, which removed a component in agreement with earlier reports for q gamma charge. In contrast, detubulated fibers had nonlinear capacitances resembling those of q beta charge, and were not affected by adding tetracaine. These findings are discussed in terms of a preferential localization of tetracaine-sensitive (q gamma) charge in transverse tubule membrane, in contrast to a more even distribution of the tetracaine-resistant (q beta) charge in both transverse tubule and surface membranes. These results suggest that q beta and q gamma are due to different molecules and that the movement of q gamma in the transverse tubule membrane is the voltage-sensing step in excitation-contraction coupling.     

Slow Sedimentation and Deformability of Charged Lipid Vesicles

The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity.