The influence of charge on phosphatidic acid bilayer membranes

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, $\text{1,2-}\text{dihexadecyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid, 1,3-dimyristoylglycerol-2-phosphoric acid and $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycerol-3-phosphoric}$ acid.The phase transition temperatures ($\text{T}{}_{\mathrm{<mtext>t</mtext>}}$) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 ($\text{p}\text{K}{}_1$). The regions between the first and the second $\text{p}\text{K}$ of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H⁺ concentration results in an abrupt change of the transition temperature. The slope of the $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$ vs. pH diagram beyond $\text{p}\text{K}{}_2$ becomes very steep. This is the     

The influence of charge on bilayer membranes. Calorimetric investigations of phosphatidic acid bilayers.

The pH-dependence of the phase transition of dimyristoyl phosphatidic acid and dihexadecyl phosphatidic acid has been investigated using differential scanning calorimetry. Varying the pH induces different degrees of ionization of the polar head group. The changes in transition temperature with pH as observed by calorimetry are in good agreement with those obtained by measuring the changes in light scattering, whereas the transition temperatures reported by the fluorescent probe N-phenylnaphthylamine do not always coincide with those determined from calorimetry [1]. The observed maximum of the transition temperature at pH 3.5 corresponds to a minimum in the transition enthalpy vs. pH diagram. At this pH a particular stable bilayer phase is formed. Full protonation of phosphatidic acids leads to suspensions of mycrocrystals. The transition enthalpy approaches the value of the melting enthalpy of crystalline anhydrous phosphatidic acid. The decrease in the transition enthalpy at high pH values is due to a change in the hydrocarbon chain interactions induced by the doubly charged head groups. The cooperativity of the transition varies with the degree of ionization of the head group, being lower for doubly charged phosphatidic acids.     

Phospholipase C-delta3 binds with high specificity to phosphatidylinositol 4,5-bisphosphate and phosphatidic acid in bilayer membranes.

In order to acquire an understanding of phospholipase C-delta3 (PLC-delta3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC-delta3 to large, unilamellar phospholipid vesicles (LUVs). PLC-delta3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC-delta3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 x 105 m-1. High binding of PLC-delta3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC-delta3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC-delta3 to PA and PtdSer vesicles was 1.6 x 104 m-1 and 9.4 x 102 m-1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC-delta3 to the phospholipid membrane. Binding of PLC-delta3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration-dependent increase in activity of PLC-delta3. The stimulatory effect of PA on PLC-delta3 was calcium-dependent. At Ca2+ concentrations lower than 1 microm, no effect of PA on the activity of PLC-delta3 was observed. PA enhanced PLC-delta3 activity by increasing the Vmax and lowering Km for PtdInsP2. As the mol fraction of PA increased from 0-40 mol% the enzyme Vmax increased 2.3-fold and Km decreased threefold. Based on the results presented, we assume that PA supports binding of PLC-delta3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium-dependent interaction with the C2 domain of PLC-delta3. We propose that binding of PLC-delta3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC-delta3 activity.     

Pressure effects on mechanisms of charge transport across bilayer membranes

Ion transport across diphytanoylphosphatidylcholine/decane bilayer membranes was measured as a function of hydrostatic pressure over the range 0.1-100 MPa (1-1000 atm). Carrier-mediated K+ conductance decreased with increasing pressure, yielding positive activation volumes of 45 A3 per complex for valinomycin mediated transport, and 74 A3 per complex in the case of nonactin. Comparison with the known pressure dependence of the viscosity of bulk alkane liquids supports the view that the rate limiting step for carrier-mediated transport is the translocation of the carrier-cation complex across an essentially fluid hydrocarbon membrane core. The parameters characterizing transient conductance by the hydrophobic anions, dipicrylaminate and tetraphenylborate, by contrast, were found to be insensitive to pressure over the range available. This was also the case for the steady-state conductance observed at elevated concentrations of both tetraphenylborate and the hydrophobic cation, tetraphenylarsonium. The quasi-stationary conductance observed at elevated concentrations of dipicrylaminate did, however, decrease significantly with increasing pressure, indicating a positive activation volume of 20 A3 per ion. Alternative explanations of this more complex response of hydrophobic ions to pressure are considered. Ancillary measurements of specific membrane capacitance revealed an increase of about 10% with an increase of pressure to 100 MPa, yielding an estimated membrane compressibility on the order of 10(-9) m2 X N-1, comparable to that of bulk liquid hydrocarbons.     

Interaction between plasma lipoproteins and bilayer lipid membranes. The role of surface charge

Interactions of planar BLM with different thickness and surface charge were analysed theoretically. Drawing together of the membranes is accompanied with the appearance of intramembrane potential jumps which may cause destruction and breakdown of the membranes. The theory is extrapolated to the interaction between spherical lipoprotein particles and planar BLM. Experimentally calculated (by means of ESR) surface charges of lipoproteins of low density (LLD) (--0,3 . 10(-2) C/m2) and lipoproteins of high density (LHD) (--2 . 10(-2) C/m2) enabled calculation of the interaction energy between the particles and BLM as well as of the values of intramembrane potential jumps. The latter cause local reconstructions of the membranes in the contact region and fusion of the particles with them. The earlier obtained experimental data were proved by the finding that LHD adsorption as compared with LLD is impeded due to the existence of a high energetic barrier. These peculiarities of the particles manifested during their interactions with BLM seem to be one of the factors responsible for atherogenic function of LLD and antiatherogenic one of LHD.     

Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes.

We purified phosphatidic acid phosphatase (EC 3.1.3.4) 2300-fold from porcine thymus membranes. The enzyme was solubilized with beta-octyl glucoside and Triton X-100 and fractionated with ammonium sulfate. The purification was then achieved by chromatography in the presence of Triton X-100 with Sephacryl S-300, hydroxylapatite, heparin-Sepharose, and Affi-Gel Blue. The final enzyme preparation gave a single band of M(r) = 83,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The native enzyme, on the other hand, was eluted at M(r) = 218,000 in gel filtration chromatography with Superose 12 in the presence of Triton X-100. The enzyme was judged to be specific to phosphatidic acid, since excess amounts of dicetylphosphate or lysophosphatidic acid did not inhibit the enzyme activity. In this respect, the enzyme was inhibited by 1,2-diacylglycerol but not by 1- or 2-monoacylglycerol and triacylglycerol. The enzyme required Triton X-100 or deoxycholate for its activity. Although the enzyme appeared to be an integral membrane protein, we could not detect its phospholipid dependencies. The activity was independent of Mg2+, and other cations were strongly inhibitory. The specific enzyme activity was 15 mumol/min/mg of protein when assayed using phosphatidic acid as Triton X-100 mixed micelles. The Km for the surface concentration of phosphatidic acid was 0.30 mol%. The enzyme was inhibited by sphingosine and chloropromazine, and less potently, by propranolol and NaF. The enzyme was insensitive to thio-reactive reagents like N-ethylmaleimide.     

The lipid charge density at the bilayer surface modulates the effects of melittin on membranes

The influence of melittin on two DMPA membrane systems at pH 4.2 and 8.2 has been investigated by solid-state 31P and 2H NMR, as a function of temperature and peptide concentration. Melittin promotes greater morphological changes for both systems in the fluid phase, the effect being larger at pH 4.2. Close inspection of fatty acyl chain dynamics suggests that some parallels can be drawn between the DMPA/melittin at pH 8.2 and PC/melittin systems. In addition, at pH 8.2 a direct neutralization at the interface of one of the lipid negative charges by a positive charge of the peptide occurs, as can be monitored by 31P NMR at the molecular level. For the system at pH 4.2 and at high temperature, a lipid-to-peptide molar ratio of 30 is sufficient to transform the whole system into an isotropic phase, proposed to be inverted micelles. When the system is cooled down towards the gel phase one observes an intermediate hexagonal phase in a narrow range of temperature.     

Phospholipase A1 acting on phosphatidic acid in porcine platelet membranes

1-[14C]Palmitoyl-2-[3H]arachidonoyl-sn-glycerol 3-phosphate was hydrolyzed to form [14C]palmitic acid and 2-[3H]arachidonoyl-glycerophosphate by porcine platelet membranes. This phospholipase A1 activity was relatively specific for phosphatidic acid; the addition of several other phospholipids in equimolar amounts did not have a significant effect on the hydrolysis of radiolabeled phosphatidic acid, and the specific activity for phosphatidic acid hydrolysis was 20-fold higher than that of the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol under the conditions used. This phospholipase A1 acting on phosphatidic acid has properties different from those reported for other phospholipases and lipases present in platelets.     

Non-protein-mediated transfer of phosphatidic acid between microsomal and mitochondrial membranes

The transfer of phosphatidic acid between rat liver microsomes loaded with [32P]-phosphatidic acid and rat liver mitochondria was studied in the absence of added lipid transfer proteins. It was found that during 1 h at 37 degrees C in the medium containing 100 mM KCl, 20-30% of phosphatidic acid but only 2.5% of phosphatidylcholine were transferred. This spontaneous transfer of phosphatidic acid remained the same after pretreatment of microsomes and mitochondria with 125 mM KCl or microsomes alone with 1 mM Tris, pH 8.6, procedures reported to remove adsorbed lipid transfer proteins. This transfer was insensitive to thiol-blocking reagents. The initial rate of this non-protein-mediated transfer of phosphatidic acid was virtually independent of the concentration of the acceptor membranes (mitochondria), thus indicating that it occurs by diffusion of the phospholipid through the aqueous phase rather than by membrane collision. About 80% of phosphatidic acid synthesized in the outer mitochondrial membrane was recovered in the inner membrane after a 1-h incubation, pointing to a high rate of the intermembrane transfer of this phospholipid within intact mitochondrion.     

Monocarboxylic acid permeation through lipid bilayer membranes

Summary The membrane permeability coefficients for the homologous monocarboxylic acids, formic through hexanoic, as well as benzoic and salicylic, were determined for egg phosphatidylcholine-decane planar bilayer membranes. The permeabilities of formic, acetic and propionic acid were also determined for solvent-free phosphatidylethanolamine bilayers. Permeability coefficients were calculated from tracer fluxes measured under otherwise symmetrical conditions, and precautions were taken to ensure that the values were not underestimated due to unstirred layer effects. The relation between the nonionic (HA) permeability (P ^m ) and the hexadecane/water partition coefficient (K _p ) was: log ^m =0.90 log K_p+0.87 (correlation coefficient=0.996). Formic acid was excluded from the analysis because its permeability was sixfold higher than predicted by the other acids. The permeabilities for solvent-free membranes were similar to those for decanecontaining membranes. The exceptionally high permeability of formic acid and the high correlation of the other permeabilities to the hexadecane/water partition coefficient is a pattern that conforms with other nonelectrolyte permeabilities through bilayers. Similarly, the mean incremental free energy change per methylene group (G-CH₂-) was –764 cal mol^–1, similar to other homologous solutes in other membrane systems. However, much less negative G values (–120, to –400 cal mol^–1) were previously reported for fatty acids permeating bilayers and biological membranes. These values are due primarily to unstirred layer effects, metabolism and binding to membranes and other cell components.     

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V_th) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V_th ∼ 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV ≤ V_th ≤ 290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V_th ∼ 250 mV) followed by a noticeable destabilization (V_th ∼ 100 mV at 20%). Interestingly, the presence of a model protein, α-hemolysin, dramatically disfavours membrane poration and V_th shows a 4-fold increase (∼ 800 mV) from a protein density in the membrane of 24 × 10^− 3 proteins/μm². In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.     

Penetration of ascorbic acid into bilayer phospholipid membranes

Using the EPR method, the temperature dependencies of the rates of ascorbic acid-induced reduction of nitroxyl radicals carrying the nitroxyl fragment in different positions of the fatty acid chain [N(4-methylidene++-1-oxyl-2,2,5,5-tetramethyl-3-imidazolidine hydrazine)]myristic acid (I) and 1-oxyl-2,2-dimethyloxazolidine derivatives of 5-ketostearic (II) and 12-ketostearic (III) acids incorporated into egg phosphatidylcholine liposomal membranes were studied. The reduction rates, activation energy and shape of kinetic curves were found to be dependent on the mode of liposome preparation (ultrasonication or reverse phase evaporation), label type and chemical composition of the membrane (with regard to the presence or absence of stearic acid). The coefficients of partition and diffusion of ascorbic acid through the membrane lipid bilayer were calculated from the rates of transbilayer (flip-flop) diffusion of I and ascorbate penetration inside the liposomes containing Fremi salt nitroxyl radical. The experimental results formed the basis for a hypothesis on the dependence of the rate of membrane-embedded spin probe reduction on the ascorbate distribution pattern inside the lipid bilayer.     

Neurogranin binds to phosphatidic acid and associates to cellular membranes

Neurogranin (Ng) is a 78-amino-acid-long protein concentrated at dendritic spines of forebrain neurons that is involved in synaptic plasticity through the regulation of CaM (calmodulin)-mediated signalling. Ng features a central IQ motif that mediates binding to CaM and is phosphorylated by PKC (protein kinase C). We have analysed the subcellular distribution of Ng and found that it associates to cellular membranes in rat brain. In vitro binding assays revealed that Ng selectively binds to PA (phosphatidic acid) and that this interaction is prevented by CaM and PKC phosphorylation. Using the peptide Ng-(29-47) and a mutant with an internal deletion (Ng-IQless), we have shown that Ng binding to PA and to cellular membranes is mediated by its IQ motif. Ng expressed in NIH-3T3 cells accumulates at peripheral regions of the plasma membrane and localizes at intracellular vesicles that can be clearly visualized following saponin permeabilization. This distribution was affected by PLD (phospholipase D) and PIP5K (phosphatidylinositol 4-phosphate 5-kinase) overexpression. Based on these results, we propose that Ng binding to PA may be involved in Ng accumulation at dendritic spines and that Ng could modulate PA signalling in the postsynaptic environment.     

Electric field increases the phase transition temperature in the bilayer membrane of phosphatidic acid

The effect of the electric field on the phase transition temperature (Tc) of acidic 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) and zwitterion, i.e. 1,2-dipalmitoyl-rac-3-phosphocholine and 1,2-distearoyl-rac-glycero-3-phosphocholine (DPPC and DSPC), lipids has been investigated. The phase transition was detected using the jump-like increase effect in the conductance of the planar bilayer membrane. A voltage increase to 150 mV has been shown to increase the phase transition temperature in a bilayer lipid membrane (BLM) of phosphatidic acids (DPPA and thion-DPPA) by 8-12 degrees C while the transition temperature in the bilayer of zwitterion lipids (DPPC and DSPC) increases insignificantly. The increasing of Tt in BLM of acidic lipids is attributed to the voltage-induced changes in the molecule packing density.     

Cooperative lipid-protein interaction. Effect of pH and ionic strength on polymyxin binding to phosphatidic acid membranes.

The binding of polymyxin-B to charged dipalmitoyl phosphatidic acid membranes has been studied as function of the external pH and of the ionic strength of the buffer solution. The phase transition curves were obtained by measuring the fluorescence depolarization of diphenyl hexatriene incorporated into the membrane with temperature. The molecular process of polymyxin binding was elucidated: 1. At an ionic strength of I greater than or equal to 0.1 mol/l a three step phase transition curve is found. A high-temperature step corresponds to the non-bound lipid. A lowered phase transition concerns to protein-bound lipid domains. This again is splitted into two steps. An inner core of the domain is characterized by a lipid-protein complex which is stabilized through hydrophobic and electrostatic interactions between polymyxin and the charged lipid. This core is surrounded by an outer belt of only hydrophobically bound molecules. This part shows a lower phase transition temperature than the inner core. 2. The binding curves of polymyxin to phosphatidic acid membranes depend strongly on the ionic strength of the water phase. The cooperativity of the binding process increases with increasing ionic strength and reaches a constant value at I greater than 0.2 mol/l. The maximum fraction of bound lipid decreases with increasing ionic strength. 3. The pH of the water phase strongly influences the cooperative binding process. At pH 6 a loss of cooperativity is observed at low ionic strength. Increasing the ion concentration to I = 0.3 mol/l recuperates the cooperativity of the binding process. At pH 3.0 no cooperative binding is obtained even at high ionic strength.     

The influence of surface charge on the kinetics of ion-translocation across biological membranes

Rate equations have been developed which describe the concentration dependence for ion-translocation across charged membranes for those cases in which the translocation process can be considered to be formally equivalent with an enzymic process of a Michaelis-Menten type. We have limited ourselves to those cases in which the ion-translocational step through the membrane is electroneutral. In addition it is assumed that the sites on the membrane involved in the ion-translocation process can not move through the membrane when these sites are not occupied by ions.It is shown that in general deviations from Michaelis-Menten kinetics may be expected. In case of monovalent ion-translocation across oppositely charged membranes apparent negative homotrope cooperative effects may occur, whereas for ion-translocation across equally charged membranes apparent positive homotrope cooperative effects may be found. When the bulk aqueous phase also contains polyvalent ions both types of effects may occur both in the case of ion-translocation across oppositely charged membranes as well as with ion-translocation across a membrane of which the sign of the surface charge is the same as that of the ion translocated.Under limited conditions, also apparent single Michaelis-Menten kinetics may be observed. In these cases, however, the apparent K_m generally is no linear function of the concentration of a competing ion. It is shown that even when an ion does not bind to the translocation sites the K_m is affected by increasing concentrations of this ion, a phenomenon which is not expected when the membrane is not charged. The effects of divalent ions, added to the bulk aqueous phase as 1-1-electrolytes, upon the K_m are discussed in connection with in literature reported effects of Ca⁺⁺ upon the rate of uptake of several monovalent ions into plant cells.