Dilauroylphosphatidic Acid Research Articles

Destabilizing effect of perfluorodecanoic acid on simple membrane models.

Perfluoroalkyl acids (PFA) are amphiphilic surfactants widely used in industry with several commercial applications. An important feature of these compounds is their non-biodegradability and their tendency to bio-accumulate in the environment, which has led to these compounds being considered among the most persistent pollutants worldwide. Many studies have provided evidence of their toxic effect on humans and wildlife. For this reason, more and more efforts have been made to better understand the effect of these compounds on living organisms. The aim of the present study is to understand how the electrostatic interactions and film compactness of biological membrane models modulate their interaction with PFA, more specifically with perfluorodecanoic acid (PFD). Langmuir isotherms and Brewster angle microscopy (BAM) are used to evaluate the effect of PFD on lipid membrane models (air/water monolayers and vesicles), analyzing the behavior of PFD : lipid mixtures. The lipids used in this study are distearoyl phosphatidic acid (DSPA), dilauroyl phosphatidic acid (DLPA) and distearoyl phosphatidylethanolamine (DSPE). PFD induces an increase in the mean molecular area per lipid in monolayers, mainly at lower surface pressures. BAM images demonstrate that PFD mixes with DLPA, inducing a decrease in gray level, while it forms a non-miscible mixture with DSPA, segregating PFD domains. Insertion studies of PFD within monolayers and dynamic light scattering experiments demonstrate that PFD can penetrate into monolayers and bilayers above 30 mN m-1, which is the lateral pressure value accepted for a cellular bilayer.

Electron transfer kinetics and morphology of cytochrome c at the biomimetic phospholipid layers

Abstract Electrochemistry of cytochrome c (cyt c) at biomimetic phospholipid layers was studied in a phosphate buffer solution, which were formed with dilauroyl phosphatidic acid (DLPA, C12:0), dipalmitoyl phosphatidic acid (DPPA, C16:0), distearoyl phosphatidic acid (DSPA, C18:0), and palmitoyl–oleoyl phosphatidic acid (POPA, C16:0–18:1). The lipid-layers formed firstly at the air/water interface were immediately transferred onto the electrode surface using the Langmuir–Blodgett (LB) technique. The electrochemical properties of cyt c at the lipid covered electrodes depended on the orientation, number of layers of phospholipids, tail (or head) group down, and vice versa. Atomic force microscopy (AFM) images of cyt c adsorbed on the POPA monolayer (showing the head group diameter of POPA to be ca. 0.7 nm) formed on highly oriented pyrolytic graphite (HOPG) displayed uniform surface morphology of lipid layer and clumps of aggregated cyt c molecules with a minimum size corresponding to four cyt c molecules. The heterogeneous electron transfer rate constants, k0 values, of cyt c were determined to be 1.02 × 10−3, 0.98 × 10−3, and 0.67 × 10−3 cm/s for the lipid monolayer in the tail down orientation (X-type) of POPA, DLPA, and DPPA, and 0.67 × 10−3 and 0.50 × 10−3 cm/s for the head down orientation (Z-type) of POPA and DLPA monolayers, respectively.

Preparation of Bionanocapsules by the Layer-by-Layer Deposition of Polypeptides onto a Liposome

Hollow nanoparticles (bionanocapsules) were prepared using liposomes as a template for the layer-by-layer deposition of biopolymers. We carried out the alternative deposition of poly-l-lysine (PLys) and poly-l-aspartic acid (PAsp) onto a negatively charged liposome, which was prepared from dilauroyl phosphatidic acid (DLPA) and dimyristoylphosphatidylcholine (DMPC). Efficient separation of nanocapsules from unbound polymers was achieved by ultrafiltration. ζ-Potentials of nanocapsules changed between positive and negative charges at each deposition. FE-TEM revealed that the liposome was covered with a thin polymeric layer. A fluorescent probe, 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), was encapsulated into nanocapsules. The release of HPTS was suppressed by the polymer deposition, and the release rate was tunable by coverage of the first PLys layer and the ratio of DLPA.

An X-Ray Absorption Spectroscopy Study of the Zinc Environment in Langmuir-Blodgett Phospholipid Multilayers

For the first time x-ray absorption spectroscopy was used to investigate the Zn environment in Langmuir-Blodgett multilayers. The multilayers were taken as a model of the multilamellar structure of the myelin sheath, the membrane surrounding the nerve axon, which plays a crucial role for signal transduction along the axon. The layers were assembled from the phospholipid dilauroylphosphatidic acid, both in the presence and in the absence of myelin basic protein. The analysis of the extended x-ray absorption fine structure and of the near edge regions of the x-ray absorption spectra at the Zn K-edge provided an accurate description of the local structure showing that the Zn ions are bound to the heads of the phospholipid molecules. The myelin basic protein induces a distortion on the Zn local environment due to a steric constraint but does not substitute the phosphate headgroups. These findings represent an important step in understanding the interplay among myelin basic protein, Zn, and the lipids of the myelin sheath.

Synthesis and characterization of a dianionic carbohydrate-based phospholipid

A novel carbohydrate-based phospholipid containing a phosphatidic acid head group, bis-(2,3-lauroyl)-1-methoxy-5-ribo-phosphatidic acid (DLRPA), was synthesized and characterized by 1H NMR, 13C NMR, and 31P NMR. This molecule is an analog of dilauroyl phosphatidic acid (DLPA). The T m of DLRPA decreases with increasing pH in a similar pattern to DLPA, as determined by MDSC. From this thermotropic behavior, the apparent p K as of DLRPA are estimated to be 4 and 8.

Total External Reflection X-ray Absorption Spectroscopy Reveals a Zinc Coordination Shell in Phospholipid Langmuir−Blodgett Films

We have studied the local environment of zinc in phospholipid Langmuir-Blodgett films with extended X-ray absorption fine structure spectroscopy in total reflection conditions (reflEXAFS). Mono- and multilayer films made of dilauroyl-phosphatidic acid (DLPA) and monolayer films made of dimyristoyl-phosphatidylcholine (DMPC) were deposited on quartz slides from ZnCl2 solutions, and their molecular order was checked by means of X-ray reflectivity measurements. A very good signal-to-noise ratio in the reflEXAFS spectra of both multi- and monomolecular films was obtained. The theoretical fits to the experimental data show that zinc coordinates four atoms about 0.20 nm distant in both acidic DLPA and neutral DMPC films, while in water solutions, zinc coordinates six atoms about 0.21 nm distant.

Synthesis and biological evaluation of a PtdIns(4,5)P2 and a phosphatidic acid affinity matrixDedicated to the memory of the late Professor Roy Gigg.

Analogues of dipalmitoyl phosphatidic acid (PA), dilauroyl PA and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] were synthesised and immobilised onto a solid support, Affi-Gel 10. Using them as affinity matrices, a number of known proteins as well as a set of novel proteins were found to bind specifically to PA.

Modulation of phospholipase A2 by electrostatic fields and dipole potential of glycosphingolipids in monolayers

Phospholipase A2 activity against mixed monolayers of dilauroylphosphatidic acid or dilauroylphosphatidylcholine with glycosphingolipids can be reversibly modulated by external constant electrostatic fields. The changes of enzymatic activity are correlated to the depolarization or hyperpolarization of the film caused by specific dipolar properties of glycosphingolipids. Hyperpolarizing fields enhance the enzymatic activity against pure dilauroylphosphatidic acid while depolarizing fields induce a decrease of activity. Compared to the pure substrate, the interface of mixed films containing neutral glycosphingolipids or gangliosides is already partially depolarized and the magnitude of activation induced by an external hyperpolarizing field is decreased; conversely, depolarizing fields cause an increased inhibition of activity. Differing from gangliosides, sulfatides bring about a hyperpolarization of the mixed lipid monolayer and external hyperpolarizing or depolarizing fields cause enhanced activation and reduced inhibition, respectively. The effects of glycosphingolipids depend on their relative proportion in the monolayer. Results were similar with dilauroylphosphosphatidylcholine but the field effects were less than half of those found with dilauroylphosphatidic acid. Our work shows that the activity of phospholipase A2 in addition to responding reversibly to external electrostatic fields, is directly modulated by the polarity and magnitude of the lipid polar head group dipole moments.—Maggio, B. Modulation of phospholipase A2 by electrostatic fields and dipole potential of glycosphingolipids in monolayers. J. Lipid Res. 1999. 40: 930–939.

Binding of Local Anesthetic Tetracaine to Phosphatidylinositol/Dipalmitoylphosphatidylcholine Mixed Vesicles

Abstract We measured the binding of local anesthetic tetracaine to phosphatidylinositol (PI), dipalmitoylphoshpatidylcholine (DPPC) and PI/DPPC mixed vesicles by an equilibrium dialysis method at different pH (5.5, 7.0, 8.0) at 35 °C. The results obtained for the pure phospholipid vesicles show that tetracaine binds to PI with the polyhydroxy headgroup, rather than to twitter ionic DPPC. For mixed vesicles, the higher is the ratio of the PI in mixed vesicles, the higher is the binding number. The change in pH slightly influences the binding to PI, while it strongly influences the binding to DPPC. We calculated the binding parameters which characterize the binding isotherms based on Langmuir-type and Hill equations. The binding parameters for pure PI and DPPC vesicles well reproduce the binding isotherms for PI/DPPC mixed vesicles with different mixing ratios. The results for pure PI were compared with those previously obtained for dilauroylphosphatidic acid (DLPA) in order to examine the influence of inositol in the head group of PI on the binding of tetracaine to phospholipid vesicles.

Poly(ethylene glycol)-conjugated surfactants promote or inhibit aggregation of phospholipids

The calcium-induced aggregation of dilauroyl phosphatidic acid (DLPA) suspensions, with or without added poly(ethylene oxide) (PEO)-conjugated surfactants containing 4 to 30 ethylene oxide subunits, were monitored by turbidity measurement and quasi-elastic light scattering (QLS). The aggregation was inhibited (protected) by the incorporated PEO surfactant for most samples, while a window for promotive effect was found for samples with low surface coverage by the PEO moiety of the incorporated surfactant. Promotion occurs only when the aggregation is slow and at a low level. The promotion is explained by the synergistic effect of PEO and divalent calcium cations when the steric repulsion is weak. The promotion/protection crossover is a display between the PEO/calcium synergistic effect and the steric repulsion.

Kinetic and Pharmacological Characterization of Phospholipases A2 from Bothrops neuwiedii Venom

Two phospholipases A2 (PLA2) (EC 3.1.1.4) were purified from Bothrops neuwiedii venom (isoenzymes P-1 and P-2). The molecular weights of P-1 and P-2 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 15,000 and 16,200 and the isoelectric points were 4.8 and 4.6, respectively. The N-terminal 14-amino-acid sequences determined were Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly and Ser-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly for P-l and P-2, respectively. Since both show sequence almost identical to that of a PLA2 from Crotalus atrox it was tentatively classified as being of group II. The enzymatic activity of P-l and P-2 toward lipid monolayers was studied. The hydrolysis of dilauroylphosphatidylcholine (dlPC) shows a broad optimum between 7 and 18 mN m−1 and a cut-off pressure of 22 mN m−1. The activity toward dlPC displays a maximum at pH 8 and is dependent on the presence of Ca2+ with an apparent Kd of 0.1 mM, for both enzymes. P-1 and P-2 are heat-stable enzymes, unable to hydrolyze dilauroylphosphatidic acid monolayers. The enzymes are not lethal to mice at doses up to 5 μg/g body weight by intraperitoneal injection and they do not show myotoxic (up to 40 μg) or hemolytic activity (up to 8.5 μg/ml). Both lack anti-coagulant activity, determined by absence of changes in the recalcification time of platelet poor plasma (up to 100 μg/ml), and are not able to induce platelet aggregation (up to 50 μg/ml). However, both isoenzymes exhibit an important edema-inducing activity that is not altered at short times by the irreversible chemical inactivation of the hydrolytic activity with phenacyl bromide. P-l and P-2 are able to release arachidonic acid from membrane phospholipids of neutrophils, property that is lost by the inactivation of the enzyme. This suggests that the edema-inducing activity of the active but not the inactive forms may be partly due to arachidonic acid-derived mediators. The edema-inducing activity of the active or inactive forms of the enzymes is inhibited by antagonists of histamine, suggesting that histamine plays an important role in both the active and the inactive B. neuwiedii PLA2s-induced edema. The results suggest that the inflammatory and the catalytic activity of these enzymes constitute separate properties.

Binding of Local Anesthetic Tetracaine to Phospholipid Mixed Vesicles

Abstract We measured the binding isotherms of a local anesthetic, tetracaine, to dilauroylphosphatidic acid (DLPA) and dilauroylphosphatidylcholine (DLPC) mixed vesicles along with their pure vesicles. The binding isotherms were constructed by the equilibrium dialysis method at different pH (5.5, 7.0, 8.0) at 35 °C. The binding isotherms were analyzed based on the equations combined with Langmuir and Hill-type isotherms, and the parameters characterizing the binding isotherms were evaluated. The thus-obtained binding parameters for pure vesicles well reproduce the binding isotherms for the mixed vesicles by taking account of the mixing ratios. This indicates that DLPA and DLPC also provide independent binding sites for tetracaine, as for previously reported dibucaine. The estimated values of the binding parameters for these two anesthetics, i.e., tetracaine and dibucaine, were compared in terms of the strength of the anesthetic action as well as the molecular structure.

The transition of α-helix to β-structure of poly( d-lysine) induced by phosphatidic acid vesicles and its kinetics at alkaline pH

Static and dynamic circular dichroism (CD) measurements were carried out for poly( l-lysine) in suspensions of dilauroylphosphatidic acid (DLPA) vesicles at alkaline pH (8–11.5). The static experiments demonstrated that the α-helix of poly( l-lysine) induced by deprotonation in alkaline solutions is transformed to β-structure by the addition of DLPA vesicles. Stopped-flow CD measurements for such order-to-order transition revealed that the rate determining step is the unfolding process of α-helix to random coil. Previously, we have reported the conformational change of poly( l-lysine) induced by DLPA vesicles at neutral pH, where the β-structure transition from random coil was observed. Thus two types of transition of poly( l-lysine) are observed depending on bulk pH, i.e., from random coil to β-structure and from α-helix to β-structure. So far the phospholipid-induced conformations of poly( l-lysine) were interpreted in terms of counterbalance between the positively charged terminals of the lysyl chains and the negative headgroups of the phospholipid in vesicle. However, present work indicates the direct interaction other than electrostatic interaction between the lysyl chain and phosphate groups of the lipid.

Binding of Dibucaine to Phospholipid Mixed Vesicles

Abstract Binding isotherms of a local anesthetic, dibucaine, to phospholipid vesicles were measured using a dialysis method. Two types of phospholipids, dilauroylphosphatidic acid (DLPA) and dilauroylphosphatidylcholine (DLPC), were used to prepare vesicles. The binding isotherms for pure DLPA and DLPC vesicles and their mixtures were analyzed with the Hill equation. The binding parameters obtained for pure phospholipid vesicles reproduce the binding isotherms for the mixed vesicles by taking account of the mixing ratio; this indicates the presence of independent binding sites, corresponding to the DLPA and DLPC molecule, in the mixed vesicle.

Concerted modulation by myelin basic protein and sulfatide of the activity of phospholipase A2 against phospholipid monolayers.

The effect of myelin basic protein (MBP) on the activity of phospholipase A2 (PLA2, EC 3.1.1.4) against monolayers of dilauroylphosphatidylcholine (dlPC) or dilauroylphosphatidic acid (dlPA) containing different proportions of sulfatide (Sulf) and galactocerebroside (GalCer) was investigated. MBP was introduced into the interface by direct spreading as an initial constitutive component of the lipid-protein film or by adsorption and penetration from the subphase into the preformed lipid monolayers. The effect of MBP on PLA2 activity depends on the type of phospholipid and on the proportion of MBP at the interface. At a low mole fraction of MBP, homogeneously mixed lipid-protein monolayers are formed, and the PLA2 activity against dlPC is only slightly modified while the degradation of dlPA is markedly inhibited. This is probably due to favorable charge-charge interactions between dlPA and MBP that interfere with the enzyme action. The PLA2 activity against either phospholipid is increased when the mole fraction of MBP exceeds the proportion at which immiscible surface domains are formed. GalCer has little effect on the modulation by MBP of the phospholipase activity. The effect of Sulf depends on its proportions in relation to MBP. The individual effects of both components balance each other, and a finely tuned modulation is regulated by the interactions of MBP with Sulf or with the phospholipid.

Abnormal aggregation behavior of acidic phospholipid vesicles in the very low concentration range of divalent cations

Aggregation behavior of dilauroylphosphatidic acid (DLPA) vesicles was investigated in the presence of divalent cations (Mg 2+, Ca 2+, Ba 2+, and Cd 2+) at extremely low concentration range (below 1 m M). Ca 2+ induced the vesicle aggregation, which starts after an induction period of several hours. The induction period was shortened by the increase of Ca 2+ concentration to 0.15 m M, above which it disappeared. The size of aggregates formed by the vesicle aggregation showed a bell-shaped dependence on Ca 2+ concentration below 0.05 m M; it also depended on both lipid concentration and cationic species. These experimental findings suggest that the aggregation of DLPA vesicles occurring at low concentration range of divalent cations proceeds by the intervesicular salt-linkage mechanism rather than by the Derjanguin-Landau-Verwey-Overbeek mechanism.

Monolayer characterstics and thermal behaviour of phosphatidic acids

The monolayer and thermal behaviour of different phosphatidic acids are presented. At neutral pH and 22°C dilauroylphosphatidic acid and unsaturated phosphatidic acids form liquid-expanded monolayers, while dipalmitoyl- and distearoylphosphatidic acid form condensed monolayers. Dimyristoylphosphatidic acid undergoes a transition from the liquid-expanded to the condensed state. With long-chain saturated and unsaturated phosphatidic acids little change in molecular area is observed between pH 2 and 7. In contrast, the short chain saturated phosphatidic acids, dilauroyl- and dimyristoylphosphatidic acids, undergo a condensation in the pH range 2 to 7. This is so in spite of the fact that the phosphoric acid group dissociates and the phosphatidic acid molecule attains one negative charge over this pH range. This finding is interpreted to indicate that the electrostatic repulsion between negatively charged phosphatidic acid molecules is compensated for or even outweighed by other intermolecular forces. Hydrogen bonding at the lipid/water interface is supposed to play a major role. All phosphatidates studied exhibit a significant expansion in the pH range 7 to 12. The second apparent p K of the primary phosphate group of phosphatidic acids is 8.6 and the expansion observed in this pH range is therefore due to electrostatic repulsion. At neutral pH the ether analogues of saturated phosphatidic acids have monolayer properties similar to those of the ester compounds. Considering the total pH range of 2 to 12 studied the force-area curves of the ether analogues are more condensed compared to the ester compounds. Synthetic phosphatidates and their ether analogues give reversible sharp crystal(gel)-to-liquid crystal transitions while the naturally occurring egg phosphatidate gives a broad, asymmetric one. The transition temperature T m of saturated phosphatidates increases with increasing hydrocarbon chain length and at a given chain length T m decreases markedly with unsaturation. The T m values of the ether analogues are about 10°C higher and the ΔH values are 10–15% lower than those of the corresponding esters.

Effect of sulfatide and gangliosides on phospholipase C and phospholipase A 2 activity. A monolayer study

The effect of sulfatide and gangliosides G M1, G D1a and G T1b on the activity of phospholipase C from Clostridium perfringens on dilauroylphosphatidylcholine and of porcine pancreatic phospholipase A 2 on dilauroylphosphatidic acid was studied in lipid monolayers containing different proportions of glycolipids under zero-order kinetics at various constant surface pressures. The presence of sulfatide in the monolayer increases the activity of phospholipase C at high surface pressures. Gangliosides shift the cut-off pressure to lower values and inhibit the action of phospholipase C. In mixed monolayers with dilauroylphosphatidic acid, sulfatide at a molar fraction of 0.5 increases the activity of phospholipase A 2 at surface pressures below 18 mN/m and shows an inhibitory effect at higher pressures. Ganglioside G M1 at a molar fraction of 0.25 completely inhibits the enzyme above 20 mN/m and markedly reduces its activity at lower pressures. Gangliosides G D1a and G T1b abolish the enzyme activity at all pressures at molar fractions of 0.25 and 0.15, respectively. The modified velocity of the enzymatic reaction in the presence of glycosphingolipids is not due to an irreversible alteration of the catalytic activity.

Stopped-flow kinetic study on aggregation of dilauroylphosphatidic acid vesicles induced by divalent cations

Aggregation kinetics of dilauroylphosphatidic acid (DLPA) vesicles induced by divalent cations (Mg 2+, Ca 2+, Sr 2+, and Ba 2+) was studied by a stopped-flow rapid mixing technique with turbidity detection. The aggregation rate was evaluated in terms of the initial velocity of turbidity increase, V 0. With the increase of cation concentrations above a threshold, V 0 increased and reached a maximum value, V 0,max. No significant difference in the relation between V 0 and the cation concentration was observed among the divalent-cation species. The threshold concentration and the concentration at which V 0,max is attained were independent of the lipid concentration. From the temperature dependence of V 0,max; the activation energy for the aggregation was estimated to be 3.9 kcal/mole, which corresponds to the activation energy for the diffusion-controlled reaction in aqueous media. These experimental results for the divalentcation-induced aggregation of DLPA vesicles are well explained on the basis of the Derjaguin-Landau-Verwey-Overbeek theory for electrically stabilized colloids.

Interactions of neutral and anionic glycosphingolipids with dilauroylphosphatidylcholine and dilauroylphosphatidic acid in mixed monolayers

The effect of short hydrocarbon chains of phospholipids on the interactions with glycosphingolipids having different oligosaccharide chains was studied for systems containing dilauroylphosphatidylcholine or dilauroylphosphatidic acid mixed in different proportions with galactosylceramide, sulfatide, or gangliosides G M1, G D1a or G T1b. Lipid monolayers at the air-NaCl interface in the presence or absence of CaCl 2 were studied. The mean molecular area and surface potential per molecule at different surface pressures and the excess free energy of mixing were compared with the corresponding parameters obtained for mixtures of the same glycosphingolipids with dipalmitoylphosphatidylcholine and dipalmitoylphosphatidic acid. In addition to the well-known influence of the polar head group of the glycosphingolipids and phospholipids on the intermolecular interaction, the present work shows that the organization and surface potential of these interfaces are markedly influenced by the type of hydrocarbon chain of the phospholipid. The surface behavior is modified by the presence of Ca 2+ which, in addition, induces immiscibility in some of these mixtures.