Binding of spin-labeled local anesthetics to phosphatidylcholine and phosphatidylserine liposomes

A homologous series of spin-labeled local anesthetics, 2-[N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-p-alkoxybenzoates were shown to bind to phosphatidylcholine and phosphatidylserine liposomes. Under similar conditions, 70% of the ethoxy homolog (R2C) of these spin-labeled local anesthetics bound to synthetic dipalmitoyl lecithin while 98% bound to phosphatidylserine liposomes. Five percent of R2C's bound signal could be released by 4 mm calcium from phosphatidylserine liposomes, but calcium had no effect on R2C bound to synthetic lecithin. The butoxy (R4C) and hexyloxy (R6C) homologs bound to phosphatidylcholine in the order R6C > R4C. All of R6C and all of R4C were bound to phosphatidylserine liposomes, while only 90% of R6C bound to synthetic dipalmitoyl lecithin. Calcium was incapable of displacing bound R4C or R6C from either phosphatidylcholine or phosphatidylserine liposomes. The results are discussed in light of anesthetic binding by electrostatic and Van der Waal's forces to phospholipids.     

Binding of cytochrome b5 to cholesterol-containing phosphatidylcholine vesicles

Cytochrome b₅ was found to bind readily to sonicated vesicles containing as much as 0.8 mol cholesterol per mol egg phosphatidylcholine. This observation conflicts with the suggestion of Enomoto and Sato ((1977) Biochim. Biophys. Acta 466, 136–147) that cholesterol prevents binding of this protein to erythrocyte membranes.     

Calcium association with phosphatidylserine. Modification by cholesterol and phosphatidylcholine in monolayers and bilayers

We have examined the association of Ca2+ with phosphatidylserine/cholesterol and phosphatidylserine/dimyristoylphosphatidylcholine mixed monolayers using a surface radiocounting technique. No Ca2+ association with pure monolayers of the uncharged molecules was observed. The Ca2+/phosphatidylserine surface ratio was approximately 1:2 in expanded monolayers of the pure anionic lipid and in phosphatidylserine/phosphatidylcholine mixtures. An increase in surface-associated Ca2+ to a number ratio of 1:1 was observed in phosphatidylserine/cholesterol films when the mole fraction of cholesterol was raised to 0.5 and above and the phospholipid number density held constant. We interpret these findings as a prevention of intermolecular salt formation by the sterol. Further support is provided by particle electrophoresis.     

Lipid lateral heterogeneity in phosphatidylcholine/phosphatidylserine/diacylglycerol vesicles and its influence on protein kinase C activation.

To test the hypothesis that the activation of protein kinase C (PKC) is influenced by lateral heterogeneities of the components of the lipid bilayer, the thermotropic phase behavior of dimyristoylphosphatidylcholine (DMPC)/dimyristoylphosphatidylserine (DMPS)/dioleoylglycerol (DO) vesicles was compared with the activation of PKC by this system. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to monitor the main transition (i.e., the gel-to-fluid phase transition) as a function of mole fraction DO (chi(DO)) in DMPC/DO, DMPS/DO, and [DMPC/DMPS (1:1, mol/mol)]/DO multilamellar vesicles (MLVs). In each case, when chi(DO) < or approximately 0.3, DO significantly broadened the main transition and shifted it to lower temperatures; but when chi(DO) > approximately 0.3, the main transition became highly cooperative, i.e., narrow, again. The coexistence of overlapping narrow and broad transitions was clearly evident in DSC thermograms from chi(DO) approximately 0.1 to chi(DO) approximately 0.3, with the more cooperative transition growing at the expense of the broader one as chi(DO) increased. FTIR spectroscopy, using analogs of DMPC and DMPS with perdeuterated acyl chains, showed that the melting profiles of all three lipid components in [DMPC/DMPS (1:1, mol/mol)]/DO MLVs virtually overlay when chi(DO) = 0.33, suggesting that a new type of phase, with a phospholipid/DO mole ratio near 2:1, is formed in this system. Collectively, the results are consistent with the coexistence of DO-poor and DO-rich domains throughout the compositions chi(DO) approximately 0.1 to chi(DO) approximately 0.3, even at temperatures above the main transition. Comparison of the phase behavior of the binary mixtures with that of the ternary mixtures suggests that DMPS/DO interactions may be more favorable than DMPC/DO interactions in the ternary system, especially in the gel state. PKC activity was measured using [DMPC/DMPS (1:1, mol/mol)]/DO MLVs as the lipid activator. At 35 degrees C (a temperature above the main transition of the lipids), PKC activity increased gradually with increasing chi(DO) from chi(DO) approximately 0.1 to chi(DO) approximately 0.4, and activity remained high at higher DO contents. In contrast, at 2 degrees C (a temperature below the main transition), PKC activity exhibited a maximum between chi(DO) approximately 0.1 and chi(DO) approximately 0.3, and at higher DO contents activity was essentially constant at 20-25% of the activity at the maximum. We infer from these results that the formation of DO-rich domains is related to PKC activation, and when the lipid is in the gel state, the coexistence of DO-poor and DO-rich phases also contributes to PKC activation.     

Interactions of chromogranin A-derived vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine

Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.     

Mixed monolayers of straight-chain/branched-chain phospholipids. I. Mixed monolayers of distearoyl phosphatidylcholine and diisoeicosanoyl phosphatidylcholine

The temperature dependence of the force/area isotherms of monolayer of distearoyl phosphatidylcholine (DSPC), diisoeicosanoyl phosphatidylcholine (DIEPC) and a complete mixed compositional range of these two lecithins are reported. The isotherms for DSPC closely resemble those previously reported for dipalmitoyl phosphatidylcholine but are shifted to higher temperatures by 16 degrees C. The isotherms of DIEPC, an iso-branched lecithin, show differences from these obtained for similar straight-chain lecithins in that the full condensed isotherms are more expanded, the fully expanded isotherms are more condensed and therefore the liquid expanded (LE)/liquid condensed (LC) intermediate region is significantly reduced. This means that the condensed state is more disordered and the expanded state is less disordered than the corresponding states in straight-chain lecithins. Data for the mixed films are interpreted in terms of surface pressure/mole fraction phase diagrams and both energies and entropies of compression associated with the LE/LC transition. The phase diagrams at 34.1 degrees C, 35.8 degrees C and 38.5 degrees C are all of the negative azeotropic type with the surface pressure minimum point shifting with temperature. The thermodynamic analysis indicates that from 34.1 degrees C to 38.5 degrees C the driving force for mixing changes from the entropy to the energy of the transition. It would seem that at the lower temperature the packing of the distearoyl lecithin is perturbed by the diisoeicosanoyl lecithin, while at higher temperatures the very high entropy of pure or nearly pure diisoeicosanoyl lecithin results in other mixtures having less entropy than would be expected on an ideal mixing basis.     

Calcium association with phosphatidylserine Modification by cholesterol and phosphatidylcholine in monolayers and bilayers

We have examined the association of Ca²⁺ with phosphatidylserine/cholesterol and phosphatidylserine/ dimyristoylphosphatidylcholine mixed monolayers using a surface radiocounting technique. No Ca²⁺ association with pure monolayers of the uncharged molecules was observed. The Ca²⁺/phosphatidylserine surface ratio was approximately 1:2 in expanded monolayers of the pure anionic lipid and in phosphatidylserine/phosphatidylcholine mixtures. An increase in surface-associated Ca²⁺ to a number ratio of 1:1 was observed in phosphatidylserine/cholesterol films when the mole fraction of cholesterol was raised to 0.5 and above and the phospholipid number density held constant. We interpret these findings as a prevention of intermolecular salt formation by the sterol. Further support is provided by particle electrophoresis     

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.     

Detection of phase separation in fluid phosphatidylserine/phosphatidylcholine mixtures.

The nonideal mixing of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine, (16:0, 18:1)PS, and 1,2-didodecenoyl-sn-glycero-3-phosphocholine, (12:1, 12:1)PC, in fluid lamellar model membranes was studied by measuring binding of aqueous Ca2+ ions and by x-ray diffraction. A region of two-phase coexistence was found by invariance of the aqueous concentration and by the appearance of two sets of lamellar spacings. The phases were identified as fluid from the diffuse x-ray diffraction in the wide-angle region. The width of the two-phase coexistence region was greater at higher ionic strength. In 800 mM KCl, the phase boundaries were at PS mole fraction 0.5 and 0.8. In 100 mM KCl, the phase boundaries were at PS mole fraction 0.52 and 0.62. Monte Carlo simulations of the lateral distributions of these PS/PC mixtures show pronounced clustering of the lipids.     

Free energy potential for aggregation of mixed phosphatidylcholine/phosphatidylserine lipid vesicles in glucose polymer (dextran) solutions.

The energetics of lipid vesicle-vesicle aggregation in dextran (36,000 mol wt) solutions have been studied with the use of micromechanical experiments. The affinities (free energy reduction per unit area of contact) for vesicle-vesicle aggregation were determined from measurements of the tension induced in an initially flaccid vesicle membrane as it adhered to another vesicle. The experiments involved controlled aggregation of single vesicles by the following procedure: two giant (approximately 20 micron diam) vesicles were selected from a chamber on the microscope stage that contained the vesicle suspension and transferred to a second chamber that contained a dextran (36,000 mol wt) salt solution (120 mM); the vesicles were then maneuvered into position for contact. One vesicle was aspirated with sufficient suction pressure to create a rigid sphere outside the pipette; the other vesicle was allowed to spread over the rigid vesicle surface. The aggregation potential (affinity) was derived from the membrane tension vs. contact area. Vesicles were formed from mixture of egg lecithin (PC) and phosphatidylserine (PS). For vesicles with a PC/PS ratio of 10:1, the affinity showed a linear increase with concentration of dextran; the values were on the order of 10(-1) ergs/cm2 at 10% by weight in grams. Similarly, pure PC vesicle aggregation was characterized by an affinity value of 1.5 X 10(-1) ergs/cm2 in 10% dextran by weight in grams. In 10% by weight in grams solutions of dextran, the free energy potential for vesicle aggregation decreased as the surface charge (PS) was increased; the affinity extrapolated to zero at a PC/PS ratio of 2:1. When adherent vesicle pairs were transferred into a dextran-free buffer, the vesicles did not spontaneously separate. They maintained adhesive contact until forceably separated, after which they would not read here. Thus, it appears that dextran forms a "cross-bridge" between the vesicle surfaces.     

The intrinsic pKa values for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in monolayers deposited on mercury electrodes.

The intrinsic pKa values of the phosphate groups of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and of the phosphate and carboxyl groups of phosphatidylserine (PS) in self-organized monolayers deposited on a hanging mercury drop electrode were determined by a novel procedure based on measurements of the differential capacity C of this lipid-coated electrode. In view of the Gouy-Chapman theory, plots of 1/C at constant bulk pH and variable KCl concentration against the reciprocal of the calculated diffuse-layer capacity Cd,0 at zero charge exhibit slopes that decrease from an almost unit value to vanishingly low values as the absolute value of the charge density on the lipid increases from zero to approximately 2 microC cm-2. The intrinsic pKa values so determined are 0.5 for PE and 0.8 for PC. The plots of 1/C against 1/Cd,0 for pure PS exhibit slopes that pass from zero to a maximum value and then back to zero as pH is varied from 7.5 to 3, indicating that the charge density of the lipid film passes from slight negative to slight positive values over this pH range. An explanation for this anomalous behavior, which is ascribed to the phosphate group of PS, is provided. Interdispersion of PS and PC molecules in the film decreases the "formal" pKa value of the latter group by about three orders of magnitude.     

Interaction of gentamicin with phosphatidylserine/phosphatidylcholine mixtures in adsorption monolayers and thin liquid films: morphology and thermodynamic properties

Gentamicin possesses strong adverse actions like oto and nephrotoxicity. The latter is a result of strong gentamicin–acid phospholipid interactions, resulting in cell fusion, fission, etc., ions as calcium interact with gentamicin and effectively deter its toxicity. In this work, the interactions of gentamicin and Ca²⁺ with phosphatidylserine/phosphatidylcholine (PS/PC) mixtures of different ratio are experimentally characterized. Special attention is paid to bridge thermodynamic and morphological properties of adsorption monolayers and thin liquid films (TLFs) composed of these lipid mixtures. Our results show that gentamicin decreases the stability of common black TLFs formed of pure PS coupled with suppression of lipid surface adsorption to the monolayers at the air–water interface; also, gentamicin reveals effects of lowering of lipid spreading on the interface and significant loss of material during monolayer cycling, increase of condensed phase, and organization of dense net-like domain monolayer texture. Gentamicin addition results in opposite effects for films formed of DPPC/PS (95:5) mixture. It increases the stability of Newton black TLFs formed by DPPC/PS correlated with faster and stronger surface adsorption and better surface spreading; also, gentamicin lowers the amount of condensed phase and organization of domains of smaller size. We also showed that Ca²⁺ itself decreases the stability of common black TLFs formed of PS accompanied with weaker surface adsorption, formation of higher amounts of condensed phase and organization of domains. In our experiments, Ca²⁺ softens, even deters, the effects of gentamicin on both PS and DPPC/PS films.     

Nanometer scale organization of mixed surfactin/phosphatidylcholine monolayers.

Mixed monolayers of the surface-active lipopeptide surfactin-C(15) and of dipalmitoyl phosphatidylcholine (DPPC) were deposited on mica and their nanometer scale organization was investigated using atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS). AFM topographic images revealed phase separation for mixed monolayers prepared at 0.1, 0.25, and 0.5 surfactin molar ratios. This was in agreement with the monolayer properties at the air-water interface indicating a tendency of the two compounds to form bidimensional domains in the mixed systems. The step height measured between the surfactin and the DPPC domains was 1.2 +/- 0.1 nm, pointing to a difference in molecular orientation: while DPPC had a vertical orientation, the large peptide ring of surfactin was lying on the mica surface. The N/C atom concentration ratios obtained by XPS for pure monolayers were compatible with two distinct geometric models: a random layer for surfactin and for DPPC, a layer of vertically-oriented molecules in which the polar headgroups are in contact with mica. XPS data for mixed systems were accounted for by a combination of the two pure monolayers, considering respective surface coverages that were in excellent agreement with those measured by AFM. These results illustrate the complementarity of AFM and XPS to directly probe the molecular organization of multicomponent monolayers.     

Free energy potential for aggregation of erythrocytes and phosphatidylcholine/phosphatidylserine vesicles in Dextran (36,500 MW) solutions and in plasma.

The free energy potential (affinity) for aggregation of human red blood cells and lipid vesicles in Dextran solutions and blood plasma has been quantitated by measuring to what extent a vesicle is encapsulated by the red cell surface. The free energy reduction per unit area of contact formation (affinity) was computed from the observation of the fractional extent of encapsulation at equilibrium with the use of a relation based on the elastic compliance of the red cell membrane as it is deformed to adhere to the vesicle surface. Micromanipulation methods were used to select and transfer single lipid vesicles (2-3 X 10(-4) cm diameter) from a chamber that contained the vesicle suspension to a separate chamber on the microscope stage that contained red cells in an EDTA buffer with Dextran or whole plasma. The vesicle and a red cell were maneuvered into close proximity and contact allowed to take place without forcing the cells together. To evaluate the effects of surface charge density and steric interactions on aggregation, vesicles were made from mixtures of egg phosphatidylcholine (PC) and bovine phosphatidylserine (PS) over a range of mole ratios (PC/PS)from (1:0) to (1:1); the vesicles were formed by rehydration in buffer. The Dextran solutions were made with a sharp-cut fraction of 36,500 MW in a concentration range of 0-10% by weight in grams (wt/wt).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of membrane phase behavior as a tool to detect extrinsic protein-induced domain formation: binding of prothrombin to phosphatidylserine/phosphatidylcholine vesicles

The temperature-composition phase diagram of mixed dimyristoylphosphatidylserine (DMPS) and dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles was determined in the presence and absence of bound bovine prothrombin by monitoring the phospholipid order-disorder phase separation using diphenylhexatriene (DPH) fluorescence anisotropy. The shape of the membrane temperature-composition diagram was essentially unaltered by the binding of prothrombin in the presence of Ca2+ although the two-phase (gel/fluid) region was slightly narrowed and shifted by 1-10 degrees C to higher temperatures. This result does not support the popular idea that extensive domains rich in negatively charged phospholipid are induced in response to prothrombin binding. Instead of implying domain formation, our results demonstrate that the observed increase in melting temperature associated with binding of prothrombin to acidic phospholipid membranes can be accounted for by the observed altered membrane order both in the fluid and in the solid lamellar phases. The membrane order in the liquid-crystalline phase increased with increased acidic lipid content, and much more so for DMPS than for dipentadecanoylphosphatidylglycerol (DC15PG). These results demonstrate that simple shifts in membrane phase behavior cannot be properly interpreted to prove the existence of charged lipid domains. In addition, we report the unexpected observation that prothrombin increased the anisotropy of DPH in DMPS/DMPC vesicles in the liquid-crystalline phase in the absence of Ca2+ as well as in its presence. This effect was seen to a lesser extent and only at a much higher charged-lipid content for DC15PG/DMPC vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.     

Bioconversion of phosphatidylcholine to phosphatidylserine using immobilized enzyme mini-columns

Phospholipase-d, immobilized on controlled porosity glass beads, was used for the preparation of phosphatidylserine. The synthesis of phosphatidylserine was monitored using immobilized mini-reactors of choline oxidase and serine dehydratase isolated from rat liver. The immobilized enzymes showed good stability, and no deterioration in enzyme activity was recorded after use for 4 months.