Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Effects of phospholipid composition on the metabolism of triacylglycerol, cholesteryl ester and phosphatidylcholine from lipid emulsions injected intravenously in rats

Lipid emulsions were prepared with a similar size and lipid composition to natural lymph chylomicrons, but in which the surface phospholipid was either egg phosphatidylcholine, dioleoyl-, dimyristoyl-, dipalmitoyl- or 1-palmitoyl-2-oleoylphosphatidylcholine (EYPC, DOPC, DMPC, DPPC or POPC). When injected into the bloodstream of conscious rats, the emulsions containing EYPC or POPC were metabolized similarly to natural chylomicrons, consistent with rapid lipoprotein lipase-mediated hydrolysis of triacylglycerols, followed by hepatic uptake of the remnants derived from the emulsions. Phospholipids from the injected emulsions were removed more slowly and became associated with the high-density lipoprotein fractions of the plasma. Emulsions containing DPPC were metabolized differently. Triacylglycerols disappeared very slowly from plasma, indicating lack of hydrolysis by lipoprotein lipase, and phospholipid radioactivity did not transfer to high-density lipoprotein. With emulsions containing DMPC, the plasma removal rates for emulsion triacylglycerols and cholesteryl esters were fast, but phospholipid radioactivity failed to transfer to the high-density lipoprotein fractions of plasma. With DOPC emulsions, clearances were slower than EYPC or POPC emulsions, but transfer to high-density lipoproteins was efficient. Therefore, an unsaturated chain at the glycerol 2-position was necessary for rapid hydrolysis by lipoprotein lipase and for efficient transfer of phospholipids to high-density lipoproteins. With an unsaturated chain at the glycerol 2-position, a saturated chain at the glycerol 1-position optimized the rate of remnant removal from the plasma.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

The molecular organization of nerve membranes

Summary The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 226 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.The preceding papers of this series were published inArchives of Biochemistry and Biophysics.     

Phospholipid miscibility in ternary mixtures

The gel to liquid-crystalline phase transition of aqueous dispersions of phospholipid mixtures was investigated by means of the repartition of the spin label 2,2,6,6-tetramethylpiperidine-I-oxyl between aqueous space and lipid hydrocarbon region. The dimyristoylphosphatidylcholine (DMPC)/dibehenoylphosphatidylcholine (DBPC) and dipalmitoylphosphatidylcholine (DPPC)/DBPC phase diagrams indicate gel phase immiscibility, whereas the distearoylphosphatidylcholine (DSPC)/DBPC phase diagram indicates non-ideal gel phase miscibility at low DBPC molar fractions. Aqueous dispersions of DMPC/DPPC/DBPC ternary mixtures show two distinct phase transitions, the first associated with the melting of a DMPC/DPPC phase and the second with the melting of a DBPC phase. Aqueous dispersions of DMPC/DSPC/DBPC ternary mixtures show to phase transitions at low DSPC molar fractions; the first is probably associated with the melting of a DMPC/DSPC phase, and the second with the melting of a DBPC/DSPC phase. At high DSPC molar fractions, only one phase transition is observed; this suggests that all the lipids are mixed in gel state membranes.     

Propofol, a general anesthetic, promotes the formation of fluid phase domains in model membranes

The molecular site of anesthetic action remains an area of intense research interest. It is not clear whether general anesthetics act through direct binding to proteins or by perturbing the membrane properties of excitable tissues. Several studies indicate that anesthetics affect the properties of either membrane lipids or proteins. However, gaps remain in our understanding of the molecular mechanism of anesthetic action. Recent developments in membrane biology have led to the concept of small-scale domain structures in lipid and lipid--protein coupled systems. The role of such domain structures in anesthetic action has not been studied in detail. In the present study, we investigated the effect of anesthetics on lipid domain structures in model membranes using the fluorescent spectral properties of Laurdan (6-dodecanoyl-2-dimethylamino naphthalene). Propofol, a general anesthetic, promoted the formation of fluid domains in model membranes of dipalmitoyl phosphatidyl choline (DPPC) or mixtures of lipids of varying acyl chains (DPPC:DMPC dimyristoyl phosphatidyl choline 1:1). The estimated size of these domains is 20--50 A. Based on these studies, we speculate that the mechanism of anesthetic action may involve effects on protein--lipid coupled systems through alterations in small-scale lipid domain structures.     

Reassembled plasma low density lipoproteins. Phospholipid-cholesterol ester-apoprotein B complexes

Reassembled low density lipoprotein (LDL) complexes have been prepared by the interaction of lipid-free sodium deoxycholate-solubilized apoprotein B (apoB) of native human LDL with preformed, 200 A in diameter, microemulsions of cholesteryl oleate (CO), surface-stabilized by either egg yolk phosphatidylcholine ( EYPC ) or dimyristoyl phosphatidylcholine (DMPC). Gel chromatography of PC/CO/apoB complexes shows co-elution of the complex at 43% PC, 43% CO, and 14% apoB. Negative stain electron microscopy shows the particles to be circular, homogeneous, and approximately 200 A in diameter. PC/CO/apoB complexes exhibit beta-migration on agarose gels and show one high molecular weight protein band on 3.0% sodium dodecyl sulfate-polyacrylamide gels. Differential scanning calorimetry and x-ray scattering show the lipids in the complexes to undergo at least two specific thermal transitions depending on lipid composition, one associated with the core-located cholesterol esters similar to LDL and the protein-free microemulsions and the other from the phospholipid forming the surface monolayer. In addition, particle disruption-protein unfolding/denaturation occur irreversibly at 80-85 degrees C. At 4 degrees C, the secondary structure of apoB on complexes of EYPC /CO/apoB is similar to that of native LDL. For complexes of DMPC/CO/apoB, the secondary structure shows less alpha-helix which correlates with the difference in surface lipid environment. The reassembled complexes of PC/CO/apoB provide a defined system in which the components may be varied systematically in order to study the molecular organization, molecular interactions, and metabolism of LDL.     

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.     

Interaction of furosemide with lipid membranes

Summary The interaction of furosemide with different phospholipids was investigated. Its influence on the lipid structure was inferred from its effect on the phase transition properties of lipids and on the conductance of planar bilayer membranes. The thermotropic properties of dipalmitoyl phosphatidylcholine, phosphatidylethanolamine (natural), dipalmitoyl phosphatidylethanolamine, brain sphingomyelin, brain cerebrosides and phosphatidylserine in the presence and absence of furosemide were investigated by differential scanning calorimetry,. The modifying effect of furosemide seems to be strongest on phosphatidylethanolamine (natural) and sphingomyelin bilayers. The propensity of furosemide to decrease the electrical resistance of planar lipid membranes was also studied and it is shown that the drug facilitates the transport of ions. Partition coefficients of furosemide between lipid bilayers and water were measured.Abbreviations DSC differential scanning calorimetry - PLM planar lipid membranes - DPPC dipalmitoyl phosphatidylcholine - DMPC dimyristoyl phosphatidylcholine - PE phosphatidyl ethanol     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions.

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

Interaction of the pore-forming protein equinatoxin II with model lipid membranes: A calorimetric and spectroscopic study.

The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry. EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition. This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10. For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule. The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements. Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG). The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII. The second transition involves the formation of alpha-helical structure. Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association.     

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between model triacylglycerol-rich lipoproteins and high-density lipoproteins in rat, rabbit and man

There are inverse relationships between HDL cholesterol and plasma triacylglycerol concentrations in normal and in hypertriglyceridemic individuals. To investigate the interactions between triacylglycerol-rich lipid particles and HDL, a lipid emulsion model of the triacylglycerol-rich lipoproteins was prepared. When emulsion particles were incubated with rat high-density lipoproteins (HDL) in the presence of lipid transfer activity (d greater than 1.21 g/ml fractions) from rabbit or human plasma there was a rapid bi-directional exchange of cholesteryl oleate (CO) and phospholipid (PL) labels between lighter and heavier fractions of HDL and emulsion particles. The transfers of CO and PL labels between both light and heavy fractions of HDL and the emulsion particles were increased with increasing amounts of emulsion added to the incubations. Incubation with the d greater than 1.21 g/ml fraction from rat plasma resulted in only a small exchange of CO whereas PL exchange was similar to rabbit and human plasma. Retinyl palmitate label was not transferred from emulsion particles to the HDL fractions even in the presence of lipid transfer activity from rabbit or human plasma. The present study shows that the transfer protein-mediated exchanges of surface and core lipids between HDL and the triacylglycerol-rich lipoproteins are affected by the quantity of triacylglycerol-rich particles in the system. This mechanism may contribute to the inverse relationships between plasma triacylglycerol concentrations and HDL concentrations in normal and hypertriglyceridemic individuals.     

Minimal peptide length for interaction of amphipathic alpha-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature- and ionic strength-induced conformational changes in the lipid head group region of liposomes as suggested by zeta potential data.

Neutral liposomes composed of DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine) are found to exhibit non-zero zeta potentials in an electric field even when they are dispersed in solution at pH 7.4. A model for the orientation of lipid head groups is proposed to explain the observed non-zero zeta potentials. The dependence of the zeta potential on temperature and ionic strength is analyzed via this model to obtain the information on the direction of the lipid head group in the liposome surface region. The direction of the lipid head group is found to be sensitive to the temperature and the ionic strength of the medium. At low ionic strengths, the phosphatidyl groups are located at the outer portion of the head group region. At constant temperature, as the ionic strength increases, the choline group approaches the outer region of the bilayer surface while the phosphatidyl group hides behind the surface. At the phase transition temperature of the lipid, the phosphatidyl group lies in the outer-most region of the surface and the choline group is in the inner-most region.     

The effects of bilirubin on the thermal properties of phosphatidylcholine bilayers.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.     

Interactions of surfactin with membrane models.

Surfactin, an acidic cyclic lipopeptide produced by strains of Bacillus subtilis, is a powerful biosurfactant possessing biological activities. Interactions of ionized surfactin (two negative charges) with lecithin vesicles have been monitored by changes in its CD spectra. These changes are more important in the presence of Ca2+ ions. We have studied the penetration of ionized surfactin into lipid monolayers. Using dimyristoyl phospholipids, the surfactin penetration is more important in DMPC than in DMPE monolayers and is greatly reduced in DMPA monolayers because of electrostatic repulsion. The surfactin penetration is lowered when the acyl chain length of the phospholipids increases. The exclusion pressure varies from 40 mN m-1 for DMPC to 30 mN m-1 for DPPC and 18 mN m-1 for egg lecithin. The presence of Ca2+ ions, which neutralize the charges of both surfactin and lipids in the subphase, leads to an important change of the penetration process that is enhanced in the case of acidic, but also of long chain (higher than C14) zwitterionic phospholipids (DPPC and lecithin). From compression isotherms of mixed surfactin/phospholipid monolayers, it appears that surfactin is completely miscible with phospholipids. The present study shows that surfactin penetrates spontaneously into lipid membranes by means of hydrophobic interactions. The insertion in the lipid membrane is accompanied by a conformation change of the peptide cycle.     

Triolein-cholesteryl oleate-cholesterol-lecithin emulsions: structural models of triglyceride-rich lipoproteins

The organization of lipids within emulsions composed of triolein (TO), cholesteryl oleate (CO), cholesterol (C), and egg yolk phosphatidylcholine (L) was examined. CO was substituted for TO in a series of emulsions to obtain TO:CO ratios comparable to the triglyceride:cholesterol ester ratios observed in subfractions of triglyceride-rich lipoproteins. The weight fraction of TO in the surface phase (0.02-0.05) was independent of the TO content of the emulsions. However, the weight fraction of CO in the surface phase depended upon the percentage of CO in the emulsions and was less than 0.004 even when 13.7% CO was present in the emulsion. When CO was substituted for TO, the percent of the total particle C which was carried in the droplet oil phase was increased. The interparticle equilibration of lipids was studied in subfractions of sonicated emulsions with particle sizes comparable to triglyceride-rich lipoproteins. The TO:CO ratios of the subfractions of a given emulsion were constant and independent of size, but the C:L ratio decreased in particles of smaller diameter. However, the surface C:L ratio was the same in all particles from a given emulsion. The size dependence of the C:L ratios was attributed to the partitioning of C into the oil cores of the emulsions. Because large droplets have the greatest core:surface mass ratios, more of their total particle C is carried in the core.     

Interaction of melittin with glycosphingolipids and phospholipids in mixed monolayers at different temperatures. Effect of the lipid physical state

The influence of the liquid-expanded or liquid-condensed state of the lipid interface induced by changes of temperature on the lipid-protein interactions and their two-dimensional miscibility was studied for mixtures of melittin with different phospholipids (DPPC, DMPC, DOPC egg PC) and gangliosides (G_M1, G_D1a) in mixed monolayers at the air/145 mM NaCl interface. The critical amount of melittin at which a phase separation takes place in the mixed film increases as the glycosphingolipid or phospholipid is more liquid-expanded. The lipid-protein interaction increases the stability of both melittin and the lipid. The interaction of melittin with gangliosides is thermodynamically more favorable as these are more liquid-expanded. The interaction of melittin with phospholipids, on the other hand, is more favorable when the lipids are in the liquid-condensed state even if these films show lateral immiscibility at a lower proportion of protein compared to lipids in the liquid-expanded state. Hydration-dehydration effects in the polar head group region are likely to participate in these lipid-protein interactions.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Pressure and temperature dependence of the intramolecular excimer formation rate.

We have measured the pressure dependence of the intramolecular excimer formation rate, K(p), for di-(1'-pyrenedecanoyl)-phosphatidylcholine (dipy10PC) probes in single-component lipid multilamellar vesicles (MLV) as a function of temperature. Apparent volumes of activation (V(a)) for intramolecular excimer formation are obtained from the slopes of plots of log K(p) versus P. For liquid-crystalline saturated lipid MLV (DMPC and DPPC), these plots are linear and yield a unique V(a) at each temperature, whereas for unsaturated lipids (POPC and DOPC) they are curvilinear and V(a) appears to decrease with pressure. The isothermal pressure induced phase transition is marked by an abrupt drop in the values of K(p). The pressure to temperature equivalence values, dPm/dT, estimated from the midpoint of the transitions, are 47.0, 43.5, and 52.5 bar degree C-1 for DMPC, DPPC, and POPC, respectively. In liquid-crystalline DMPC, V(a) decreases linearly as a function of temperature, with a coefficient -dVa/dT = 0.65 +/- 0.11 ml degree C-1 mol-1. Using a modified free volume model of diffusion, we show that this value corresponds to the thermal expansivity of DMPC. Both the apparent energy and entropy of activation, Ea and delta Sa, increase with pressure in DMPC, whereas both decrease in POPC and DOPC. This difference is attributed to the sensitivity of the dynamics and/or packing of the dipy10PC probes to the location of the cis-double bonds in the chains of the unsaturated host phospholipids. Finally, the atmospheric pressure values of Ea and delta Sa for the four host MLV examined are shown to be linearly related. The relevance of this finding with respect to the structure of the excimers formed by the dipy10PC probes is briefly discussed.