The pH-dependence in the partitioning behaviour of (RS)-[3H]propranolol between MDCK cell lipid vesicles and buffer

PURPOSE: The pH-dependent partitioning of (RS)-[3H]propranolol between unilamellar vesicles of MDCK cell lipids and buffer was determined. METHODS: Partitioning studies were performed by means of equilibrium dialysis at 37 degrees C between pH 7 and 11 at a molar propranolol/lipid ratio in the membrane of 10(-6). RESULTS: The partition-pH diagram was bell-shaped. The highest apparent partition coefficient was 1797 at pH 9.7, the lowest was 805 at pH 6.9. Curve fitting with a combination of Henderson-Hasselbalch equations revealed an inflection point at the apparent pKa of propranolol, i.e. 9.7, and two additional pKa values at pH 7.7 and 10.0. The first one corresponds to the pKa of free fatty acids (FFA) within lipid bilayers and the other one to the pKa of phosphatidylethanolamine (PhE). The true partition coefficients (P) of the neutral as well as the ionised solute were fitted for each ionisation status of the membrane. The highest P, i.e. 2123, was calculated for neutral propranolol in the membrane with deprotonated FFA and protonated PhE. CONCLUSIONS: The partitioning behaviour of (RS)-[3H]propranolol in a complex membrane/buffer system can be described when considering ionisation changes of drug and lipids.     

Vasculopathy and insulin resistance in the JCR:LA-cp rat

The fluorescent dye FM1-43 labels nerve terminals in an activity-dependent fashion and has been found increasingly useful in exploring the exo- and endocytosis of synaptic vesicles and other cells by fluorescence methods. The dye distributes between the aqueous phase and the lipid membrane but the physical-chemical parameters characterizing the adsorption/partition equilibrium have not yet been determined. Fluorescence spectroscopy alone is not sufficient for a detailed elucidation of the adsorption mechanism since the method can be applied only in a rather narrow low-concentration window. In addition to fluorescence spectroscopy, we have therefore employed high sensitivity isothermal titration calorimetry (ITC) and deuterium magnetic resonance (2H-NMR). ITC allows the measurement of the adsorption isotherm up to 100 microM dye concentration whereas 2H-NMR provides information on the location of the dye with respect to the plane of the membrane. Dye adsorption/partition isotherms were measured for neutral and negatively-charged phospholipid vesicles. A non-linear dependence between the extent of adsorption and the free dye concentration was observed. Though the adsorption was mainly driven by the insertion of the non-polar part of the dye into the hydrophobic membrane interior, the adsorption equilibrium was further modulated by an electrostatic attraction/repulsion interaction of the cationic dye (z=+2) with the membrane surface. The Gouy-Chapman theory was employed to separate electrostatic and hydrophobic effects. After correcting for electrostatic effects, the dye-membrane interaction could be described by a simple partition equilibrium (Xb=Kcdye) with a partition constant of 103-104 M-1, a partition enthalpy of DeltaH=-2.0 kcal/mol and a free energy of binding of DeltaG=-7.8 kcal/mol. The insertion of FM1-43 into lipid membranes at room temperature is thus an entropy-driven reaction following the classical hydrophobic effect. Deuterium nuclear magnetic resonance provided insight into the structural changes of the lipid bilayer induced by the insertion of FM1-43. The dye disturbed the packing of the fatty acyl chains and decreased the fatty acyl chain order. FM1-43 also induced a conformational change in the phosphocholine headgroup. The -P-N+ dipole was parallel to the membrane surface in the absence of dye and was rotated with its positive end towards the water phase upon dye insertion. The extent of rotation was, however, much smaller than that induced by other cationic molecules of similar charge, suggesting an alignment of FM1-43 such that the POPC phosphate group is sandwiched by the two quaternary FM1-43 ammonium groups. In such an arrangement the two cationic charges counteract each other in a rotation of the -P-N+ dipole.     

Structural phase transitions involved in the interaction of phospholipid bilayers with octyl glucoside

The transitional stages induced by the interaction of the nonionic surfactant octyl glucoside (OcOse) on phosphatidylcholine liposomes were studied by means of transmission electron microscopy (TEM), light scattering and permeability changes. A linear correlation was observed between the effective surfactant/lipid molar ratio (Re; three-stage model proposed for liposome solubilization) and the OcOse concentration in the initial and final interaction stages, despite showing almost a constant value during bilayer saturation. The bilayer/aqueous phase partition coefficient (K) decreased in the subsolubilizing interaction steps and increased during solubilization. Thus, whereas a preferential distribution of surfactant monomers in the aqueous phase with respect to the lipid bilayers took place in the initial interaction steps, a larger association of OcOse molecules with these lipids in bilayers occurred during solubilization. The initial steps of bilayer saturation (50-70% permeability) were attained for a lower free surfactant (Sw) than that for its critical micellar concentration (cmc). When Sw reached the OcOse cmc, solubilization started to occur (Resat). Large unilamellar vesicles began to form as the OcOse exceeded 60 mol/100 mol, exhibiting for 65 mol/100 mol (50% permeability) vesicles of approximately 400 nm. TEM pictures for 100% permeability (72 mol/100 mol) and Resat still showed unilamellar vesicles, albeit that the Resat TEM picture showing traces of smaller structures. Exceeding surfactant amounts led to a decrease in static light scattering; the vesicle-size curve began to show a bimodal distribution. The TEM picture showed tubular structures together with bilayer fragments. Thereafter, the open structures were gradually affected by the surfactant and the scattered intensity gradually decreased to a constant low value.     

Conservative treatment of stress urinary incontinence in women: a systematic review of randomized clinical trials

The role played by the ceramides in the sublytic interactions of sodium dodecyl sulfate (SDS) with liposomes modeling the stratum corneum (SC) lipid composition was studied. The surfactant/lipid molar ratios (Re) and the bilayer/aqueous phase partition coefficients (k) were determined by monitoring the changes in the fluorescence intensity of liposomes due to the 5(6) carboxyfluorescein (CF) released from the interior of vesicles. The presence in liposomes of higher and lower ceramide proportions than that existing in the SC lipids led to a fall and to a rise in the sublytic activity of SDS on these structures. However, the SDS partitioning into liposomes (or affinity with these bilayer structures) increased as the proportion of Cer increased up to achieve almost a constant value for a Cer proportion similar to that in the SC lipids (about 40%). Thus, at low Cer proportions the ability of SDS molecules to alter these bilayer structures was higher than that for liposomes approximating the SC lipid composition despite their reduced partitioning into liposomes. These findings are in agreement with the recently reported dependencies of the level of ceramides in skin lipids and function barrier abnormalities and could explain in part these dependencies. The fact that the free surfactant concentration needed to achieve the two interaction levels investigated was lower than the surfactant critical micellar concentration (CMC) indicates that permeability alterations were mainly ruled by the action of surfactant monomers, regardless of the liposome lipid composition.     

Direct imaging by cryo-TEM shows membrane break-up by phospholipase A2 enzymatic activity

Phospholipid hydrolysis to free fatty acid and 1-lyso-phospholipid by water-soluble phospholipase A2 (PLA2) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis. Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles. This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA2-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA2 activity and the morphology of the lipid suspension.     

Transport of long-chain native fatty acids across lipid bilayer membranes indicates that transbilayer flip-flop is rate limiting

Evidence from a number of laboratories suggests that membrane proteins may meditate the transport of physiologic fatty acids (FA) across cell membranes. However, studies using lipid membranes indicate that FA are capable of spontaneous flip-flip, raising the possibility that rapid transport through the lipid phase obviates the need for a transport protein. Determining the rate-limiting steps for transport of FA across lipid membranes, therefore, is central to understanding FA transport across cell membranes. The transport of long-chain FA across lipid membranes, from the aqueous compartment on one side of the lipid bilayer to the aqueous phase on the other side, has not been measured previously. In this study, we have used the fluorescent probe ADIFAB to monitor the time course of FA movement from the outer to the inner aqueous compartments and from the lipid membrane to the outer aqueous compartment of lipid vesicles. These two measurements, together with measurements of the lipid:aqueous partition coefficients, allowed the determination of the rate constants for binding (kon), flip-flop (kff), and dissociation (koff) for the transport of long-chain natural FA across lipid vesicles. These rates were determined using large unilamellar vesicles (LUV) of approximately 1000 A diameter, prepared by extrusion and giant unilamellar vesicles (GUV), prepared by detergent dialysis, that are >/=2000 A diameter. The results of these studies for vesicles composed of egg phosphatidylcholine (EPC) and cholesterol reveal kff values that range from 3 to 15 s-1 for LUV and from 0.1 to 1.0 s-1 for GUV, depending upon temperature and FA type. For these same vesicles, dissociation rate constants range from 4 to 40 s-1 for LUV and from 0.3 to 2.5 s-1 for GUV. In all instances, the rate constant for flip-flop is smaller than koff, and because the rate of binding is greater than the rate of transport, we conclude that flip-flop is the rate-limiting step for transport. These results demonstrate that (1) kff and koff are smaller for GUV than for LUV, (2) the rate constants increase with FA type according to oleate (18:1) < palmitate (16:0) < linoleate (18:2), and (3) the barrier for flip-flop has a significant enthalpic component. Comparison of the flip-flop rates determined for GUV with values estimated from previously reported metabolic rates for cardiac myocytes, raises the possibility that flip-flop across the lipid phase alone may not be able to support metabolic requirements.     

Hydrophobicity and sorption of chlorophenolates to lipid membranes

We have studied sorption of ionized species of chlorophenols and pentahalophenols to lipid membranes using egg-phosphatidylcholine (egg-PC) vesicles and measuring their zeta-potential as a function of aqueous concentration of the phenolates. The zeta-potential isotherms can be understood in terms of a sorption model that is a combination of the Gouy-Chapman model of the electrical double layer at the membrane-water interface and the Langmuir model for sorption. Two intrinsic sorption parameters were determined: the linear partition coefficient beta m, which relates the membrane surface density of the phenolates to their aqueous concentration and the area of the adsorption site, Ps. The linear partition coefficient is the measure of the affinity of phenolates to the lipid membrane. It depends strongly on the molecular structure: 2,6-dichlorophenolate beta m = (0.45 +/- 0.08) x 10(-7); m; 3,5-dichlorophenolate beta m = (0.22 +/- 0.02) x 10(-6) m; 2,4,6-trichlorophenolate beta m = (0.63 +/-0.06) x 10(-6) m; 2,4,5-trichlorophenolate beta m = (0.11 +/- 0.01) x 10(-5) m; 2,3,5,6-tetrachlorophenolate beta m = (0.56 +/- 0.07) x 10(-5) m; 2,3,4,5-tetrachlorophenolate beta m = (0.55 +/- 0.06) x 10(-5) m; pentachlorophenolate beta m = (0.34 +/- 0.05) x 10(-4) m; pentafluorophenolate beta m = (1.00 +/- 0.13) x 10(-7) m and pentabromophenolate beta m = (0.19 +/- 0.04) x 10(-3) m. Ps was found to be independent of phenolate structure, Ps = 3.3 +/- 0.1 nm2. The membrane affinity of chlorophenolates was compared with the octanol-water partition coefficients of un-ionized chlorophenols. It was shown that the free energy of transfer of chlorophenolates from water into the lipid membrane can be divided into non-electrostatic and electrostatic contributions. The no-nelectrostatic contribution corresponds to the hydrophobicity parameter alpha = 3.94 +/- 0.0.08 kcal per nm2 of molecular surface area. The electrostatic contribution contains a term inversely proportional to the molecular radius of the phenolate ion which has the physical meaning of the work of transfer of the phenolate ion from water into the membrane. The polarity of the sorption region of egg-PC membranes is given in terms of the dielectric constant and was estimated to be 12.4 (range 10.5-13.4).     

Partition in two-polymer aqueous phases reflects differences between membrane surface properties of erythrocytes, ghosts and membrane vesicles

Partition of cells (defined as the percentage of added cells in the top phase) in dextran-polyethylene glycol aqueous two-phase systems is an extremely sensitive indicator of cell surface properties. By appropriate choice of polymer concentrations and salt composition and concentration one can, to some extent, select the membrane properties (e.g. charge (Walter, H. (1975) in Methods in Cell Biology (Prescott, D.M., ed.), Vol. 9, pp. 25-50, Academic Press, New York), lipid composition (Walter, H, Krob, E.J. and Brooks, D.E. (1976) Biochemistry, 15, 2959-2964)) that determine the partition behavior of cells (or membranes). In the present experiments we have obtained by partition information on separability, alteration and ensuing heterogeneity during preparation of human erythrocytes, ghosts and rightside-out and inside-out vesicles from human erythrocyte membranes. A phase system in which partition is greatly (although not exclusively) dtermined by membrane charge was used. It was found that: 1. The partition coefficients (and hence the membrane surfaces) of ghosts and of rightside-out vesicles differ from those of the erythrocytes from which they are derived. 2. Rightside-out and inside-out vesicles have different countercurrent distribution patterns with the rightside-out vesicles having the higher partition coefficient (probably reflecting higher membrane charge of the latter). 3. Rightside-out vesicles are highly heterogeneous membrane populations as evidenced by broader than theoretical countercurrent distribution curve which, in some experiments, even split into two populations outright. This heterogeneity is particularly interesting since the enzyme markers used to establish vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed), Vol. 2, pp. 245-281, Plenum Press, New York) indicate "pure" rightside-out preparations. 4. Inside-out vesicles could, under no conditions tested, be rendered totally free of rightside-out vesicles according to the enzyme markers used to indicate vesicle sidedness (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed), Vol. 2, pp. 245-281, Plenum Press, New York). A discussion of factors involved in the partition of the vesicles and ghosts together with analogies to the partition behavior of stored or in vitro modified human red blood cells is presented.     

The composition of lipids in intestinal digesta of young pigs receiving diets containing tallow and tallow fatty acids

Three semipurified diets containing a low level of fat or 10 percent of either beef tallow or beef tallow free fatty acids were fed to young pigs. Jejunal digesta was sampled 1.5, 2.5, 3.5, and 4.5 h after feeding by aspiration through tubes leading from the jejunal lumen to the exterior. The samples were forced through Millipore filters (1 x 10(-7) m pore size) to separate aqueous phase and oil phase lipid. The total and aqueous phase lipid was separated into triglyceride, monoglyceride, and free fatty acid, and the fatty acid composition of each fraction determined. The concentration of aqueous phase lipid was not influenced by diet, although the concentration of the oil phase lipid was generally higher for the addition of fat to the diets; the increase was greater for the beef tallow free fatty acid diet than for the beef tallow diet. Free fatty acids were the predominant component of the aqueous phase lipid along with some monoglyceride and traces of triglyceride. The major component of the oil phastions of triglyceride and monoglyceride. These must have been derived from endogenously secreted lipid in the case of the tallow fatty acid diet. Thus, the lower digestibility of completely hydrolyzed beef tallow than of conventional beef tallow was not due to an absence of monoglyceride in the intestinal lumen. The proportion of stearic acid in the jejunal digesta was greater than in the dietary lipid, whereas there were lower proportions of palmitic and oleic acids in the jejunal digesta than in the diet; the effect being most pronounced for the tallow free fatty acid diet. The ratio of oleic to palmitic acid in the aqueous phase was less than in the lipid phase suggesting preferential uptake of oleic acid from the micelle by the intestinal mucosa.     

Entropy-driven binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles

This study employed large unilamillar vesicles composed of purchased stratum corneum lipids to investigate the binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles. The partition coefficients of amino acids/dipeptides between the stratum corneum lipid vesicles and the acetate buffer were determined by HPLC. In addition, the binding/partition enthalpy of amino acids/dipeptides with the stratum corneum lipid vesicles was derived by directly measuring the binding/partition heat with isothermal titration calorimetry. According to the binding/petition Gibbs free energy and the binding/partition enthalpy, all the binding/partition of amino acids/dipeptides with the stratum corneum lipid vesicles is endothermic, implying an entropy-driven binding/partition. Also, the equilibrium binding/partition results demonstrate that the partition coefficients of amino acids/dipeptides do not correlate with the transdermal permeability. This finding suggests that either the interaction between the penetrants and the lipid bilayer between corneocytes may not be a determining step or that the paracellular path is not a dominant route of transdermal penetration.     

Permeability of acetic acid across gel and liquid-crystalline lipid bilayers conforms to free-surface-area theory

Solubility-diffusion theory, which treats the lipid bilayer membrane as a bulk lipid solvent into which permeants must partition and diffuse across, fails to account for the effects of lipid bilayer chain order on the permeability coefficient of any given permeant. This study addresses the scaling factor that must be applied to predictions from solubility-diffusion theory to correct for chain ordering. The effects of bilayer chemical composition, temperature, and phase structure on the permeability coefficient (Pm) of acetic acid were investigated in large unilamellar vesicles by a combined method of NMR line broadening and dynamic light scattering. Permeability values were obtained in distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dilauroylphosphatidylcholine bilayers, and their mixtures with cholesterol, at various temperatures both above and below the gel-->liquid-crystalline phase transition temperatures (Tm). A new scaling factor, the permeability decrement f, is introduced to account for the decrease in permeability coefficient from that predicted by solubility-diffusion theory owing to chain ordering in lipid bilayers. Values of f were obtained by division of the observed Pm by the permeability coefficient predicted from a bulk solubility-diffusion model. In liquid-crystalline phases, a strong correlation (r = 0.94) between f and the normalized surface density sigma was obtained: in f = 5.3 - 10.6 sigma. Activation energies (Ea) for the permeability of acetic acid decreased with decreasing phospholipid chain length and correlated with the sensitivity of chain ordering to temperature, [symbol: see text] sigma/[symbol: see text](1/T), as chain length was varied. Pm values decreased abruptly at temperatures below the main phase transition temperatures in pure dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine bilayers (30-60-fold) and below the pretransition in dipalmitoylphosphatidylcholine bilayers (8-fold), and the linear relationship between in f and sigma established for liquid-crystalline bilayers was no longer followed. However, in both gel and liquid-crystalline phases in f was found to exhibit an inverse correlation with free surface area (in f = -0.31 - 29.1/af, where af is the average free area (in square angstroms) per lipid molecule). Thus, the lipid bilayer permeability of acetic acid can be predicted from the relevant chain-packing properties in the bilayer (free surface area), regardless of whether chain ordering is varied by changes in temperature, lipid chain length, cholesterol concentration, or bilayer phase structure, provided that temperature effects on permeant dehydration and diffusion and the chain-length effects on bilayer barrier thickness are properly taken into account.     

Effect of protein aggregation in the aqueous phase on the binding of membrane proteins to membranes

Analysis of the binding of hydrophobic peptides or proteins to membranes generally assumes that the solute is monomeric in both the aqueous phase and the membrane. Simulations were performed to examine the effect of solute self-association in the aqueous phase on the binding of monomeric solute to lipid vesicles. Aggregation lowered the initial concentration of monomeric solute, which was then maintained at a relatively constant value at the expense of the aggregated solute, as the lipid concentration was increased. The resultant binding isotherm has a more linear initial portion rather than the classic hyperbolic shape. Although this shape is diagnostic of solute self-association in the aqueous phase, various combinations of values for the membrane partition coefficient and the solute self-association constant will generate similar isotherms. Data for cytochrome b5 were analyzed and, when the self-association constant was estimated by gel filtration, a unique value for the membrane partition coefficient was obtained. Thus, to obtain a true partition coefficient the state of the solute in the aqueous phase must be known. If the concentration of the monomeric solute species in the aqueous phase can be independently determined, then, even with heterogeneous aggregates, the true partition coefficient can be obtained.     

Interaction of glucose oxidase with phospholipid vesicles

The interactions between glucose oxidase and phospholipid vesicles were investigated. The investigations were carried on molecules adsorbed on the outer surfaces as well as entrapped in the interior of the vesicles . The adsorption of glucose oxidase on the surfaces of egg egg licithin vesicles, containing varying amounts of cholesterol and stearoylamine was measured by determining the free fraction of glucose oxidase detected in the filtrates. In general an enhancement of enzymic activity was observed upon interaction with the vesicles. The enhancement depends on the lipid composition of the vesicles and the surface concentration of the adsorbed glucose oxidase. It reached a maximal value at a surface concentration of 1.4-10(11) molecules/cm2 (approximately 7.1 - 10(4) A2/molecule) on pure phosphatidylcholine vesicles and about 6.5 - 10(10) molecules/cm2 (approximately 16 - 10(4) A2/molecule) when the vesicles contained cholesterol or cholesterol and stearoylamine. CD measurements indicated that the change in enzymic activity of the adsorbed glucose oxidase was accompanied by conformational modification of the enzyme. In order to entrap glucose oxidase into the vesicles, the lipid was sonicated in the presence of the enzyme. After removal of the free and adsorbed enzyme the amount of the entrapped enzyme was determined by measuring its activity after disintegration of the vesicles with Triton. The enzymic activity of the entrapped glucose oxidase served as a measure for the permeability of the bilayer membrane of the lipid vesicles to glucose. Addition of insulin to the suspension of vesicles containing the entrapped glucose oxidase increased the permeability of glucose by up to 9 - 10(-8) cm/s. This value is the lowest estimate based on the assumption that one glucose oxidase molecule was entrapped in every vesicle.     

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.     

The formation and annealing of structural defects in lipid bilayer vesicles

It is shown that sonication of phospholipid-water dispersions below the crystalline leads to liquid crystalline phase transition temperature (Tc) produces bilayer vesicles with structural defects within the bilayer membrane, which permit rapid permeation of ions and catalyze vesicle-vesicle fusion. These structural defects are annihilated simply by annealing the vesicle suspension above Tc. The rate of annealing was found to be slow, of the order of an hour for T = 3 degrees C above Tc, but annealing is complete within 10 min for T = 10 degrees C above Tc. It is proposed that these structural defects are fault-dislocations in the bilayer structure, which arise from a population defect in the distribution of the lipid molecules between the outer and inner monolayers, when small bilayer fragments reassemble to form the small bilayer vesicles during the sonication procedure. Such a population defect can only be remedied by lipid transport via the inside in equilibrium outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 degrees C above the phase transition.     

Interactions of long-chain fatty acids and albumin: determination of free fatty acid levels using the fluorescent probe ADIFAB

Equilibrium binding of long-chain fatty acids (FA) with albumin from human serum (HSA), bovine serum (BSA), and murine serum (MSA) has been studied by measuring the equilibrium levels of free fatty acids (FFA). FFA levels were measured directly, using a new fluorescent probe composed of acrylodan-derivatized intestinal fatty acid binding protein (ADIFAB). Measurements of [FFA] were done as a function of the ratio of total FA to total albumin (v) for v values between 0 and 6, at pH 7.4 and 37 degrees C. Under conditions observed in normal human physiology (v < or = 2), [FFA] values of the most abundant serum FA (palmitate, stearate, oleate) in equilibrium with human or bovine albumin are less than 15 nM. These values are considerably smaller than the generally quoted values of [FFA] in equilibrium with albumin: more than 20-fold for palmitate and more than 50-fold for oleate. FFA levels were found to increase monotonically with for all three albumins and all FA. In most cases [FFA] increased, for the same chain length, with increasing degree of acyl chain unsaturation, suggesting that FA aqueous solubility may play a significant role in the equilibrium between FA association with albumin and the aqueous phase. [The highest FFA levels (approximately 3000 nM), for example, were observed for linoleate (18:3) at the maximum v value (6).] Although aqueous-phase solubility of the FA may be important in understanding the interaction between FA and albumin, protein structure, as reflected in differences among the three albumins, also significantly affects the equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

The myristoyl moiety of myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein is embedded in the membrane

Members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) family are involved in several cellular processes such as secretion, motility, mitosis, and transformation. In addition to their ability to bind calmodulin and to cross-link actin filaments, reversible binding to the plasma membrane is most certainly an important component of the so far unknown functions of these proteins. We have therefore investigated the binding of murine MARCKS-related protein (MRP) to lipid vesicles. The partition coefficient, Kp, describing the affinity of myristoylated MRP for acidic lipid vesicles (20% phosphatidylserine, 80% phosphatidylcholine) is 5-8 x 10(3) M-1, which is only 2-4 times larger than the partition coefficient for the unmyristoylated protein. Interestingly, the affinity of MRP for acidic lipid membranes is 20-30-fold smaller than reported for murine MARCKS (Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214-28219). Since only a marginal binding could be observed with neutral phosphatidylcholine vesicles, we propose that electrostatic interactions are the major determinant of the binding of MRP to pure lipid membranes. Although the myristoyl moiety does not contribute drastically to the binding of MRP to vesicles, photolabeling experiments with a photoreactive phospholipid probe show that the fatty acid is embedded in the bilayer. The same membrane topology was found for bovine brain MARCKS. Since the relatively low affinity of MRP for vesicles is insufficient to account for a stable anchoring of the protein to cellular membranes, insertion of the myristoyl moiety into the bilayer might favor the interaction of MRP with additional factors required for the binding of the protein to intracellular membranes.     

A short, psychiatric, case-finding measure for HIV seropositive outpatients: performance characteristics of the 5-item mental health subscale of the SF-20 in a male, seropositive sample

Free thin liquid films (foam films) formed from aqueous dispersions of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylethanolamine with covalently bound poly-(ethylene glycol) of molecular weight 2000 (DPPE-PEG-2000) were studied by the thin liquid film microinterferometric technique of Scheludko and Exerowa in the temperature range 14-36 degrees C. The surface tension kinetics of the dispersions were studied in order to ensure equilibration of the foam films. These measurements showed that the rate of surface coverage depends slightly on the temperature and does not reach equilibrium values within reasonable time intervals for the dispersions containing only one amphiphile (DPPE-PEG-2000). The destruction of the vesicles at the air/(aqueous dispersion) interface was much faster for the dispersions containing DMPC/DPPE-PEG-2000 mixtures above 23 degrees C, the temperature of the chain-melting phase transition of the main lipid component (DMPC). The dependence of the equilibrium thickness of the foam films on the electrolyte concentration was measured for 1 and 9 mol% DPPE-PEG-2000 at 28 degrees C in the range 10-3 to 0.5 M NaCl. These results indicate that at the low electrolyte concentrations the electrostatic and van der Waals interactions are dominant similar to the foam films stabilized with DMPC alone. At the high electrolyte concentrations the steric repulsion of the PEG layers becomes dominant. The temperature-composition dependence of the bilayer thickness was measured for the foam bilayers at 0.14 M NaCl. The data for the foam bilayer thickness and the comparison with the phase diagrams of PC/PE-PEG dispersions, show that the DMPC/DPPE-PEG-2000 foam bilayers are able to exist in two phase states characterised by different conformations (mushroom and brush) of the grafted polymer.     

Purification of the stress protein alpha B-crystallin and its differentially phosphorylated forms

The subsolubilizing alterations caused by a series of alkyl glucosides (alkyl chain lengths ranging from C8 to C12) in unilamellar phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios (RE) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the increase of the fluorescence intensity of liposome suspensions due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous phase. Given that the free surfactant concentrations was always lower than the critical micelle concentration (CMC) of the surfactant tested we may assume that the surfactant-liposome interactions were mainly ruled by the action of surfactant monomers. In general terms, the decrease in the surfactant alkyl chain length (or the rise in the surfactant CMC) resulted in an increase in the ability of these surfactants to alter the permeability of liposomes and, inversely, in an abrupt decrease in their affinity with these bilayers structures. The overall balance of these opposite tendencies shows that at the two interaction levels studied (50 and 100% of CF release) the nonyl and the octyl glucoside showed, respectively, the highest ability to alter the release of the CF trapped in bilayers (lowest RE values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values).     

The noneffect of a large linear hydrocarbon, squalene, on the phosphatidylcholine packing structure

The interaction of squalene with liposomes and monolayers of dipalmitoyl phosphatidylcholine (DPL) has been studied by differential scanning calorimetry, Raman spectroscopy, and surface potential measurements. Mole ratios of squalene to DPL up to 9 to 1 were studied. In contrast to small, nonpolar molecules, which profoundly influence the structure of lipid bilayers as detected by changes in both their thermodynamic phase transition parameters and membrane fluidity, this large, nonpolar, linear hydrocarbon is devoid of such influences. It is clear from our data that a large nonpolar molecule such as squalene, having no polar group that might anchor it to the aqueous interface, cannot intercalate between the acyl chains either below or above the phase transition of DPL. This behavior is not compatible with models that treat the bilayer interior as a bulk hydrocarbon, and suggests that great caution should be exercised in extrapolating partition coefficients based on bulk hydrocarbon measurements to lipid bilayers.