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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple, rapid, and almost quantitative technique is described for the preparation of 1-40 ml of homogeneous unilamellar liposomes from dilute or concentrated aqueous suspensions of egg phosphatidylcholine. Aqueous suspensions of lipid are placed with the chamber of a French pressure cell at room temperature and rapidly extruded at 20,000 psi through the small orifice. A single pass transforms more than 70% of the extruded lipid into a homogeneous population of single-wall bilayer vesicles; more than 90% is transformed by recycling the lipid through the French pressure cell. About 95% of these liposomes range between 150-300 A in diameter (mean 200 A). The liposomes are stable for days to months when stored under nitrogen at 0.4 degrees C and can be prepared at 0 degrees, 25 degrees, or 37 degrees C. The liposomes appeared unaltered by repeated passages through the French pressure cell and no degradation of the phospholipid was detected after ten consecutive cycles at 20,000 psi in the absence of a nitrogen atmosphere. The method is especially useful for trapping small molecular weight substances because the concentration of both lipid and solute can be made quite high. Cholesterol up to 45 mole % can be incorporated into larger liposomes of egg phosphatidylcholine (mean diameter 315 A). Other phospholipids and different lipid mixtures can also be transformed into unilamellar vesicles with this method which has the advantage that additional steps of ultracentrifugation, column chromatography, dialysis, and concentrating procedures are usually unnecessary. Multilayered liposomes of small size (980 A mean diameter; > 95% between 500-1,500 A) are produced at lower pressure (3,000 psi). The latter are separated by gel permeation chromatography from a second population of homogeneous vesicles of even smaller size (580 A mean diameter; > 95% between 300-900 A) that contain two bilayer shells.
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PMID:Unilamellar liposomes made with the French pressure cell: a simple preparative and semiquantitative technique. 719 33

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 degrees C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97-106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis-Menten function dependent on the catalytic properties of the enzyme (kcat approximately equal to 9200 min-1, Km approximately equal to 0.12 mM), then level off to a slower rate determined by the desorption equilibrium constant (KD approximately equal to 0.01 mM) and desorption rate constant (kD approximately equal to 0.15 min-1). The relaxation time, tau, for the transition to the desorption-limited reaction is approximately 0.5 min; this large value of tau probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lysophosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decrease to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.
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PMID:Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine. 745 86

To determine the effect of cholesterol and lipid packing on the solubility of membrane proteins in bilayers, cytochrome b5 incorporation into phosphatidylcholine (PC) liposomes was determined as a function of bilayer curvature (SUVs versus LUVs), fatty acyl chain composition, and cholesterol content. The equilibrium affinity constant for the formation of a 1:1 b5/PC complex, Kp, and the number of PC's per "site" at saturation, n, were determined from binding isotherms, which were obtained by measuring the increase in intrinsic tryptophan fluorescence. With LUVs, n was also determined directly by gel filtration. The following results were obtained: (1) Both Kp and the saturating level of b5 binding, s (n-1), are significantly greater for SUVs than for LUVs. In LUVs, a binding site must consist of several surrounding lipid layers. (2) Cholesterol reduces Kp and s by factors that range from 1 to > 100. Binding inhibition is highly sensitive to the liposome size and to the fatty acyl composition of the PC; the latter correlates with the condensing effects on PC: C1satC2mono > C1satC2di approximately natural mixtures > C1unsat-C2unsat. (3) With POPC LUVs, the binding inhibition was 3.6-, 1.4- and 17-fold within the ranges of 0-20, 20-33, and 33-50 mole percent cholesterol, respectively. (4) The equilibrium binding constant to SUVs is greater for liposomes that are prepared from natural PC mixtures than for vesicles of a single synthetic phospholipid. The reductions in b5 binding correlate with reductions in bilayer free volume, which were calculated from monolayer studies of the lipid mixtures. The sensitivity of liposome saturability to bilayer curvature, fatty acyl chain composition, and cholesterol content may account for the disparate results among previous studies of cholesterol-protein interactions. A more significant implication is that in biological membranes with high levels of cholesterol, subtle variations in the fatty acyl chain composition could substantially affect the solubility and physical states of integral membrane proteins.
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PMID:Effect of cholesterol, fatty acyl chain composition, and bilayer curvature on the interaction of cytochrome b5 with liposomes of phosphatidylcholines. 789 81

To comprehend the renal defect underlying idiopathic Fanconi syndrome in the Basenji dog, we have focused on delineating the lipid profiles of renal brush border membranes isolated from affected and normal Basenji dogs to establish any physical or compositional changes underlying previously observed transport and membrane-fluidity changes. The lipid composition was studied with respect to total lipid, cholesterol, and phospholipid content, cholesterol to phospholipid ratio, distribution of the major phospholipid classes, and fatty acid composition. Total phospholipid of the isolated renal brush border membranes from Fanconi syndrome dogs analyzed by 31P nuclear magnetic resonance showed no difference compared with that of normal dogs. Examination of total fatty acids in both membranes using gas-liquid chromatography analysis of fatty acid methyl esters showed no difference in the mole percents of the major fatty acids. Our data suggest that changes in bulk membrane fluidity of the Fanconi syndrome dog renal brush border as measured by 1,6-diphenyl-1,3,5-hexatriene cannot be attributed to phospholipid and fatty acid compositional change. In the membranes isolated from affected dog kidney, the cholesterol content determined by gas-liquid chromatography analysis was 66 mol% higher than in membranes isolated from normal dog kidney. This correlates with the higher cholesterol to phospholipid molar ratio of 0.82 +/- 0.08 in the affected animal as compared with 0.58 +/- 0.04 in the normal. Cholesterol content and its microdomain in the membrane bilayer may be important in modulating transport functions. Increased membrane cholesterol content may affect the conformational motility of membrane transport proteins and thus affect their function.
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PMID:Renal brush border membrane lipid composition in Basenji dogs with spontaneous idiopathic Fanconi syndrome. 808 81

Recently, the influence of acyl structure on galactosylceramide's (GalCer) interfacial phase behavior was studied [Ali, S., Smaby, J. M., & Brown, R.E. (1993) Biochemistry 32, 11696-11703]. Here, we show that acyl structure is a key parameter controlling GalCer's ability to interact with cholesterol. Different chain-pure GalCer species containing saturated (24:0, 18:0, or 10:0), or unsaturated (24:1 delta 15, 22:1 delta 13, or 18:2 delta 9, 12) acyl chains were synthesized. After measurement of the force-area behavior of mixed cholesterol/GalCer films at 24 degrees C, the average molecular area and average compressibility were determined as a function of film composition. Cholesterol exerts only a slight condensing effect when the GalCer species are liquid-ordered [liquid-condensed], with maximum condensation occurring near 0.25 mole fraction. However, cholesterol exerts a marked condensing effect on liquid-disordered (liquid-expanded) GalCer species regardless of whether the acyl chain is saturated or unsaturated. Maximum condensation occurs at cholesterol mole fractions between 0.3 and 0.4. We also compared cholesterol's relative condensing effect on liquid-expanded GalCer versus sphingomyelin. Cholesterol's condensation of either bovine brain or egg sphingomyelin is 25-30% greater than that observed with different liquid-expanded GalCer species. Aside from average area behavior, we assessed cholesterol's interfacial interactions with the various sphingolipids by determining the average compressibility as a function of composition. The compressibility of condensed GalCer derivatives changes very little upon addition of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cholesterol's interfacial interactions with galactosylceramides. 813 Feb 3

We recently demonstrated that cationic lipids, added in monomer or micellar form, bind to DNA, resulting in the formation of a hydrophobic complex. This complex can serve as a well-defined intermediate in the preparation of DNA-lipid particles (DLPs) with many potential applications for delivery of polynucleotides in vitro and in vivo. To develop a better understanding of the factors governing complex formation, we have characterized the cationic lipid/DNA binding reaction. This was evaluated by measuring DNA and cationic lipid (DODAC) complex formation using the Bligh and Dyer extraction procedure. Efficient recovery of DNA (> 95%) in the organic phase was achieved when sufficient monocationic lipids interact with DNA phosphate groups. The rate of binding depends on the amount of DNA or cationic lipid present in the system. The time required to generate the hydrophobic complex was increased when < 10 micrograms of DNA or < 40 nmol of DODAC was present. Surprisingly, the rate of complex formation was contingent on the incubation period after partitioning the DNA/lipid mixture into organic and aqueous phases. These results suggest that the cationic lipid/DNA complex forms at the aqueous/organic interface and that DNA/lipid binding is dependent on multivalent interactions at this interface. A Scatchard analysis of DNA/DODAC binding demonstrated that the binding reaction exhibits a high degree of positive cooperativity. The apparent dissociation constant (Kn), using data obtained under conditions where DODAC binding to DNA approached saturation, indicated a high-affinity reaction (Kn > 10(-11) mol L-1). At this point, approximately 8400 mol of DODAC was bound per mole of DNA, which is equivalent to a charge ratio (+/-) of 0.585 for the 7.2 kb plasmid used and suggests that formation of the hydrophobic complex occurs at a stage prior to charge neutralization. The influence of other lipids on DNA/cationic lipid binding at the aqueous/organic interface was also studied. Cholesterol and DOPC had little effect on DNA/DODAC binding while the anionic lipids LPI, DOPS, and DMPG inhibited complex formation. The zwitterionic lipid DOPE, however, had a concentration-dependent effect on cationic lipid binding that was also dependent on the mixing order. We believe that this approach for evaluating lipid/DNA binding provides an effective procedure for assessing factors which control the dissociation of lipids from DNA and may be beneficial in the selection of lipids for effective use in gene transfection studies.
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PMID:Cationic lipid binding to DNA: characterization of complex formation. 863 36

The cause and effect relationship between membrane cholesterol and gallbladder muscle contractility was examined by altering membrane cholesterol to phospholipid mole ratio using cholesterol-rich or cholesterol-free liposomes. Gallbladder single muscle cells, from prairie dogs that were fed either a regular or high-cholesterol (1.2%) diet, were isolated enzymatically with collagenase. Plasma membranes of gallbladder muscle were purified in sucrose gradient. Cholesterol was measured using the cholesterol oxidase method. Phospholipids were measured with the method of G.R. Bartlett (J. Biol. Chem. 234: 466-468, 1959). The results of this experiment are 1) after high-cholesterol feeding, cholesterol contents and cholesterol/ phospholipid mole ratio in plasma membranes of gallbladder muscle increased 90%, and muscle cell contraction in response to cholecystokinin octapeptide decreased 58%; 2) similar changes were observed when normal gallbladder muscle cells were incubated with cholesterol-rich liposomes for 2 h; and 3) the changes induced either in vivo or in vitro were reversed when muscle cells were subsequently incubated with cholesterol-free liposomes for 2-6 h. We conclude that gallbladder muscle may incorporate excess cholesterol into its plasma membrane when exposed to a cholesterol-rich environment, that excess membrane cholesterol impairs muscle contractility, and that these changes appear to be reversible.
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PMID:Membrane cholesterol alters gallbladder muscle contractility in prairie dogs. 876 Jan 7

The mixing behaviour of plant oils (ricebran, saffola and clove) with water in presence of amphiphiles (Triton X-100, Tween-60, Aerosol OT, Igepal, Na-oleate, ethanol and cinnamic alcohol) in various ternary and quaternary combinations has been studied. The phase behaviour at different mass proportions and temperature has been investigated in the absence and presence of additives such as NaCl, glucose, urea and cholesterol. Of all the combinations studied, those with ethanol plus sodium oleate as amphiphile have shown maximum extent of single phase microemulsion formation. The presence of urea in the aqueous medium has further increased the monophasic extent whereas NaCl has decreased it. Cholesterol in oil and glucose in water have apparently shown inert effects. The effects of the additives on the formation of biphasic or triphasic formulations, on the other hand, have been found to be distinct and well-dependent on [H2O]/[amphiphile] mole ratio and temperature. Spectral measurements of I3- in the aqueous micropool in microemulsion of clove oil/(ethanol + Na-oleate)/water have shown the microenvironment to be physicochemically different from bulk water.
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PMID:Biological microemulsions V: mutual mixing of oils, amphiphiles and water in ternary and quaternary combinations. 882 91

Why agonist-induced activation of the nicotinic acetylcholine receptor (nAcChoR) fails completely in the absence of cholesterol is unknown. Affinity-purified nAcChoRs from Torpedo reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine/1, 2-dioleoyl-sn-glycero-3-phosphate/steroid bilayers at mole ratios of 58:12:30 were used to distinguish between three regions of the membrane where cholesterol might act: the lipid bilayer, the lipid-protein interface, or sites within the protein itself. In the bilayer, the role of fluidity has been ruled out and certain neutral lipids can substitute for cholesterol [C. Sunshine, M.G. McNamee, Biochim. Biophys. Acta 1191 (1994) 59-64]; therefore, we first tested the hypothesis that flip-flop of cholesterol across the membrane is important; a plausible mechanism might be the relief of mechanical bending strain induced by a conformation change that expands the two leaflets of the bilayer asymmetrically. Cholesterol analogs prevented from flipping by charged groups attached to the 3-position's hydroxyl supported channel opening, contrary to this hypothesis. The second hypothesis is that interstitial cholesterol binding sites exist deep within the nAcChoR that must be occupied for channel opening to occur. When cholesterol hemisuccinate was covalently 'tethered' to the glycerol backbone of phosphatidylcholine, channel opening was still supported. Thus, if there are functionally important cholesterol sites, they must be very close to the lipid-protein interface and might be termed periannular.
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PMID:Where does cholesterol act during activation of the nicotinic acetylcholine receptor? 954 86

We recently reported the equilibrium maximum solubility of cholesterol in a lipid bilayer, chi*chol, to be 0.66 in four different phosphatidylcholines, and 0.51 in a phosphatidylethanolamine (Huang, J.,J.T. Buboltz, and G. W. Feigenson. 1999. Biochim. Biophys. Acta. in press). Here we present a model of cholesterol-phospholipid mixing that explains these observed values of chi*chol. Monte Carlo simulations show that pairwise-additivity of nearest-neighbor interactions is inadequate to describe all the chi*chol values. Instead, if cholesterol multibody interactions are assigned highly unfavorable energy, then jumps occur in cholesterol chemical potential that lead to its precipitation from the bilayer. Cholesterol precipitation is most likely to occur near three discrete values of cholesterol mole fraction, 0.50, 0.57, and 0.67, which correspond to cholesterol/phospholipid mole ratios of 1/1, 4/3, and 2/1, respectively. At these solubility limits, where cholesterol chemical potential jumps, the cholesterol-phospholipid bilayer mixture forms highly regular lipid distributions in order to minimize cholesterol-cholesterol contacts. This treatment shows that dramatic structural and thermodynamic changes can occur at particular cholesterol mole fractions without any stoichiometric complex formation. The physical origin of the unfavorable cholesterol multibody interaction is explained by an "umbrella model": in a bilayer, nonpolar cholesterol relies on polar phospholipid headgroup coverage to avoid the unfavorable free energy of cholesterol contact with water. Thus, at high cholesterol mole fraction, this unfavorable free energy, not any favorable cholesterol-phospholipid interaction, dominates the mixing behavior. This physical origin also explains the "cholesterol condensing effect" and the increase in acyl chain order parameter in cholesterol-phospholipid mixtures.
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PMID:A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. 1009 8


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