Determination of the distribution coefficient for rifabutin in a liposome-water system by fluorescence measurements

Fluorescence quenching was used to determine the distribution coefficient K _d for a tuberculostatic rifabutin in a liposome-water system at pH 6.4 and 7.4. Liposomes were large unilamellar vesicles composed of phosphatidylcholine or its mixtures with cholesterol or cardiolipin and containing a fluorescent label (anthryl phosphatidylcholine with the fluorophore in the hydrophobic region). The K _d values calculated in the Stern-Volmer model are comparable for phosphatidylcholine and phosphatidylcholine/cholesterol at both pH, and testify to rifabutin hydrophobicity (logK _d ≈ 2.4–2.6). Inclusion of negatively charged cardiolipin increases the K _d by more than an order of magnitude at pH 6.4, and ionization of the second phosphate at pH 7.4 produces an additional increase. These results demonstrate the large contribution of electrostatic forces into the interaction of rifabutin with model membranes.     

Preferential lipid association and mode of penetration of apocytochrome c in mixed model membranes as monitored by tryptophanyl fluorescence quenching using brominated phospholipids

The fluorescence of the single tryptophan residue at position 59 in apocytochrome c, the biosynthetic precursor of the inner mitochondrial membrane protein cytochrome c, was studied in small unilamellar vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with or without specifically Br-labelled acyl chains at the sn-2 position. The protein has a very high affinity for PS-containing vesicles (dissociation constant Kd less than 1 microM). From the relative quenching efficiency by the brominated phospholipids, it could be concluded that the protein specifically associates with the PS component in mixed vesicles and that maximal quenching occurred with phospholipids in which the bromine was present at the 6,7-position of the 2-acyl chain suggesting that (part of) the bound protein penetrates 7-8 A deep into the hydrophobic core of the bilayer.     

Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family

Human placental anticoagulant protein-I (PAP-I) is a member of the lipocortin/calpactin/annexin family of Ca2+-dependent phospholipid binding proteins. PAP-I was labeled with fluorescein 5-isothiocyanate (1 mol/mol); this derivative had anticoagulant activity identical to the unlabeled protein and could be used to measure Ca2+-dependent binding to phospholipid vesicles through changes in fluorescence quenching. At 1.2 mM Ca2+, 0.50 M ionic strength, pH 7.4, 25 degrees C, fluorescein-labeled PAP-I bound to phospholipid vesicles containing 80% phosphatidylcholine, 20% phosphatidylserine with a Kd of 1.2 +/- 0.2 nM (mean +/- S.D.). At an ionic strength of 0.15 M, the Kd decreased to less than 0.1 nM. Prothrombin and factor Xa both competed with fluorescein-labeled PAP-I for binding to anionic phospholipid vesicles, but with affinities at least 1000-fold weaker than PAP-I. PAP-I bound only weakly (Kd greater than 2 x 10(-5) M) to neutral or anionic phospholipid monomers, and this binding was not calcium-dependent. These results show that the affinity of PAP-I for anionic phospholipid surfaces is sufficient to explain its potency as an in vitro anticoagulant.     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Fusion of membrane vesicles bearing only the influenza hemagglutinin with erythrocytes, living cultured cells, and liposomes

Membrane vesicles, bearing only the influenza viral hemagglutinin glycoprotein, were reconstituted following solubilization of intact virions with Triton X-100. The viral hemagglutinin glycoprotein was separated from the neuraminidase glycoprotein by agarose sulfanilic acid column. The hemagglutinin glycoprotein obtained was homogenous in gel electrophoresis and devoid of any neuraminidase activity. A quantitative determination revealed that the hemolytic activity of the hemagglutinin vesicles was comparable to that of intact virions. Incubation of fluorescently labeled hemagglutinin vesicles with human erythrocyte ghosts (HEG) or with liposomes composed of phosphatidylcholine/cholesterol or phosphatidylcholine/cholesterol/gangliosides, at pH 5.0 but not at pH 7.4, resulted in fluorescence dequenching. Very little, if any, fluorescence dequenching was observed upon incubation of fluorescently labeled HA vesicles with neuraminidase or glutaraldehyde-treated HEG or with liposomes composed only of phosphatidylcholine. Hemagglutinin vesicles were rendered non-hemolytic by treatment with NH2OH or glutaraldehyde or by incubation at 85 degrees C or low pH. No fluorescence dequenching was observed following incubation of non-hemolytic hemagglutinin vesicles with HEG or liposomes. These results clearly suggest that the fluorescence dequenching observed is due to fusion between the hemagglutinin vesicles and the recipient membranes. Incubation of hemagglutinin vesicles with living cultured cells, i.e. mouse lymphoma S-49 cells, at pH 5.0 as well as at pH 7.4, also resulted in fluorescence dequenching. The fluorescence dequenching observed at pH 7.4 was inhibited by lysosomotropic agents (methylamine and ammonium chloride) as well as by EDTA and NaN3, indicating that it is due to fusion of hemagglutinin vesicles taken into the cells by endocytosis.     

Factors contributing to the distribution of cholesterol among phospholipid vesicles

The distribution of cholesterol between vesicles of different lipid composition at equilibrium has been determined. Small, sonicated unilamellar vesicles and large unilamellar vesicles were incubated at a defined temperature, and aliquots were then obtained at selected times for analysis. Inclusion of a small amount of phosphatidylserine or phosphatidylinositol in the membrane does not appreciably affect the distribution of cholesterol at equilibrium by these measurements. A membrane in the gel state is a poor acceptor of cholesterol. The length of the hydrocarbon chain on the phospholipid may also play a role. Bovine brain sphingomyelin dramatically slows the kinetics of cholesterol transfer, and the equilibrium distribution of cholesterol among vesicles containing sphingomyelin is therefore not observable in these experiments. Data obtained with vesicles containing phosphatidylethanolamine indicate a preference of cholesterol for vesicles composed of phosphatidylcholine compared to vesicles consisting primarily of phosphatidylethanolamine, at equilibrium. Experiments with a chaotropic agent indicate that the nature of the surface of the phosphatidylethanolamine bilayer, and its hydration, are important factors in the distribution of cholesterol among membranes in which phosphatidylethanolamine is present. These data suggest that membrane lipid content may play a role in the distribution of cholesterol among the membranes of a cell.     

Kinetic analysis of the interaction of the phosphatidylcholine exchange protein with unilamellar vesicels and multilamellar liposomes.

The mode of action of the phosphatidylcholine exchange protein from bovine liver has been studied by using unilamellar vesicles and multilamellar liposomes both of which membranes contain phosphatidylcholine and phosphatidic acid. The protein-mediated exchange of phosphatidylcholine between vesicles and liposomes fit the kinetic model presented in a previous study [V.D. Besselaar et al. (1975) Biochemistry, 1j, 1852]. Kinetic analysis of the rates of exchange indicate that the apparent dissociation constant of the exchange protein-vesicle complex decreases with an increasing phosphatidic acid content of the vesicles. Both vesicles and liposomes of 10 mol% phosphatidic acid show the same dissociation constant; on the other hand, both the formation and the disruption of the protein-membrane complex was 50--100-times higher for the vesicles than for the liposomes. This implies that the exchange protein can discriminate between vesicles and liposomes. Equilibrium gel chromatography of a column of Bio Gel A-5m confirmed that the exchange protein binds more strongly to vesicles of an increased phosphatidic acid content. The protein-mediated exchange of phosphatidylcholine in the vesicle-liposome system demonstrates a pH optimum at 4.0 to 5.5. The kinetic analysis at pH 5.0 as compared to pH 7.4 indicates that the enhanced exchange at pH 5.0 can solely be accounted for by altered interaction of the exchange protein with the liposomes.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Cholesterol transfer from small and large unilamellar vesicles

The rates of transfer of [14C]cholesterol from small and large unilamellar cholesterol/egg yolk phosphatidylcholine vesicles to a common vesicle acceptor were compared at 37 degrees C. The rate of exchange of cholesterol between vesicles of identical cholesterol concentrations (20 mol%) did not differ from the rate of transfer from donor vesicles containing 20 mol% cholesterol to egg yolk PC vesicles. Further, the rate of transfer of [14C]cholesterol from vesicles containing 15 mol% dicetyl phosphate (to confer a negative charge) was not different from the rate of transfer from neutral vesicles. However, the half-time for transfer of [14C]cholesterol from large unilamellar donor vesicles was about 5-times greater (10.2 h, 80 nm diameter) than from small unilamellar vesicles (2.3 h, 23 nm diameter). These data suggest that increased curvature in small unilamellar vesicles reduces cholesterol-nearest neighbor interactions to allow a more rapid transfer of cholesterol into the aqueous phase.     

Free fatty acids modulate intermembrane trafficking of cholesterol by increasing lipid mobilities: novel 13C NMR analyses of free cholesterol partitioning

Cholesterol and free fatty acids in membranes modulate major biological processes, and their cellular metabolism and actions are often coordinately regulated. However, effects of free fatty acid on cholesterol-membrane interactions have proven difficult to monitor in real time in intact systems. We developed a novel (13)C NMR method to assess effects of free fatty acids on molecular interactions of cholesterol within--and transfer between--model membranes. An important advantage of this method is the ability to acquire kinetic data without separation of donor and acceptor membranes. Large unilamellar phospholipid vesicles (LUV) with phosphatidylcholine/cholesterol ratios of 4:1 served as cholesterol donors. Small unilamellar vesicles (SUV) made with phosphatidylcholine were acceptors. The (13)C(4)-cholesterol peak is narrow in SUV, but very broad in LUV, spectra; the increase in intensity of this peak over time monitored transfer. Oleic acid and other long chain free fatty acids [saturated (C12-18) and unsaturated (C18)] dose-dependently increased mobilities of lipids in LUV (phospholipid and cholesterol) and cholesterol transfer rates, whereas short (C8-10) and very long (C24) chain free fatty acids did not. Decreasing pH from 7.4 to 6.5 (+/-oleic acid) had no effect on cholesterol transfer, and 5 mol % fatty acyl-CoAs increased transfer rates, demonstrating greater importance of the fatty-acyl tail over the headgroup. In LUV containing sphingomyelin, transfer rates decreased, but the presence of oleic acid increased transfer 1.3-fold. These results demonstrate free fatty acid-facilitated cholesterol movement within and between membranes, which may contribute to their multiple biological effects.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Ph-Sensitive Liposomes: Structural Characterization and Possible Therapeutic Applications

Abstract

The definition of liposomal preparations where sensitivity to moderate drops of pH (i.e. from 7.4 to 6.8) can be induced by the presence of plasma itself has been investigated. Liposome stability was monitored using 5,6-carboxyfluorescein (CF). We used sulfatide as the pH sensitive molecule on the basis of our previous studies in which we demonstrated an enhanced anti-tumor activity against a transplantable metastatic tumor model for ADM entrapped liposomes containing sulfatide. The amount of CF released at pH 6.8 in the presence of 50% plasma from small unilamellar vesicles (SUV), composed of egg phosphatidylcholine (EPC) and bovine brain sulfatide (CS) (4:1 m/m), was 3-fold that at pH 7.4, whereas no significant differences were observed when the same liposomes were incubated in buffer at 7.4 and 6.8 respectively. The plasma dependent pH sensitivity of these liposomes seems to specifically depend on the presence of sulfatide in the bilayer since neither cholesterol 3 sulfate (Choi 3S) nor galactocerebroside, are able to induce pH sensitivity in EPC liposomes. Of all the plasma components considered, VLDL seemed preferentially involved in the pH sensitivity induced by CS since they promoted an almost complete release of CF from EPC-CS liposomes at pH 6.8.     

Effect of cholesterol on interaction of dibucaine with phospholipid vesicles: a fluorescence study

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid membranes.     

Partition of amphiphilic molecules into phospholipid vesicles and human erythrocyte ghosts: measurements by ultraviolet difference spectroscopy

Molar partition coefficients for chlorpromazine and methochlorpromazine between phospholipid vesicles or human erythrocyte ghosts and buffer are determined by ultraviolet difference spectroscopy. The partition coefficients between small unilamellar egg phosphatidylcholine vesicles and buffer at pH 7.4 are 4.4 X 10(5) for chlorpromazine and 0.8 X 10(5) for methochlorpromazine, determined with 10 microM amphiphile. An increase in the partition of chlorpromazine into vesicles is seen as the pH is increased to the pKa of chlorpromazine at 9.2. Chlorpromazine also partitions preferentially into fluid-phase phospholipid compared to solid-phase phospholipid. Molar partition coefficients between unsealed human erythrocyte ghosts and buffer at pH 8.0 with 10 microM amphiphile are determined to be 6.5 X 10(5) for chlorpromazine and 2.5 X 10(5) for methochlorpromazine. Difference spectroscopy is an equilibrium technique that does not require separation of bound from free amphiphile, as do many other methods of determining membrane-buffer partition coefficients. This method is useful for any amphiphile that has an appreciable absorbance below its critical micelle concentration and whose absorbance is sensitive to environment.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles.

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37 degrees C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20 degrees C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20 degrees C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freeze-thaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Determination of the sidedness of carbocyanine dye labeling of membranes

We have found that 2,4,6-trinitrobenzenesulfonate (TNBS) is an effective quencher of the fluorescence of the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanines (CNdiI's). This quenching appears to occur by complex formation rather than a collisional mechanism. By use of this quenching, we have studied the transbilayer asymmetry of CNdiI labeling for large unilamellar membranes of egg phosphatidylcholine and the plasma membranes of human red blood cells and of ram epididymal spermatozoa. When CNdiI is added to membranes by ethanolic injection, only the outer leaflet labels. We have further shown that in large unilamellar vesicles of egg phosphatidylcholine, the CNdiI's do not appreciably "flip-flop" to the inner leaflet for at least 4 h at temperatures between 4 and 37 degrees C.     

Studies of synthetic peptide analogs of the amphipathic helix. Correlation of structure with function

The four peptide analogs of the amphipathic helix whose interactions with dimyristoyl phosphatidylcholine were described in the preceding paper were compared with apolipoproteins (apo) A-I and A-II in ability to displace native apolipoprotein from high density lipoprotein (HDL) and in ability to activate lecithin:cholesterol acyltransferase. The rank order of the ability of the four peptide analogs to displace apo-A-I from intact HDL was 18A-Pro-18A greater than 18A greater than des-Val10-18A greater than reverse-18A, the same order suggested in the preceding paper for relative lipid affinities. Modified HDL from which 40% of the apo-A-I had been displaced by 18A was indistinguishable from unmodified HDL in its ability to act as a lecithin:cholesterol acyltransferase substrate. This suggests that the easily displaced apo-A-I molecules in polydisperse HDL are relatively ineffectual as lecithin:cholesterol acyltransferase activators and/or 18A replaces the lecithin:cholesterol acyltransferase activity lost. The peptide analog 18A-Pro-18A was found to be a powerful activator of lecithin:cholesterol acyltransferase when incubated with unilamellar egg phosphatidylcholine (PC) vesicles, reaching 140% of the activity of apo-A-I at a 1:1.75 peptide-to-egg PC ratio. In another experiment, it was found that discoidal egg PC complexes of 18A-Pro-18A, 18A, and des-Val10-18A, formed by cholate dialysis, had 30-45% of the activity of apo-A-I/egg PC discoidal complexes, also formed by cholate dialysis, at the same peptide/lipid weight ratio. Examination of the structures formed when the 18A-Pro-18A peptide was incubated with unilamellar egg PC vesicles indicated that the ability of 18A-Pro-18A to exceed apo-A-I in lecithin:cholesterol acyltransferase activating ability is due to the spontaneous conversion by 18A-Pro-18A of egg PC vesicles to small protein annulus-bilayer disc structures. Apo-A-I, apo-A-II, nor any of the other three peptide analogs of the amphipathic helix studied were able to convert a significant fraction of egg PC unilamellar vesicles to discoidal structures.     

Movement of cholesterol between vesicles prepared with different phospholipids or sizes

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.     

Membrane proteins exposed on the external side of the intestinal brush-border membrane have fusogenic properties

The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.