Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.     

Interactions of triglycerides with phospholipids: incorporation into the bilayer structure and formation of emulsions

Interactions of carbonyl 13C-enriched triacylglycerols (TG) with phospholipid bilayers [egg phosphatidylcholine (PC), dipalmitoylphosphatidylcholine (DPPC), and an ether-linked phosphatidylcholine] were studied by 13C NMR spectroscopy. Up to 3 mol % triolein (TO) or tripalmitin (TP) was incorporated into DPPC vesicles by cosonication of the TG and DPPC at approximately 50 degrees C. NMR studies were carried out in a temperature range (30-50 degrees C) in which pure TO is a liquid whereas pure TP is a solid. In spectra of DPPC vesicles with TG at 40-50 degrees C, both TO and TP had narrow carbonyl resonances, indicative of rapid motions, and chemical shifts indicative of H bonding of the TG carbonyls with solvent (H2O) at the aqueous interfaces of the vesicle bilayer. Below the phase transition temperature of the DPPC/TG vesicles (approximately 36 degrees C), most phospholipid peaks broadened markedly. In DPPC vesicles with TP, the TP carbonyl peaks broadened beyond detection below the transition, whereas in vesicles with TO, the TO carbonyl peaks showed little change in line width or chemical shift and no change in the integrated intensity. Thus, in the gel phase, TP solidified with DPPC, whereas TO was fluid and remained oriented at the aqueous interfaces. Egg PC vesicles incorporated up to 2 mol % TP at 35 degrees C; the TP carbonyl peaks had line-width and chemical shift values similar to those for TP (or TO) in liquid-crystalline DPPC. TO incorporated into ether-linked PC had properties very similar to TO in ester-linked PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

The polar nature of 7-ketocholesterol determines its location within membrane domains and the kinetics of membrane microsolubilization by apolipoprotein A-I

7-Ketocholesterol is an oxidized derivative of cholesterol with numerous physiological effects. In model membranes, 7-ketocholesterol and cholesterol were compared by physical measures of bilayer order and polarity, formation of detergent resistant domains (DRM), phase separation, and membrane microsolubilization by apolipoprotein A-I. In binary mixtures of a saturated phosphatidylcholine (PC), dipalmitoyl-PC (DPPC), and cholesterol or 7-ketocholesterol, the sterols modulate bilayer order and polarity and induce DRM formation to a similar extent. Cholesterol induces formation of ordered lipid domains (rafts) in tertiary mixtures with dioleoyl-PC (DOPC) and DPPC, or DOPC and sphingomyelin (SM). In tertiary mixtures, cholesterol increased lipid order and reduces bilayer polarity more than 7-ketocholesterol. This effect was more pronounced when the mixtures were in a miscible liquid-disordered (L(d)) phase. Substitution of 7-ketocholesterol for cholesterol dramatically reduced the extent of DRM formation in DOPC/DPPC and DOPC/SM bilayers and ordered lipid phase separation in mixtures of a spin-labeled PC with DPPC and with SM. Compared to cholesterol, 7-ketocholesterol decreased the rate for the microsolubilization of dimyristoyl-PC multilamellar vesicles by apolipoprotein A-I. The membrane effects of 7-ketocholesterol were dependent on the phospholipid matrix. In L(d) phase phospholipids, a model for 7-ketocholesterol indicates that the proximity of the 7-keto and 3beta-OH groups puts both polar moieties at the lipid-water interface to tilt the sterol nucleus to the plane of the bilayer. 7-Ketocholesterol was less effective in forming ordered lipid domains, in decreasing the level of bilayer hydration, and in forming phase boundary bilayer defects. Compared to cholesterol, 7-ketocholesterol can differentially modulate membrane properties involved in protein-membrane association and function.     

Cytochrome b5 induced flip-flop of phospholipids in sonicated vesicles

Cytochrome b5 induced flip-flop of phosphatidylethanolamine (PE) in sonicated vesicles prepared from a 9:1 mixture of phosphatidylcholine (PC) to phosphatidylethanolamine was determined as follows. First, vesicles having a nonequilibrium distribution of PE across the bilayer were prepared by amidinating the external amino groups with isethionyl acetimidate. Amidinated cytochrome b5 was then added, and after the protein was completely bound, the rate of appearance of fresh PE on the outer surface was determined by removing aliquots at timed intervals and titrating the external amino groups with trinitrobenzenesulfonic acid. The results show an initial rapid phase of flip-flop (especially in the presence of salt) followed by a very slow phase, at 25 degrees C. Similar results were obtained when cytochrome b5 was introduced into the amidinated vesicles by spontaneous transfer from PC donor vesicles. These results indicate that the accumulation of the transferable ("loose") form of cytochrome b5 on the outer surface of a vesicle causes a transient, global destabilization of the bilayer that is relieved by lipid flip-flop. We speculate that this mechanism may be a significant driving force for the transfer of amphipathic molecules across membranes.     

Dependence of the conformation of the polar head groups of phosphatidylcholine on its packing in bilayers. Nuclear magnetic resonance studies on the effect of the binding of lanthanide ions.

Proton magnetic resonance spectra of vesicles of various sizes composed of egg phosphatidylcholine (PC) with varying concentrations of cholesterol differed in the apparent line width of the signal of the methylene protons of PC (delta v1/2). They also varied in the extent of lanthanide-induced shifts of the 31P and 1H NMR signals of the corresponding nuclei of the polar head groups located on the outer surface of the vesicles (delta delta). The differences in the lanthanide-induced shifts of the 31P signals are fully accounted for by the ratio between the externally added lanthanide and the number of PC head groups available for interaction with the lanthanide ions. This was not the case ofr the changes in the 1H NMR spectra. Here delta delta decreased with increasing delta v1/2, suggesting that the packing of the PC paraffinic chaings in the bilayer affects the conformation of the polar head groups; tightening of the packing probably results in a more extended conformation of the head groups. This conclusion is also supported by the larger effect lanthanides have on the 1H chemical shift of the choline head groups on the outer surface of small unilamellar vesicles as compared to groups on the inner, tighter packed layer.     

Hydrolysis of a phospholipid in an inert lipid matrix by phospholipase A2: a 13C NMR study

A new approach to study phospholipase A2 mediated hydrolysis of phospholipid vesicles, using 13C NMR spectroscopy, is described. [13C]Carbonyl-enriched dipalmitoylphosphatidylcholine (DPPC) incorporated into nonhydrolyzable ether-linked phospholipid bilayers was hydrolyzed by phospholipase A2 (Crotalus adamanteus). The 13C-labeled carboxyl/carbonyl peaks from the products [lyso-1-palmitoylphosphatidylcholine (LPPC) and palmitic acid (PA)] were well separated from the substrate carbonyl peaks. The progress of the reaction was monitored from decreases in the DPPC carbonyl peak intensities and increases in the product peak intensities. DPPC peak intensity changes showed that only the sn-2 ester bond of DPPC on the outer monolayer of the vesicle was hydrolyzed. Most, but not all, of the DPPC in the outer monolayer was hydrolyzed after 18-24 h. There was no movement of phospholipid from the inner to the outer monolayer over the long time periods (18-24 h) examined. On the basis of chemical shift measurements of the product carbonyl peaks, it was determined that, at all times during the hydrolysis reaction, the LPPC was present only in the outer monolayer of the bilayer and the PA was bound to the bilayer and was approximately 50% ionized at pH approximately 7.2. Bovine serum albumin extracted most of the LPPC and PA from the product vesicles, as revealed by chemical shift changes after addition of the protein. The capability of 13C NMR spectroscopy to elucidate key structural features without the use of either shift reagents or separation procedures which may alter the reaction equilibrium makes it an attractive method to study this enzymatic process.     

Solid-state NMR study of membrane interactions of the pore-forming cytolysin, equinatoxin II

Equinatoxin II (EqtII) is a pore-forming protein from Actinia equina that lyses red blood cell and model membranes. Lysis is dependent on the presence of sphingomyelin (SM) and is greatest for vesicles composed of equimolar SM and phosphatidylcholine (PC). Since SM and cholesterol (Chol) interact strongly, forming domains or “rafts” in PC membranes, ³¹P and ²H solid-state NMR were used to investigate changes in the lipid order and bilayer morphology of multilamellar vesicles comprised of different ratios of dimyristoylphosphatidylcholine (DMPC), SM and Chol following addition of EqtII. The toxin affects the phase transition temperature of the lipid acyl chains, causes formation of small vesicle type structures with increasing temperature, and changes the T₂ relaxation time of the phospholipid headgroup, with a tendency to order the liquid disordered phases and disorder the more ordered lipid phases. The solid-state NMR results indicate that Chol stabilizes the DMPC bilayer in the presence of EqtII but leads to greater disruption when SM is in the bilayer. This supports the proposal that EqtII is more lytic when both SM and Chol are present as a consequence of the formation of domain boundaries between liquid ordered and disordered phases in lipid bilayers leading to membrane disruption.     

Structure of Sphingomyelin Bilayers: A Simulation Study

We have carried out a molecular dynamics simulation of a hydrated 18:0 sphingomyelin lipid bilayer. The bilayer contained 1600 sphingomyelin (SM) molecules, and 50,592 water molecules. After construction and initial equilibration, the simulation was run for 3.8 ns at a constant temperature of 50°C and a constant pressure of 1 atm. We present properties of the bilayer calculated from the simulation, and compare with experimental data and with properties of dipalmitoyl phosphatidylcholine (DPPC) bilayers. The SM bilayers are significantly more ordered and compact than DPPC bilayers at the same temperature. SM bilayers also exhibit significant intramolecular hydrogen bonding between phosphate ester oxygen and hydroxyl hydrogen atoms. This results in a decreased hydration in the polar region of the SM bilayer compared with DPPC. Since our simulation system is very large we have calculated the power spectrum of bilayer undulation and peristaltic modes, and we compare these data with similar calculations for DPPC bilayers. We find that the SM bilayer has significantly larger bending modulus and area compressibility compared to DPPC.     

Perturbation of phospholipid bilayers by DDT

The localization of the effects of DDT (5–50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23°C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54°C or egg PC at 23°C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.     

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.     

Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers.

The distribution of ganglioside in supported lipid bilayers has been studied by atomic force microscopy. Hybrid dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylethanolamine (DPPE) and (2:1 DPPC/cholesterol)/DPPE bilayers were prepared using the Langmuir Blodgett technique. Egg PC and DPPC bilayers were prepared by vesicle fusion. Addition of ganglioside GM1 to each of the lipid bilayers resulted in the formation of heterogeneous surfaces that had numerous small raised domains (30--200 nm in diameter). Incubation of these bilayers with cholera toxin B subunit resulted in the detection of small protein aggregates, indicating specific binding of the protein to the GM1-rich microdomains. Similar results were obtained for DPPC, DPPC/cholesterol, and egg PC, demonstrating that the overall bilayer morphology was not dependent on the method of bilayer preparation or the fluidity of the lipid mixture. However, bilayers produced by vesicle fusion provided evidence for asymmetrically distributed GM1 domains that probably reflect the presence of ganglioside in both inner and outer monolayers of the initial vesicle. The results are discussed in relation to recent inconsistencies in the estimation of sizes of lipid rafts in model and natural membranes. It is hypothesized that small ganglioside-rich microdomains may exist within larger ordered domains in both natural and model membranes.     

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle <--> micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle --> micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3-7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5-7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.     

Interactions of acyl carnitines with model membranes: a (13)C-NMR study

Esterification of fatty acids with the small polar molecule carnitine is a required step for the regulated flow of fatty acids into mitochondrial inner matrix. We have studied the interactions of acyl carnitines (ACs) with model membranes [egg yolk phosphatidylcholine (PC) vesicles] by (13)C-nuclear magnetic resonance (NMR) spectroscopy. Using AC with (13)C-enrichment of the carbonyl carbon of the acyl chain, we detected NMR signals from AC on the inside and outside leaflets of the bilayer of small unilamellar vesicles prepared by cosonication of PC and AC. However, when AC was added to the outside of pre-formed PC vesicles, only the signal for AC bound to the outer leaflet was observed, even after hours at equilibrium. The extremely slow transmembrane diffusion ("flip-flop") is consistent with the zwitterionic nature of the carnitine head group and the known requirement of transport proteins for movement of ACs through the mitochondrial membrane. The partitioning of ACs (8-18 carbons) between water and PC vesicles was studied by monitoring the [(13)C]carbonyl chemical shift of ACs as a function of pH and concentration of vesicles. Significant partitioning into the water phase was detected for ACs with chain lengths of 12 carbons or less. The effect of ACs on the integrity of the bilayer was examined in vesicles with up to 25 mol% myristoyl carnitine; no gross disruption of the bilayer was observed. We hypothesize that the effects of high levels of long-chain AC (as found in ischemia or in certain diseases) on cell membranes result from molecular effects on membrane functions rather than from gross disruption of the lipid bilayer.     

Differences in hydrocarbon chain tilt between hydrated phosphatidylethanolamine and phosphatidylcholine bilayers. A molecular packing model.

Both wide-angle and lamellar x-ray diffraction data are interpreted in terms of a difference in hydrocarbon chain tilt between fully hydrated dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE). Although the hydrocarbon chains of multilayers of DPPC tilt ty approximately 30 degrees relative to the normal to the plane of the bilayer, as previously reported by others, the hydrocarbon chains of DPPE appear to be oriented approximately normal to the plane of the bilayer. It is found that the chain tilt in DPPC bilayers can be reduced by either: (a) adding an n-alkane to the bilayer interiors or (b) adding lanthanum ions to the fluid layers between bilayers. A molecular packing model is presented which accounts for these data. According to this model, DPPC chains tilt because of the size and conformation of the PC polar head group.     

Structure in the polar head region of phospholipid bilayers: A 31P [1H] nuclear Overhauser effect study.

The structure of the head-group region of some phospholipid bilayers in vesicle form has been studied and an intermolecular association of the N-methyl protons of phosphatidylcholine (PC) with the phosphate of phosphatidylethanolamine (PE) in mixed vesicles has been identified. Observation of a 31P[1H] nuclear Overhauser effect (NOE) in the phosphorus nuclear magnetic resonances of both PC and PE in mixed vesicles demonstrates an intimate dipolar interaction between some protons and the phosphorus nuclei. Substitution of deuterium for the N-methyl protons of PC eliminated the majority of the effect and necessitated the construction of a model of the bilayer surface in which the N-methyl protons of PC could interact closely with the phosphates of neighboring PE molecules. The predominant orientation of the head group must then be parallel to the bilayer surface. The amino protons of PE do not contribute significantly to the observed NOE. A corollary of these results is that there is little if any tendency for either PC or PE in the mixed vesicles to segregate into separate domains. A decrease in NOE in sphingomyelin vesicles on going from H2O to D2O suggests that an exchangeable proton contributes to the NOE. In addition the low value of the NOE observed in D2O suggests that the head-group conformation of sphingomyelin differs from that of PC.     

Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.     

The temporal sequence of events in the activation of phospholipase A2 by lipid vesicles. Studies with the monomeric enzyme from Agkistrodon piscivorus piscivorus

The substrate dependence of the time courses of hydrolysis of both small and large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) by Agkistrodon piscivorus piscivorus monomeric phospholipase A2 is consistent with an activation process involving enzyme aggregation on the vesicle surface. The time course of hydrolysis of large unilamellar vesicles is particularly complex; a slow initial rate of hydrolysis is followed by an extremely abrupt increase in enzyme activity. The length of this slow phase is a minimum at the phase transition temperature of the vesicles. The intrinsic fluorescence intensity of the phospholipase A2 also abruptly increases (50-60%) after a latency period revealing a strong temporal correlation between enzyme activity and the increase in fluorescence intensity. The length of the latency period before the sudden increase in fluorescence intensity is directly proportional to substrate concentration at DPPC concentrations above 20-100 microM. At lower concentrations, the length of the latency period is inversely proportional to the DPPC concentration. Such biphasic substrate dependence is predicted by a previously proposed enzyme activation model involving dimerization on the surface vesicle. Simultaneous monitoring of the protein fluorescence and hydrolysis demonstrates that the magnitude of the fluorescence change and the rate of hydrolysis are in exact temporal correlation. Furthermore, simultaneous monitoring of the fluorescence of the protein and that of a lipid probe, trimethylammonium diphenylhexatriene, indicates a change in lipid vesicle structure prior to, or coincident with, the abrupt change in protein activation. These results are consistent with the hypothesis that the monomeric phospholipase A2 from A. piscivorus piscivorus initially possesses a low level of intrinsic activity toward large unilamellar DPPC vesicles and that the enzyme slowly becomes further activated on the vesicle surface via dimerization. Eventually, the vesicles undergo an abrupt transition in internal structure leading to sudden rapid activation of the enzyme.     

The influence of the molar ratio of cholesteryl hemisuccinate/dipalmitoylphosphatidylcholine on 'liposome' formation after lipid film hydration.

The morphology of the structures formed after hydration of lipid films of cholesteryl hemisuccinate/dipalmitoylphosphatidylcholine (CHEMS/DPPC) was investigated in low ionic strength solutions. The importance of addition of a charge inducing agent/geometrical structure such as CHEMS for the formation of stable vesicle dispersions upon hydration was demonstrated. The encapsulated volume measured for CHEMS/DPPC ratios below 1:50 was low. For a ratio of CHEMS/DPPC of 1:30 EM micrographs showed mainly small unilamellar vesicles, with particle sizes between 0.07 and 0.3 microns, together with a small number of much larger vesicles. For ratios of CHEMS/DPPC above 0.1 only unilamellar vesicles and no bilayer stacks were found. The results confirm the hypothesis by Hauser (Biochim. Biophys. Acta 772 (1984) 37-50), that the structures formed upon hydration of charged phospholipid films are unilamellar vesicles, while for neutral phospholipid films upon hydration bilayer stacks and multilamellar vesicles are formed. The effect of CHEMS on the liposome bilayer structure can be mainly ascribed to its charge inducing properties and presumably to a minor extent to its molecular geometry, or to a combination of both.     

Uptake of yttrium-90 into lipid vesicles

Abstract

Lipid vesicles may be safely and efficiently loaded with therapeutic dose levels of the beta emitter yttrium-90 (⁹⁰Y) by using the ability of the cation ionophore A23187 to transport yttrium across the lipid bilayer where it is chelated on the vesicle interior by diethylenetriamine pentaacetic acid (DTPA). For 100 nm diameter vesicles composed of diplamitoylphosphatidylcholine (DPPC) and cholesterol (Choi), DPPC/Chol (1:1), containing 15 mM DTPA with 40 nmoles of external yttrium, total uptake was > 95% of added yttrium within 5 min at 50° using 0.4 ng of ionophore per nmole of lipid. Background binding in these neutral vesicles accounts for less than 0.1% of the yttrium associated with the vesicles. Important operational parameters were the amount of ionophore (> 0.2 μg of ionophore per μmole of lipid was required) and also the temperature (for DPPC/Chol (1:1) vesicles uptake at 40° was essentially background but was > 95% at 50°). The presence of the polymer polyethylene glycol (PEG) on the membrane surface had no effect upon yttrium uptake. Once entrapped, vesicles did not leak any contents for several days at room temperature.     

NMR study of the interaction of retinoids with phospholipid bilayers

The interaction of three vitamin A derivatives or retinoids: all-trans-retinoic acid, 13-cis-retinoic acid and retinol with multilamellar phospholipid bilayers was studied using a combination of 2H- and 31P-NMR measurements. The following model membrane systems were used: (1) dipalmitoylphosphatidylcholine (DPPC) bilayers; (2) bilayers composed of a mixture of DPPC and bovine heart phosphatidylcholine (PC); (3) mixed PC/phosphatidylethanolamine (PE) bilayers. Only a weak interaction was observed between 13-cis-retinoic acid and DPPC membranes. Addition of all-trans-retinoic acid at a molar ratio of 1:2 to the lipid causes a small decrease (5 C degrees) in the gel to liquid crystalline phase-transition temperature of DPPC, a small increase in the order parameters of the lipid side-chains of single component bilayers and no measurable effect in the other lipid systems studied. Considerably larger perturbation in the lipid bilayer structure is introduced by addition of retinol which, at a molar ratio of 1:2 to the lipid, lowered the gel to liquid crystalline phase-transition temperature of DPPC by 21 C degrees and caused a decrease of order parameters of the lipid side-chains in all three lipid bilayer systems. These effects are consistent with intercalation of retinol molecules into the bilayer interior. The results for the mixed PC/PE bilayers indicate that the presence of retinol caused lateral separation of PE- and retinol-enriched regions.