H~+诱导心磷脂的六角形Ⅱ相和H~+的跨膜转运

本文报告H~ 能诱导心磷脂由双层排列转变为六角形Ⅱ相.含心磷脂的多层脂囊泡的~(31)P中核磁共振谱显示高场峰低场肩的双层排列特点,当pH降到2时,~(31)P核磁共振谱表现为低场峰高场肩的六角形Ⅱ相特点,表明H~ 对心磷脂多形性转变的诱导作用.用oxonol-V作为探剂.H~ 可使结合在人工脂膜上的oxonl-V的吸收峰红移和光吸收增加,表明心磷脂的六角形Ⅱ相在人工脂膜上具有H~ 的载体特性,易化H~ 的跨膜转运.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Proton magnetic resonance spectroscopic studies of the interaction of ubiquinone-10 with phospholipid model membranes

Proton magnetic resonance spectra of ubiquinone-10 and ubiquinone-10 dispersed with dipalmitoylglycerophosphocholine or egg phosphatidylcholine in aqueous medium have been obtained. The dispersions are in the form of multilamellar liposomes as judged by 31P-NMR spectra and the thermal history of the samples have ensured that ubiquinone not incorporated into the phospholipid structure only gives rise to a broad-line NMR proton spectrum. A high-resolution proton spectrum of ubiquinone is observed with upfield shifts of the O-methyl protons of the benzoquinone rings, indicating close proximity of the molecules but with an arrangement different from the pure liquid ubiquinone. Spectra obtained in the presence of the lanthanide shift reagents, dysprosium fluorooctanedionate and Dy(NO3)3, which have a preferred location in the hydrophobic and hydrophilic domains, respectively, of ubiquinone/phospholipid codispersions, are consistent with the partitioning of ubiquinone into a hydrophobic phospholipid environment remote from the aqueous phase. The type of arrangements of ubiquinone that could be accommodated within bilayers of phospholipid are discussed.     

Influence of retinoids on phosphatidylethanolamine lipid polymorphism.

The interaction of all-trans-retinoic acid and all-trans-retinol with dielaidoylphosphatidylethanolamine has been studied by differential scanning calorimetry and 31P-NMR spectroscopy. Increasing concentrations of all-trans-retinoic acid up to a mol fraction of 0.09 were found to induce shifts to lower temperatures of both the L beta to L alpha and L alpha to hexagonal-HII phase transitions, with a slight decrease in the enthalpy change of the transitions. At higher concentrations no further effects on the transitions were observed, and this is interpreted as indicative of a limited miscibility of retinoic acid with the phospholipid. 31P-NMR spectroscopy confirmed that the L alpha to hexagonal-HII phase transition was shifted to lower temperatures in the presence of retinoic acid. On the other hand increasing concentrations of all-trans-retinol up to a mol fraction of 0.166, induced a progressive shift of the L beta to L alpha and the L alpha to hexagonal-HII phase transitions to lower temperatures. At higher concentrations the main gel to liquid-crystalline phase transition was further displaced to lower temperatures and the lamellar to hexagonal-HII phase transition was not observed in the thermograms. 31P-NMR spectroscopy indicated that retinol was able of inducing the phospholipid to adopt the hexagonal-HII phase at temperatures even below the main gel to liquid-crystalline phase transition temperature of the pure phospholipid.     

Bilayer-micelle transition in phosphatidylcholine-sulfatide mixtures

Sulfatides are membrane-bound glycosphingolipids which tend to associate in micellar forms in water. In this study, combining the data obtained by several techniques, including 31P-NMR, DTA calorimetry, freeze-fracture electron microscopy, trapped volume and turbidity measurements plus enzymatic determination of outer-side "marker ganglioside", have enabled us to establish that bilayered liposomes of phosphatidylcholine formed in the presence of increasing amounts of sulfatide are stable up to 30 mol % glycolipid. Above thus, bilayered lipids progressively start to break up into micellar forms with bilayer-micelle transition complete at sulfatide concentrations above 80 mol %. The gel-to-liquid-crystalline phase transition of a dipalmitoylphosphatidylcholine-sulfatide dispersion is shown to strongly influence the equilibrium between micellar and bilayered forms, the micelles being present at higher concentrations as the fluidity of the system decreases. The possibility that such structural transitions may occur in vivo and effectively contribute to the modulation of some biological properties of the membranes is discussed.     

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

External surface area determination of lipid vesicles using trinitrobenzene sulfonate and ultraviolet/visible spectrophotometry

The lamellarity of liposomes is an important parameter to be controlled in liposomal delivery–release applications. A practical estimate of the degree of liposome lamellarity can be obtained by measuring the relative external surface area of the liposomes using a chemical assay. All such assays are based on a signal change caused by exposed marker lipids on reaction with a specific externally added reagent. However, a quantitative determination is often distorted by background reactions and contributions of internal lipid labeling. In the so-called TNBS assay, the marker lipid is phosphatidylethanolamine (PE) and the externally added reagent is TNBS (2,4,6-trinotrobenzene sulfonate). Mechanistic aspects of the TNBS assay were considered for improving the assay. Internal lipid labeling via PE flip-flop and/or TNBS permeation was minimal not only in cholesterol-containing liposomes but also in cholesterol-free liposomes if in the latter case membrane fluidity was decreased by slightly increasing the PE content. Compared with earlier versions of the TNBS assay, the amount of marker lipid and the time for analysis could be reduced considerably. The elaborated protocol was also applied to liposomes prepared from lipidic egg yolk isolates, offering a simple and inexpensive method for the development and in-process control of new liposome formation technologies.     

Short chain phospholipids in membrane protein crystallization: a 31P-NMR study of colloidal properties of dihexanoyl phosphatidylcholine

The colloidal features of short chain phospholipids can be deduced from 31P-NMR analysis by comparison with available data on phospholipid aqueous dispersion. In this study with dihexanoyl phosphatidylcholine, detergent phase separation was obtained by temperature shift and by addition of the precipitating agent polyethylene glycol. The 31P-NMR spectra indicate that the detergent micelles fuse to enter the hexagonal HII and lamellar phases. Consequences for the crystallization of membrane proteins are discussed.     

Quantitative Analysis of the Lamellarity of Giant Liposomes Prepared by the Inverted Emulsion Method

The inverted emulsion method is used to prepare giant liposomes by pushing water-in-oil droplets through the oil/water interface into an aqueous medium. Due to the high encapsulation efficiency of proteins under physiological conditions and the simplicity of the protocol, it has been widely used to prepare various cell models. However, the lamellarity of liposomes prepared by this method has not been evaluated quantitatively. Here, we prepared liposomes that were partially stained with a fluorescent dye, and analyzed their fluorescence intensity under an epifluorescence microscope. The fluorescence intensities of the membranes of individual liposomes were plotted against their diameter. The plots showed discrete distributions, which were classified into several groups. The group with the lowest fluorescence intensity was determined to be unilamellar by monitoring the exchangeability of the inner and the outer solutions of the liposomes in the presence of the pore-forming toxin α-hemolysin. Increasing the lipid concentration dissolved in oil increased the number of liposomes ∼100 times. However, almost all the liposomes were unilamellar even at saturating lipid concentrations. We also investigated the effects of lipid composition and liposome content, such as highly concentrated actin filaments and Xenopus egg extracts, on the lamellarity of the liposomes. Remarkably, over 90% of the liposomes were unilamellar under all conditions examined. We conclude that the inverted emulsion method can be used to efficiently prepare giant unilamellar liposomes and is useful for designing cell models.     

31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.     

Identification of phosphorylethanolamine in 31P-NMR spectra of human peripheral blood lymphocytes

The 31P-NMR spectrum of intact human peripheral blood lymphocytes contains a large unidentified peak in the phosphomonoester region. The pH dependency of the 31P-NMR chemical shift of this peak in perchloric acid extracts of peripheral blood lymphocytes was recorded. It was compared to the pH dependency of the chemical shift of phosphorylethanolamine, phosphorylcholine, and ribose 5-phosphate in model solutions. An excellent agreement was found between the behavior of phosphorylethanolamine and the unidentified peak. To further substantiate this assignment phosphorylethanolamine was added to extracts and the pH titrations were repeated. The added phosphorylethanolamine gave exactly the same chemical shift as the unidentified peak and no difference was observed with pH titrations. The concentration of phosphorylethanolamine in human peripheral blood lymphocytes was estimated by 31P NMR to be 2.4 mumol/10(9) cells (range 0.9-4.3/10(9) cells, n = 4).     

Light-induced membrane protein phosphorylation in the bovine rod outer segment. A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The pKa obtained from a pH titration of the 31P chemical shift was typical of serine phosphate. Therefore, these 31P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31P-NMR, and that using in vivo 31P 'spin labelling' one can study directly and selectively regions of receptors crucial to receptor function.     

多烯脂肪酸对磷脂胆固醇液晶态结构影响机理

用小角Ｘ 射线散射法、３１Ｐ 核磁共振技术和扫描隧道显微镜技术,对多烯脂肪酸多相脂质体的液晶态结构进行了研究。实验结果表明：胆固醇、多烯脂肪酸、非离子表面活性剂均对ＰＥ 脂质体的液晶态结构有明显的影响。在含有高效分散剂的磷酸缓冲溶液中制成的液晶态油酸多相脂质体和亚油酸多相脂质体均由片层六角相和片层立方相组成, 而蓖麻酸多相脂质体由立方六角相组成。     

Liposome‐Bilirubin Interaction: A Novel Strategy to Eliminate Bilirubin from Systemic Circulation

In the present study, we established the role of liposomes in removal of bilirubin from systemic circulation of the hyperbilirubinemic rats. Bilirubin has been demonstrated to possess inherent tendency to interact with liposomes through ionic as well as hydrophobic interactions. The size as well as lamellarity of the liposomes does not seem to affect their binding with bilirubin. However, the charge on the surface of liposomes plays an important role in bilirubin‐liposome interaction, e.g., bilirubin binds more extensively with positively charged liposomes as compared to the neutral or negatively charged liposomes. The present study further demonstrates that liposomes were effective in reducing the increased plasma bilirubin level in hyperbilirubinemic model animals as well. The results of the study suggest that positively charged liposome‐mediated selective homing of excess plasma bilirubin to the hepatocytes seems to offer an important strategy in management of hyperbilirubinemic conditions.     

Liposome-bilirubin interaction: a novel strategy to eliminate bilirubin from systemic circulation

In the present study, we established the role of liposomes in removal of bilirubin from systemic circulation of the hyperbilirubinemic rats. Bilirubin has been demonstrated to possess inherent tendency to interact with liposomes through ionic as well as hydrophobic interactions. The size as well as lamellarity of the liposomes does not seem to affect their binding with bilirubin. However, the charge on the surface of liposomes plays an important role in bilirubin-liposome interaction, e.g., bilirubin binds more extensively with positively charged liposomes as compared to the neutral or negatively charged liposomes. The present study further demonstrates that liposomes were effective in reducing the increased plasma bilirubin level in hyperbilirubinemic model animals as well. The results of the study suggest that positively charged liposome-mediated selective homing of excess plasma bilirubin to the hepatocytes seems to offer an important strategy in management of hyperbilirubinemic conditions.     

Effect of ConA—receptor interaction on the structure of cell membrane

Recently,the effect of ligand receptor interaction on the membrane structure of liposomes has been studied extensively,However,little is known about how it exists on biological membranes,In this paper,the effect of Concanavalin A(ConA) receptorinteratcion on the structure of cell membranes was studied by Circular DIchrosim(CD) and 31P Nuclear Magnetic Resonance(NMR).CD results of both the purified macrophage membranes and human erythrocyte hgosts(EG) showed that the conformation of membrane proteins changed after ConA binding.For further research,31P-NMR was used to detect the orgainzation of phosp[holipid molecules on macrophage membranes.After ConA binding,the tendercy to form non bilayer structure increased with the amount of ConA.The changes of 31P-NMR spectra of living macrophages might be partly due to the above stated reason too.In addition,ConA-receptor interaction also induced similar results of 31P-NMR spectra in EG.In contrast,wheat germ agglutinin (WGA),another kind of lectin,rarely showed the same influence.     

Restriction of lipid motion in membranes triggered by beta-sheet aggregation of the anti-apoptotic BH4 domain

The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state (2)H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by (31)P-NMR at 30 degrees C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static (14)N and (31)P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated beta-sheet secondary structure of BH4 with a 42 degrees tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.     

A tumor vasculature targeted liposome delivery system for combretastatin A4: Design, characterization, and in vitro evaluation

The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for αvβ3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of ∼120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with ∼80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than non-targeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.