山莨菪碱促进心磷脂脂质体膜融合

前文曾报道,山莨菪碱能诱发心磷脂脂质体形成六角形Ⅱ结构,本文采用荧光共振能量转移技术研究了山莨菪碱对心磷脂脂体融合的影响,实验结果表明,山莨菪碱能诱发心磷脂脂质体融合并能促进由C_2~(2+)诱发的融合速率.     

Mass action analysis of kinetics and extent of fusion between Sendai virus and phospholipid vesicles

The kinetics and extent of fusion between Sendai virus particles and liposomes were investigated with an assay for lipid mixing based on the relief of self-quenching of fluorescence. The measurements, which were carried out at pH 7.4 and 5.0, included liposomes of three compositions, cardiolipin (CL), CL/dioleoylphosphatidylcholine (CL/DOPC 1:1), and phosphatidylserine (PS). Liposomal lipid concentrations varied from 2.5 to 50 microM. In addition, the effect of low concentrations of the dehydrating agent poly(ethylene glycol) (PEG) on fusion between the virus and the liposomes at pH 7.4 was studied. The results were analyzed in terms of a mass action kinetic model which views the overall fusion reaction as a sequence of a second-order process of virus-liposome adhesion or aggregation, followed by the first-order fusion reaction itself. The fusion products were shown to consist of a single virus particle and several liposomes. Analytical solutions were found for the final extent of fusion and increase in fluorescence intensity following the fusion of fluorescently labeled virus particles with liposomes. The final extents of fluorescence intensity were explained by assuming an essentially irreversible binding of liposomes to inactive virus particles. The percents of active virus particles and the rate constants of fusion and aggregation were larger at pH 5 than at pH 7.4, increased when PEG was included in the medium, and varied with liposomal lipid composition according to the sequence CL greater than CL/DOPC greater than PS.(ABSTRACT TRUNCATED AT 250 WORDS)     

The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions

Small unilamellar vesicles comprised of a mixture of phosphatidylethanolamine/phosphatidylcholine/cholesterol (3 : 1 : 2) fuse to form large multilamellar vesicles on increasing the temperature from 0 to 50°C. This event is associated with the appearance of lipidic particles at the fusion sites, consistent with a role as intermediary structures during the fusion process. Further, for phosphatidylcholine/cardiolipin (1 : 1) liposomes in the presence of Mn²⁺ a direct relationship between lipidic particles and the hexagonal (H_II) phase is demonstrated which suggests that lipidic particles can also occur as intermediaries between bilayer and hexagonal (H_II) structures.     

Secondary structure and orientation of a chemically synthesized mitochondrial signal sequence in phospholipid bilayers

A pre-sequence of 25 amino acids is required for import of yeast cytochrome oxidase subunit IV into mitochondria. Structure and orientation of the 25 amino acids synthesized peptide (p25) in a lipid bilayer were investigated by infrared attenuated total reflection spectroscopy. This method allowed to overcome the difficulties related to the optical turbidity due to the light scattering on membrane fragments which prevents the use of circular dichroism. We demonstrate here that incubation of the peptide with DOPC (dioleoylphosphatidylcholine) and DOPC-CL (dioleoylphosphatidylcholine - cardiolipin) liposomes is accompanied by an increase in alpha-helical content as compared to beta structure. Polarisation measurements indicate that the amphipathic helical segment is inserted parallel to the lipid acyl chains in cardiolipin containing liposomes.     

Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin.     

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca²⁺-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1–2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal H_II phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca²⁺, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca²⁺ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.     

Fusion of phospholipid vesicles in association with the appearance of lipidic particles as visualized by freeze fracturing

The addition of Ca2+ to small unilamellar vesicles of an equimolar mixture of egg phosphatidylcholine and cardiolipin induces fusion of these vesicles in association with the appearance of lipidic particles on the fusion sites.     

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.     

Cardiolipin Regulates the Activity of the Reconstituted Mitochondrial Calcium Uniporter by Modifying the Structure of the Liposome Bilayer

Reconstitution of mitochondrial calcium transport activity requires the incorporation of membrane proteins into a lipidic ambient. Calcium uptake has been measured previously using Cytochrome oxidase vesicles. The enrichment of these vesicles with cardiolipin, an acidic phospholipid that is found only in the inner mitochondrial membrane of eukaryotic cells, strongly inhibits calcium transport, in remarkable contrast with the activation effect that cardiolipin exerts upon other mitochondrial transporters and enzymes. The relation of the inactivation of calcium transport to the physical state of the bilayer was studied by following the polarization changes of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by flow cytometry in the cardiolipin-enriched liposomes with incorporated mitochondrial solubilized proteins. Non-bilayer molecular arrangements in the cardiolipin-supplemented liposomes, detected by flow cytometry, may produce the fluidity changes observed by fluorescence polarization of DPH. Fluidity changes correlate with the abolition of calcium uptake, but have no effect on the establishment of a membrane potential in the vesicles required for calcium transport activity. Changes in the membrane structure and uniporter function are observed in the combined presence of cardiolipin and calcium leading to a modified lipid configuration.     

Fusion of influenza virus with cardiolipin liposomes at low pH: mass action analysis of kinetics and extent

The kinetics and extent of low pH induced fusion between influenza virus and large unilamellar cardiolipin liposomes were investigated with an assay for lipid mixing based on fluorescence resonance energy transfer. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of virus-liposome adhesion or aggregation followed by the first-order fusion reaction itself. The fluorescence development during the course of the fusion process was calculated by numerical integration, employing separate rate constants for the initial aggregation step and for the subsequent fusion reaction. Analytical solutions were found for several limiting cases. Deaggregation of virus--liposome aggregates was explicitly taken into account but was found to be a minor effect under the conditions studied. The calculations gave good simulations and predictions for the kinetics and extent of fusion at different virus/liposome concentrations and ratios. At pH 5.0 and 37 degrees C, very high rate constants for aggregation and fusion were obtained, and essentially all of the virus particles were involved in the fusion process. Experiments at different virus/liposome ratios showed that fusion products may consist of a single virus particle and several liposomes but not of a single liposome and several virus particles. At pH 6.0, the rate constant for aggregation was the same as at pH 5.0, but the rate constant of fusion was about 5-fold lower, and only 25-40% of the virus particles were capable of fusing with the liposomes. The analytical procedure presented enables elucidation of the crucial role of the composition of target membrane vesicles in the initial adhesion and subsequent fusion of the virus at various pH values.     

Estimation by radiation inactivation of the size of functional units governing Sendai and influenza virus fusion

The target sizes associated with fusion and hemolysis carried out by Sendai virus envelope glycoproteins were determined by radiation inactivation analysis. The target size for influenza virus mediated fusion with erythrocyte ghosts at pH 5.0 was also determined for comparison; a value of 57 +/- 15 kDa was found, indistinguishable from that reported previously for influenza-mediated fusion of cardiolipin liposomes [Gibson, S., Jung, C. Y., Takahashi, M., & Lenard, J. (1986) Biochemistry 25, 6264-6268]. Sendai-mediated fusion with erythrocyte ghosts at pH 7.0 was likewise inactivated exponentially with increasing radiation dose, yielding a target size of 60 +/- 6 kDa, a value consistent with the molecular weight of a single F-protein molecule. The inactivation curve for Sendai-mediated fusion with cardiolipin liposomes at pH 7.0, however, was more complex. Assuming a "multiple target-single hit" model, the target consisted of 2-3 units of ca. 60 kDa each. A similar target was seen if the liposomes contained 10% gangliosides or if the reaction was measured at pH 5.0, suggesting that fusion occurred by the same mechanism at high and low pH. A target size of 261 +/- 48 kDa was found for Sendai-induced hemolysis, in contrast with influenza, which had a more complex target size for this activity (Gibson et al., 1986). Sendai virus fusion thus occurs by different mechanisms depending upon the nature of the target membrane, since it is mediated by different functional units. Hemolysis is mediated by a functional unit different from that associated with erythrocyte ghost fusion or with cardiolipin liposome fusion.     

Fusion activity of influenza virus. A comparison between biological and artificial target membrane vesicles

We have investigated the pH-dependent fusion activity of influenza virus toward human erythrocyte ghosts, utilizing a recently developed fluorescence assay, which permits continuous monitoring of the fusion reaction. The rate of fusion is negligible at neutral pH but shows a sharp increase at pH values just below 5.5. This pH dependence profile closely corresponds to that of virus-induced hemolysis. Fusion is rapidly inactivated by a low-pH preincubation of the virus alone either at 37 or at 0 degrees C. The presence of ghosts during this low-pH preincubation, carried out at 0 degree C under which condition there is hardly any fusion, causes a significant protection of the viral fusion activity against inactivation. Fusion initiated at low pH can be arrested instantaneously by readjustment of the pH to neutral. The characteristics of fusion of influenza virus with ghosts deviate from those of fusion with cardiolipin liposomes (Stegmann, T., Hoekstra, D., Scherphof, G., and Wilschut, J. (1985) Biochemistry 24, 3107-3113). Fusion with ghosts is consistent with a requirement of the well-documented pH-dependent conformational change in the viral hemagglutinin, whereas fusion with cardiolipin liposomes does not exhibit a strict dependence on the conformational change. Rather, the negative surface charge on the liposomes plays a critical role, as zwitterionic liposomes containing gangliosides show fusion behavior similar to that of erythrocyte ghosts.     

心磷脂对细胞色素C氧化酶质子泵功能的影响

 用胆酸盐透析法将猪心线粒体细胞色素C氧化酶重组在含心磷脂和二肉豆寇磷脂酰胆碱的脂质体上,以还原态细胞色素C作为酶反应底物,记录脂酶体囊泡外介质液pH的变化,pH下降幅度可以反映细胞色素C氧化酶质子泵的功能。 心磷脂含量不同的细胞色素C氧化酶脂酶体质子泵功能不同。心磷脂含量在10％—40％(w/w)范围内,随心磷脂含量增高,该酶质子泵功能增强;当心磷艏含量超过50％时,该酶质子泵功能却随心磷脂含量的增加表现出下降的趋势。阿霉素可以与心磷脂紧密结合,抑制细胞色素C氧化酶的质子泵功能。然而,少量阿霉素却能增强含70％心磷脂的脂酶体的质子泵功能。     

Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes.

Membrane association of cytochrome c (cyt c) was monitored by the efficiency of resonance energy transfer from a pyrene-fatty acid containing phospholipid derivative (1-palmitoyl-2[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphocholine (PPHPC)) to the heme of cyt c. Liposomes consisted of 85 mol% egg phosphatidylcholine (egg PC), 10 mol% cardiolipin, and 5 mol% PPHPC. Cardiolipin was necessary for the membrane binding of cyt c over the pH range studied, from 4 to 7. In accordance with the electrostatic nature of the membrane association of cyt c at neutral pH both 2 mM MgCl2 and 80 mM NaCl dissociated cyt c from the vesicles completely. At neutral pH also adenine nucleotides in millimolar concentrations were able to displace cyt c from liposomes, their efficiency decreasing in the sequence ATP > ADP > AMP. In addition, both CTP and GTP were equally effective as ATP. The detachment of cyt c from liposomes by nucleotides is likely to result from a competition between cardiolipin and the nucleotides for a common binding site in cyt c. When pH was decreased to 4 there was a small yet significant increase in the apparent affinity of cyt c to cardiolipin containing liposomes. Notably, at pH 4 the above nucleotides as well as NaCl and MgCl2 were no longer able to dissociate cyt c and, on the contrary, they slightly enhanced the quenching of pyrene fluorescence by cyt c. The above results do suggest that the membrane association of cyt c at acidic pH was non-ionic and presumably due to hydrogen bonding. The pH-dependent binding of cyt c to membranes was fully reversible. Accordingly, in the presence of sufficient concentrations of either nucleotides or salts rapid detachment and membrane association of cyt c could be induced by varying pH between neutral and acidic values, respectively.     

Analysis of the hexagonal II phase and its relations to lipidic particles and the lamellar phase A freeze-fracture study

Model systems of phosphatidylethanolamine (PE) and cardiolipin (DPG), as pure components and in binary mixtures with phosphatidylcholine (PC) have been morphologically analysed. The relation between the hexagonal_II (H_II) phase and lipidic particles as well as between the H_II phase and the lamellar phase has been studied. Moreover, the periodicity of the various H_II tubes was determined. (1) The periodicity of the H_II phase of cardiolipin is dependent on the cation involved. DPG-Ca exhibits the smallest tube to tube distance when compared to Mg²⁺ and Mn²⁺. Moreover, the DPG-Ca tubes are quite straight, in contrast to the Mg²⁺ and Mn²⁺ tubes, which appear to be frequently curved. (2) H_II tubes with two distinct diameters have been observed in H_II phase containing lipid mixtures. The thickness of the H_II tube is related to the composition of the tube. In the cardiolipin-lecithin system, structural separation of the pure cardiolipin H_II phase has been suggested with Mg²⁺ and Mn²⁺, but not with Ca²⁺. (3) Models for the H_II to lamellar phase transition and for the H_II phase to the lipidic particles are presented. (4) Lipidic particles are exclusively found in lipid model systems, which contain H_II phase favouring lipids. Morphological evidence is presented which suggests these lipidic particles represent inverted micelles. These observations include: (${}_{\mathrm{<mtext>i</mtext>}}$) there is a strong topological and quantitative relation between H_II tubes and lipidic particles, (${}_{\mathrm{<mtext>ii</mtext>}}$) lipidic particles occur densely packed in conglomerates without the presence of a smooth layer.     

山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

Intermediary structures during membrane fusion as observed by cryo-electron microscopy

Lipidic phases, containing 'lipidic particles' (dioleoylphosphatidylethanolamine/cholesterol/dioleoylphosphatidylcho lin e and cardiolipin/dimyristoylphosphatidylcholine in the presence of Ca2+) have been investigated by preparing thin films from a suspension of sonicated vesicles. These thin films were vitrified and observed 'directly' by cryo-electron microscopy in their hydrated form. The thin films show various fusion products and fusion intermediates such as lipidic particles.     

山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

Stability of desmopressin loaded in liposomes

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of alpha-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against alpha-chymotrypsin digestion.     

Stability of Desmopressin Loaded in Liposomes

Abstract

Desmopressin-containing liposome formulations have been developed for intranasal administration previously. Positively charged liposomes were found to be an efficient delivery system for desmopressin. In this study, stability of the loaded desmopressin in positively charged liposomes was further investigated. Comparison of the stability of desmopressin in solution and liposomes was made. Degradation of desmopressin was shown to follow a pseudo-first-order reaction. Degradation of desmopressin in both solution and liposomes demonstrated the same kinetic behavior and exhibited no significant difference in half-lives. Similar v-shape pH-rate profile was found for desmopressin degradation in solution and liposomes. At pH 4.0, the inflection point of the v-shape pH-rate curve, the reaction rate of desmopressin was lowest and the stability was greatest. The stability of lipid ingredients of dioleoylphosphatidylcholine (DOPC), cholesterol (C), and stearylamine (S) in the liposome dispersion at pH 4.0 was studied. Results demonstrated that DOPC, C, and S were relatively stable in the liposome structure when formulated with desmopressin. The degradation of desmopressin in solution and liposomes in the presence of α-chymotrypsin was investigated. A longer half-life for desmopressin in liposomes than in solution was observed. It was suggested that desmopressin was protected by the liposomes against α-chymotrypsin digestion.