ESI-MS/MS方法检测拟南芥膜脂分子对机械伤害的响应

利用一种灵敏的、基于ESI-MS／MS（electrospray ionization tandem mass spectrometry）的脂类组学方法,测定了机械伤害诱导的拟南芥6种磷脂（phosphohpids）、2种糖脂（glycolipids）、3种溶血磷脂（lysophospholipids）和约120种脂类分子的变化,探索了膜脂响应机械伤害的基本趋势。结果表明,机械伤害后磷脂酸（phosphatidic acid,PA）和3种溶血磷脂显著升高,而叶绿体膜上的糖脂减少;在测量的1小时范围内,不同脂类水解产生的磷脂酸分子的增加速度和强度不同,反映出它们经历了不同的生化过程。具体表现为：（1）叶绿体膜脂磷脂酰甘油（phosphatidylglycero,PG）分子34：4 PG水解的产物磷脂酸分子34：4 PA的积累速度明显慢于其它磷脂酸分子;（2）磷脂酸分子34：6 PA仅有少量的积累,其可能是由叶绿体膜脂单半乳糖二酰甘油（monogalactosyldiacylglycerol,MGDG）。分子34：6 MGDG和双半乳糖二酰甘油（digalactosyldiacylglycerol,DGDG）分子34：6 DGDG水解产生,然而这两种糖脂含量明显下降,说明它们有可能还参与了其它的反应。脂类的摩尔百分组成没有剧烈的变化。     

儿茶素诱导的拟南芥根细胞膜脂变化

儿茶素是一种可以短时间内杀死植物细胞的植物毒素,由于具有强的植物毒性,儿茶素是开发除草剂的理想化合物,它可以诱导植物根系统的死亡。为了研究植物根细胞膜脂对化学胁迫的响应规律,我们运用高通量的脂类组学方法检测了拟南芥根中膜脂分子的组成,比较了儿茶素处理下拟南芥野生型（WS）及磷脂酶Dδ缺失突变体（PLDδ KO）根中膜脂分子的组成情况、膜脂含量、双键指数及碳链长度值。结果发现,儿茶素处理拟南芥根90min后,二半乳糖基二酰甘油（DGDG）、单半乳糖基二酰甘油（MGDG）、磷脂酰甘油（PG）、磷脂酰胆碱（PC）及磷脂酰肌醇（PI）的总含量在WS与PLDδ KO植株根中都显著下降,磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）在WS中下降,在PLDδ KO中上升。儿茶素处理导致PLDδ KO植株的PC/PE比值显著下降,WS植株PS碳链长度显著增加。上述结果说明儿茶素处理后,磷脂酶Dδ缺失突变体膜不稳定性增加,PLDδ KO植株对儿茶素胁迫更加敏感。     

胆固醇对二棕榈酰磷脂酰胆碱（DPPC）多层膜结构的影响

用小角Ｘ射线衍射（ＳＡＸＤ）和差示扫描量热（ＤＳＣ）方法研究胆固醇对二棕榈酸磷脂酰胆碱多层膜结构的影响。在不同胆因醇浓度及不同温度下的衍射实验和热吸收曲线实验显示纯磷脂的尖锐的凝胶－液晶相变随胆固醇含量增加而逐渐消失。当胆固醇含量约为４０（ｍｏｌ）％或更多时,衍射曲线和热吸收曲线均显示存在分相现象。     

膜脂对菠菜细胞色素b6f复合体电子传递活性的影响

利用从菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）叶绿体分离、纯化出的缺失膜脂的细胞色素ｂ６ｆ蛋白复合体（Ｃｙｔ ｂ６ｆ）制剂与从菠菜类囊体分离、纯化的膜脂进行体外重组,检测了不同膜脂对Ｃｙｔ ｂ６ｆ催化电子传递活性的影响。结果表明：被检测的５种膜脂,即单半乳糖基甘油二酯（ＭＧＤＧ）、双半乳糖基甘油二酯（ＤＧＤＧ）、磷脂酰胆碱（ＰＣ）、磷脂酰甘油（ＰＧ）和硫代异鼠李糖基甘油二酯（ＳＱＤＧ）对Ｃ     

菜豆叶片衰老期间叶绿体被膜膜脂与脂肪酸组分的变化

菜豆叶片叶绿体总脂和被膜膜脂中均含有单半乳糖甘油二脂和双半乳糖甘油二脂,在整个衰老期间两种糖脂的比值变化不大。叶绿体总脂中含有5种磷脂,脂肪酸以不饱和的亚麻酸为主,而被膜膜脂中仅含磷脂酰胆碱和磷脂酰甘油,脂肪酸以饱和的棕榈酸为主,不饱和亚油酸为次。叶片衰老过程中被膜所含两种磷脂比值明显降低,脂肪酸的不饱和指数也因亚麻酸相对含量显著减少、棕榈酸相对含量增加而降低。     

山茛菪碱对磷脂糖服混合脂质体多形性的影响

本文采用冰冻断裂电子显微镜技术研究了山茛菪碱对单葡萄糖甘油二酯与心磷脂或磷脂酰甘油混合脂质体多形性影响。山茛菪碱可促进单葡萄糖甘油二酯与心磷脂或磷脂酰甘油混合脂质体在25℃时形成脂质颗粒,当温度为55℃时山茛菪碱对上述混合脂质体形成脂质颗粒更为明显。     

山茛菪碱对磷脂糖服混合脂质体多形性的影响

本文采用冰冻断裂电子显微镜技术研究了山茛菪碱对单葡萄糖甘油二酯与心磷脂或磷脂酰甘油混合脂质体多形性影响。山茛菪碱可促进单葡萄糖甘油二酯与心磷脂或磷脂酰甘油混合脂质体在25℃时形成脂质颗粒,当温度为55℃时山茛菪碱对上述混合脂质体形成脂质颗粒更为明显。     

嗜热菌玉米黄质在不同酸碱介质中对脂质体膜的稳定性

嗜热菌玉米黄质（thermozeaxanthins,TZS)是从嗜热菌的磷脂提取物中分离得到的胡萝卜素单葡萄糖脂肪酸酯化合物,具有两亲性结构,通过检测包裹在脂质体内的荧光物质的漏出程度,来评价在不同pH缓冲液中TZS对脂质体膜的影响。脂质体由天然卵磷脂（eggPC)、大肠杆菌磷脂酰乙醇胺（E.coliPE)或不同碳链长度和饱和度的二棕榈酰磷脂酰胆碱（DPPC）、二油酰磷脂酰胆碱（DOPC）磷脂形成,结果表明：在pH5.0的缓冲液中,含0.01molTZS的脂质体比单纯由磷脂形成的脂质体要稳定,在pH8.2和9.0的环境中,其稳定作用不明显;由eggPC或E.coliPE与TZS形成的脂质体的稳定性在同样条件下要优于由DPPC或DOPC与TZS形成的脂质体。在pH6.5的缓冲液中,含有TZS的脂质体与对照脂质体的稳定性相近。TZS对脂质体的影响取决于TZS与磷脂双层膜的相互“匹配”,邓与磷脂的脂肪烃链的长度、饱和度有关。     

磷脂酰甘油合成代谢研究

从磷脂酰甘油分子的结构特点和研究方法出发,重点介绍了磷脂酰甘油合成代谢近年来的研究进展,对磷脂酰甘油在光合作用中的作用作了综述,并对磷脂酰甘油的研究问题和前景作了展望。     

ESI-MS MS方法检测拟南芥膜脂分子对机械伤害的响应

利用一种灵敏的、基于ESI-MS􊄯MS ( electrospray ionization tandem mass spectrometry) 的脂类组学方法,测定了机械伤害诱导的拟南芥6 种磷 (phospholipids) 、2 种糖脂(glycolipids) 、3 种溶血磷脂( lysophospholipids)和约120 种脂类分子的变化, 探索了膜脂响应机械伤害的基本趋势。结果表明, 机械伤害后磷脂酸( phosphatidic acid , PA) 和3 种溶血磷脂显著升高, 而叶绿体膜上的糖脂减少; 在测量的1 小时范围内, 不同脂类水解产生的磷脂酸分子的增加速度和强度不同, 反映出它们经历了不同的生化过程。具体表现为:(1 ) 叶绿体膜脂磷脂酰甘油(phosphatidylglycero , PG) 分子34∶4 PG 水解的产物磷脂酸分子34∶4 PA 的积累速度明显慢于其它磷脂酸分子; (2) 磷脂酸分子34∶6 PA 仅有少量的积累, 其可能是由叶绿体膜脂单半乳糖二酰甘油(monogalactosyldiacylglycerol, MGDG) 。分子34∶6 MGDG 和双半乳糖二酰甘油( digalactosyldiacylglycerol, DGDG) 分子34∶6 DGDG 水解产生, 然而这两种糖脂含量明显下降, 说明它们有可能还参与了其它的反应。脂类的摩尔百分组成没有剧烈的变化。     

Two cholesterol pools in Acholeplasma laidlawii membranes

Cholesterol exchange kinetics between [14C]cholesterol-labeled Acholeplasma laidlawii and Mycoplasma gallisepticum cells and phosphatidylcholine-cholesterol vesicles followed a biphasic curve, with faster exchange rates for A. laidlawii. The same biphasic curve was obtained with isolated membranes. Cholesterol exchange between lipid vesicles and A. laidlawii cells depleted of phospholipids by phospholipase A2, fitted a monophasic linear curve. The data support the hypothesis that the biphasic cholesterol exchange kinetics do not result from the transbilayer distribution of cholesterol, but reflect the presence in the membrane of two cholesterol pools associated with lipids of high and low affinity for cholesterol.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Real-time analysis of the effects of cholesterol on lipid raft behavior using atomic force microscopy

Cholesterol plays a crucial role in cell membranes, and has been implicated in the assembly and maintenance of sphingolipid-rich rafts. We have examined the cholesterol-dependence of model rafts (sphingomyelin-rich domains) in supported lipid monolayers and bilayers using atomic force microscopy. Sphingomyelin-rich domains were observed in lipid monolayers in the absence and presence of cholesterol, except at high cholesterol concentrations, when separate domains were suppressed. The effect of manipulating cholesterol levels on the behavior of these sphingomyelin-rich domains in bilayers was observed in real time. Depletion of cholesterol resulted in dissolution of the model lipid rafts, whereas cholesterol addition resulted in an increased size of the sphingomyelin-rich domains and eventually the formation of a single raftlike lipid phase. Cholesterol colocalization with sphingomyelin-rich domains was confirmed using the sterol binding agent filipin.     

A (2)H NMR study of macroscopically aligned bilayer membranes containing interfacial hydroxyl residues

The polar interface of membranes containing phosphatidylglycerol or cholesterol was studied by (2)H nuclear magnetic resonance (NMR) as a function of membrane hydration. The membranes were macroscopically aligned and hydrated with deuterium oxide. Water uptake and membrane annealing was achieved under NMR control, using a novel hydration technique. Well-resolved (2)H quadrupolar doublets were obtained from individual hydroxyl residues and from the interlamellar water. The response of the phosphatidylglycerol headgroup and of the cholesterol molecule to the spontaneous evaporation of interlamellar water could be thus monitored continuously. It is shown that the phosphatidylglycerol headgroup undergoes changes of conformation and average orientation with respect to the membrane surface and that the off-axis motion of the cholesterol molecule decreases. The deuteron exchange between hydroxyl residues and surface-associated D(2)O was determined by an inversion transfer technique. The exchange rates of the hydroxyl residues in the phosphatidylglycerol headgroup were different and depended strongly on the total hydration of the membrane. Significantly lower and almost hydration-independent rates were obtained for cholesterol. These results will be discussed with reference to earlier reports on the headgroup dynamics of phosphatidylglycerol and on the interaction of cholesterol with the membrane-water interface.     

Probes for studying cholesterol binding and cell biology

Cholesterol is a multifunctional lipid in eukaryotic cells. It regulates the physical state of the phospholipid bilayer, is crucially involved in the formation of membrane microdomains, affects the activity of many membrane proteins, and is the precursor for steroid hormones and bile acids. Thus, cholesterol plays a profound role in the physiology and pathophysiology of eukaryotic cells. The cholesterol molecule has achieved evolutionary perfection to fulfill its different functions in membrane organization. Here, we review basic approaches to explore the interaction of cholesterol with proteins, with a particular focus on the high diversity of fluorescent and photoreactive cholesterol probes available today.     

Conformational changes that effect oligomerization and initiate pore formation are triggered throughout perfringolysin O upon binding to cholesterol

Pore formation by the cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target membrane. Cholesterol was long thought to be the cellular receptor for these toxins, but not all CDCs require cholesterol for binding. Intermedilysin, secreted by Streptococcus intermedius, only binds to membranes containing the human protein CD59 but forms pores only if the membrane contains sufficient cholesterol. In contrast, perfringolysin O (PFO), secreted by Clostridium perfringens, only binds to membranes containing substantial amounts of cholesterol. Given that different steps in the assembly of various CDC pores require cholesterol, here we have analyzed to what extent cholesterol molecules, by themselves, can modulate the conformational changes associated with PFO oligomerization and pore formation. PFO binds to cholesterol when dispersed in aqueous solution, and this binding triggers the distant rearrangement of a beta-strand that exposes an oligomerization interface. Moreover, upon binding to cholesterol, PFO forms a prepore complex, unfolds two amphipathic transmembrane beta-hairpins, and positions their nonpolar surfaces so they associate with the hydrophobic cholesterol surface. The interaction of PFO with cholesterol is therefore sufficient to initiate an irreversible sequence of coupled conformational changes that extend throughout the toxin molecule.     

A comparison of the packing behavior of egg phosphatidylcholine with cholesterol and biogenically related sterols in Langmuir monolayer films

Cholesterol and selected derivatives were studied as mixed Langmuir monolayers with egg phosphatidylcholine (PC). As an extension of our earlier work, which employed binary sterol/PC mixtures, here we examined ternary mixed monolayers containing cholesterol along with an alternate sterol and PC in different molar ratios, using pressure-area isotherms. The ternary systems behaved similarly to the binary sterol/PC systems reported previously, with similar condensation noted for the sterol/PC films. To better understand how variations in sterol structure affect sterol packing in such membrane monolayers, binary mixtures containing cholestenone, cholestanol, and lanosterol with PC were also studied. Cholestanol behaved similarly to cholesterol when incorporated with PC, while cholestenone and lanosterol did not cause as much film condensation. The observed differences in molecular packing, and attributed sterol structural differences, are considered within the context of sterol/phospholipid mixtures in biological membranes.     

Single Lipid Extraction: The Anchoring Strength of Cholesterol in Liquid-Ordered and Liquid-Disordered Phases

Cholesterol is important for the formation of microdomains in supported lipid bilayers and is enriched in the liquid-ordered phase. To understand the interactions leading to this enrichment, we developed an AFM-based single-lipid-extraction (SLX) approach that enables us to determine the anchoring strength of cholesterol in the two phases of a phase-separated lipid membrane. As expected, the forces necessary for extracting a single cholesterol molecule from liquid-ordered phases are significantly higher than for extracting it from the liquid-disordered phases. Interestingly, application of the Bell model shows two energy barriers that correlate with the head and full length of the cholesterol molecule. The resulting lifetimes for complete extraction are 90 s and 11 s in the liquid-ordered and liquid-disordered phases, respectively. Molecular dynamics simulations of the very same experiment show similar force profiles and indicate that the stabilization of cholesterol in the liquid-ordered phase is mainly due to nonpolar contacts.     

Single Lipid Extraction: The Anchoring Strength of Cholesterol in Liquid-Ordered and Liquid-Disordered Phases

Cholesterol is important for the formation of microdomains in supported lipid bilayers and is enriched in the liquid-ordered phase. To understand the interactions leading to this enrichment, we developed an AFM-based single-lipid-extraction (SLX) approach that enables us to determine the anchoring strength of cholesterol in the two phases of a phase-separated lipid membrane. As expected, the forces necessary for extracting a single cholesterol molecule from liquid-ordered phases are significantly higher than for extracting it from the liquid-disordered phases. Interestingly, application of the Bell model shows two energy barriers that correlate with the head and full length of the cholesterol molecule. The resulting lifetimes for complete extraction are 90 s and 11 s in the liquid-ordered and liquid-disordered phases, respectively. Molecular dynamics simulations of the very same experiment show similar force profiles and indicate that the stabilization of cholesterol in the liquid-ordered phase is mainly due to nonpolar contacts.     

Ionic channels and nerve membrane constituents. Tetrodotoxin-like interaction of saxitoxin with cholesterol monolayers

Saxitoxin (STX) and tetrodotoxin (TTX) have the same striking property of blocking the Na⁺ channels in the axolemma. Experiments with nerve plasma membrane components of the squid Dosidicus gigas have shown that TTX interacts with cholesterol monolayers. Similar experiments were carried out with STX. The effect of STX on the surface pressure-area diagrams of lipid monolayers and on the fluorescence emission spectra of sonicated nerve membranes was studied. The results indicate a TTX-like interaction of STX with cholesterol monolayers. The expansion of the monolayers caused by 10^-6 M STX was 2.2 A²/cholesterol molecule at 25°C. From surface pressure measurements at constant cholesterol area (39 A²/molecule) in media with various STX concentrations, it was calculated that the STX/cholesterol surface concentration ratio is 0.54. The apparent dissociation constant of the STX-cholesterol monolayer complex is 4.0 x 10^-7 M. The STX/cholesterol ratio and the apparent dissociation constant are similar to those determined for TTX. The presence of other lipids in the monolayers affects the STX-cholesterol association. The interactions of STX and TTX with cholesterol monolayers suggest (a) that cholesterol molecules may be part of the nerve membrane Na⁺ channels, or (b) that the toxin receptor at the nerve membrane shares similar chemical features with the cholesterol monolayers.