脂质体重组和脂蛋白体在植物生物膜研究中的应用

脂质体是磷脂在一定条件下在水中形成的由脂质双分子层组成的内部为水相的闭合囊泡。在推动生物膜的研究进展中,它作为模式系统起着非常重要的作用,能用于研究膜蛋白的性质和功能;膜脂和膜蛋白的相互关系;膜的电化学性质等。近年来脂质体重组技术开始引入到植物学研究领域,用于对植物膜蛋白的研究。本文简要介绍了脂质体的制备和脂酶体重组的方法及其在植物生物膜研究中的应用。     

膜脂—膜蛋白的相互作用(上)

生物膜是由蛋白质,脂质以及碳水化合物等组成的超分子体系。一般认为,膜脂是膜的基本骨架,膜蛋白是膜功能的主要体现者。因此,二者的相互作用问题的探讨可以说是生物膜结构与功能研究的一个中心环节。本文先对生物膜的主要组分:膜脂和膜蛋白的概况以及生物膜结构的主要特征——流动性作一扼要介绍,然后就膜脂对膜蛋白,膜蛋白对膜脂的影响分别进行讨论,最后就二者相互作用的研究与医、农方面的联系作些介绍。一、膜脂和膜蛋白的概述 1.膜脂 (1)膜脂的组成据估计生物膜约含100种脂质。膜是一个非常复杂的体系,膜脂组成发生1—2％的变化(例如,胆固醇/磷脂的比值的改变),就足以影响细胞的存活。     

膜脂—膜蛋白的相互作用(上)

生物膜是由蛋白质,脂质以及碳水化合物等组成的超分子体系。一般认为,膜脂是膜的基本骨架,膜蛋白是膜功能的主要体现者。因此,二者的相互作用问题的探讨可以说是生物膜结构与功能研究的一个中心环节。本文先对生物膜的主要组分:膜脂和膜蛋白的概况以及生物膜结构的主要特征——流动性作一扼要介绍,然后就膜脂对膜蛋白,膜蛋白对膜脂的影响分别进行讨论,最后就二者相互作用的研究与医、农方面的联系作些介绍。     

膜脂——膜蛋白相互作用及其在医学和农业上的应用

生物膜系由蛋白质、脂质及碳水化合物等组成的复杂的超分子体系,它是各种生命现象的基础。生物膜研究是当前分子生物学和细胞生物学研究中的重大课题之一。阐明生物膜的分子结构和功能是膜研究的基础。一般认为,膜脂是膜的基本骨架,膜蛋白是膜功能的主要体现者,脂质的运动对膜蛋白     

钙库调控的钙通道Orai1体外研究方法的建立

目的:建立钙通道Orai1的体外研究方法。方法:利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果:成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论:利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。     

生物膜结构研究的一些进展

膜蛋白三维结构的解析存在很多困难.最近几年由于一些通道(如K⁺通道,Cl^-通道,水通道Aquaporin 1等)和泵（如Ca^2＋泵）的结晶获得成功,这些膜蛋白具有原子分辨率三维结构的解析才得以完成,从而基本阐明一些极性分子和离子选择性通过生物膜的分子机理.在膜脂结构方面,动物细胞质膜膜脂的分布是不均匀的.近年来已多方面证明,质膜具有一些被命名为“脂筏（lipid rafts）”和“质膜微囊（Caveolae）”的微区.它们富含鞘脂和胆固醇。简单介绍了这些脂质微区的大小、组分以及动态变化.根据研究结果,这类脂质微区含有大量信号分子,很可能具有信号传递中心的作用.此外,对脂筏在膜运送过程中的作用也进行一些评述.     

生物膜脂及脂肪酸的分析方法评述

膜脂是生物膜的基本骨架,膜蛋白是膜功能的主要体现者。生物膜脂研究是生物膜研究的一个重要组成部分。结合近几年来的新进展,从生物膜脂的分离和脂肪酸的分析角度出发,综合评述了应用于脂类物质分析的TLC、GC、HPLC、CE为代表的主要分析方法,以及CE-MS、HPLC-MS在脂类物质分析中的应用前景。     

介绍一套从水—空气界面的脂单层制备水相中平面脂双层的新装置

本文介绍了一种从水—空气界面的脂单层形成平面双分子脂膜的新装置。它可以控制膜两侧溶液的不同成分;脂双层两边的脂单层具有不同成分;还可使脂双层膜中少带或不带碳氢溶剂,因而其电容性质与生物膜更为接近。我们用它测定了不对称双分子层脂膜的电特性,进行了膜上离子通道性质及脂质体与BLM的融合等的研究。     

金属硫蛋白抗内皮素损伤膜蛋白的电子顺磁共振自旋标记研究

以大鼠红细胞膜为研究对象,以脂肪酸自旋标记物５ＮＳ,１６ＮＳ插入膜脂,蛋白自旋标记物ＭＳＬ标记膜蛋白,观察内皮素（ＥＴ）损伤后膜脂流动性、膜蛋白构象的变化及金属硫蛋白（ＭＴ）抗ＥＴ保护生物膜作用。结果表明ＥＴ（１×１０－９,１０－８及１０－７ｍｏｌ／Ｌ）对膜脂流动性无明显影响,但在５×１０－９及１×１０－７ｍｏｌ／Ｌ浓度下均改变膜蛋白构象,而且对膜蛋白构象的影响呈剂量依赖性。１×１０－５ｍｏｌ／ＬＭＴ能抵抗５×１０－９ｍｏｌ／ＬＥＴ对膜蛋白损伤,保护生物膜。     

固体载体支承的双层膜系统

固态载体支承的双层膜的各种制备过程都简便易行,较脂质体等系统有重复性好、其物化性质可严格控制等优越性,并可将膜蛋白质分子镶嵌其中,是研究生物膜的良好模型．由于其研究方法日益成熟,固态载体支承的双层膜系统越来越成为研究生物膜与膜蛋白的有利工具之一．对固态载体支承的双层膜的制备技术和研究方法进行了系统的综述,并列举了一些在膜生物物理化学领域的应用．     

Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent]

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes.     

Theoretical mimicry of biomembranes

The study of membrane proteins requires a proper consideration of the specific environment provided by the biomembrane. The compositional complexity of this environment poses great challenges to all experimental and theoretical approaches. In this article a rather simple theoretical concept is discussed for its ability to mimic the biomembrane. The biomembrane is approximated by three mimicry solvents forming individual continuum layers of characteristic physical properties. Several specific structural problems are studied with a focus on the biological significance of such an approach. Our results support the general perception that the biomembrane is crucial for correct positioning and embedding of its constituents. The described model provides a semi-quantitative tool of potential interest to many problems in structural membrane biology.     

Synthetic DNA Fragments as Useful Tools in Genetic and Protein Engineering

Within the last decade, nanoscale lipid bilayers have emerged as powerful experimental systems in the analysis of membrane proteins (MPs) for both basic and applied research. These discoidal lipid lamellae are stabilized by annuli of specially engineered amphipathic polypeptides (nanodiscs) or polymers (SMALPs/Lipodisqs®). As biomembrane mimetics, they are well suited for the reconstitution of MPs within a controlled lipid environment. Moreover, because they are water-soluble, they are amenable to solution-based biochemical and biophysical experimentation. Hence, due to their solubility, size, stability, and monodispersity, nanoscale lipid bilayers offer technical advantages over more traditional MP analytic approaches such as detergent solubilization and reconstitution into lipid vesicles. In this article, we review some of the most recent advances in the synthesis of polypeptide- and polymer-bound nanoscale lipid bilayers and their application in the study of MP structure and function.     

Selective Reconstitution of the Tonoplast H+-ATPase of the Crassulacean-Acid Metabolism Plant Kalanchoë daigremontiana

IA detergent removal technique was used to reconstitute solubilized tonoplast proteins of mesophyll cells of the CAM plant Kalanchoë daigremontiana into phosphatidylcholine liposomes. The proteoliposomes were able to hydrolyse ATP and to pump protons across the vesicle membrane. Both activities were inhibited by nitrate, an inhibitor of V-type ATPases. Freeze-fracture micrographs confirmed the incorporation of membrane proteins into liposomes. Increase of specific ATP-hydrolysis activity compared to solubilized tonoplast proteins and SDS-PAGE analysis of reconstituted proteins in comparison with the polypeptide pattern of the purified tonoplast H⁺-ATPase from the same plant source indicated a highly selective reconstitution of the tonoplast H⁺-ATPase.     

Review article. Proteoliposomes and plant transport proteins

Artifical membrane systems are being used increasingly to study the function and role of plant membrane proteins, particularly solute transporters which catalyse counter-exchange of metabolites. Performing such studies requires (i) solubilization of the protein with a suitable surfactant, (ii) functional reconstitution of the protein in a well characterized liposome system. Much of the technology has been derived from studies on non-plant systems and there are many pitfalls of which to be wary before applying it to a new protein. This short review outlines the key parameters which should be considered when attempting the study of plant membrane proteins in artificial lipid bilayers, including types of surfactants, lipid composition of vesicles, membrane permeability, and protein orientation.     

Microsystem for the single molecule analysis of membrane transport proteins

Micro-chamber arrays enable highly sensitive and quantitative bioassays at the single-molecule level. Accordingly, they are widely used for ultra-sensitive biomedical applications, e.g., digital PCR and digital ELISA. However, the versatility of micro-chambers is generally limited to reactions in aqueous solutions, although various functions of membrane proteins are extremely important. To address this issue, microsystems using arrayed micro-sized chambers sealed with lipid bilayers, referred to here as a “biomembrane microsystems”, have been developed by many research groups for the analysis of membrane proteins. In this review, I would like to introduce recent progress on the single molecule analysis of membrane transport proteins using a biomembrane microsystem, and discuss the future prospects for its use in analytical and pharmacological applications.     

Functional reconstitution of prokaryote and eukaryote membrane proteins

A new strategy for the functional reconstitution of membrane proteins is described. This approach introduces a new class of protein stabilizing agents--osmolytes--whose presence at high concentration (10-20%) during detergent solubilization prevents the inactivations that normally occur when proteins are extracted from natural membranes. Osmolytes that act in this way include compounds such as glycerol and higher polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and certain amino acids (glycine, proline, betaine). The beneficial effects of osmolytes are documented by reconstitution of a variety of prokaryote and eukaryote membrane proteins, including several proton- and calcium-motive ATPases, cation- and anion-linked solute carriers (symport and antiport), and a membrane-bound hydrolase from endoplasmic reticulum. In all cases, the presence of 20% glycerol or other osmolyte during detergent solubilization led to 10-fold or more increased specific activity in proteoliposomes. These positive effects did not depend on use of any specific detergent for protein solubilization, nor on any particular method of reconstitution, but for convenience most of the work reported here has used octylglucoside as the solubilizing agent, followed by detergent-dilution to form proteoliposomes. The overall approach outlined by these experiments is simple and flexible. It is now feasible to use reconstitution as an analytical tool to study the biochemical and physiological properties of membrane proteins.     

Lateral Microheterogeneity of Diphenylhexatriene-Labeled Choline Phospholipids in the Erythrocyte Ghost Membrane as Determined by Time-Resolved Fluorescence Spectroscopy

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane. Received: 22 July 1999/Revised: 6 January 2000     

Chromatography on cells and biomolecular assemblies

Red cells, biomembrane vesicles, proteoliposomes and liposomes non-covalently immobilized in gel particles or beads have been used as stationary phases for biomembrane affinity analyses and ion-exchange chromatographic separation. Lipid monolayers coupled to silica beads have been utilized for membrane protein purification in detergent solution and plant cell walls for group separation of macromolecules according to size and charge. Further methodological studies are essential to implement general practical application.