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。     

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。     

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.     

Membrane biogenesis

The literary data on the problem of the membrane biogenesis are generalized. The mechanisms of formation, possible ways of metabolism of biomembrane structure in cells and the ways of their degradation are considered. A conclusion has been made on the existence in the cells several types of movement as for the separate components and membrane fragments as well.     

Thermodynamics of the isothermal denaturation of lysozyme by guanidinium chloride.

A thermodynamic analysis of the isothermal denaturation of lysozyme by guanidinium chloride has been performed. The analysis is based on the equation which relates the equilibrium constant for denaturation to the preferential binding of denaturant. The equation has been derived previously by thermodynamic methods, whereas in this article a derivation based on statistical mechanics is given. By application of the equation the free energy of denaturation is first calculated and from it, by subtracting the calorimetrically-determined enthalpy of denaturation, the entropy of denaturation is determined.     

Thermodynamics of the hydrophobic effect. III. Condensation and aggregation of alkanes, alcohols, and alkylamines

Knowledge of the energetics of the low solubility of non-polar compounds in water is critical for the understanding of such phenomena as protein folding and biomembrane formation. Solubility in water can be considered as one leg of the three-part thermodynamic cycle - vaporization from the pure liquid, hydration of the vapor in aqueous solution, and aggregation of the substance back into initial pure form as an immiscible phase. Previous studies on the model compounds n-alkanes, 1-alcohols, and 1-aminoalkanes have noted that the thermodynamic parameters (Gibbs free energy, DeltaG; enthalpy, DeltaH; entropy, DeltaS; and heat capacity, DeltaC(p)) associated with these three processes are generally linear functions of the number of carbons in the alkyl chains. Here we assess the accuracy and limitations of the assumption of additivity of CH(2) group contributions to the thermodynamic parameters for vaporization, hydration, and aggregation. Processes of condensation from pure gas to liquid and aqueous solution to aggregate are compared. Hydroxy, amino, and methyl headgroup contributions are estimated, liquid and solid aggregates are distinguished. Most data in the literature were obtained for compounds with short aliphatic hydrocarbon tails. Here we emphasize long aliphatic chain behavior and include our recent experimental data on long chain alkylamine aggregation in aqueous solution obtained by titration calorimetry and van't Hoff analysis. Contrary to what is observed for short compounds, long aliphatic compound aggregation has a large exothermic enthalpy and negative entropy.     

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.     

Study of potential-dependent changes in the conformation of the beta-carotene molecule using combination scattering spectroscopy

It has been found by Raman spectroscopy that conformational changes of beta-carotene at liposomal membrane are potential-dependent. Changes of K+ or Ca+ gradient on the liposome membrane pressed the polyene chain of beta-carotene. A mechanism of potential-dependent carotenoid conformational changes in the regulation of "antenna" or "potential sensor" in biomembrane was discussed.     

Thermodynamics of phospholipid self-assembly

Negatively charged phospholipids are an important component of biological membranes. The thermodynamic parameters governing self-assembly of anionic phospholipids are deduced here from isothermal titration calorimetry. Heats of demicellization were determined for dioctanoyl phosphatidylglycerol (PG) and phosphatidylserine (PS) at different ionic strengths, and for dioctanoyl phosphatidic acid at different pH values. The large heat capacity (ΔC^o_P ∼ −400 J.mol⁻¹ K⁻¹ for PG and PS), and zero enthalpy at a characteristic temperature near the physiological range (T^∗ ∼ 300 K for PG and PS), demonstrate that the driving force for self-assembly is the hydrophobic effect. The pH and ionic-strength dependences indicate that the principal electrostatic contribution to self-assembly comes from the entropy associated with the electrostatic double layer, in agreement with theoretical predictions. These measurements help define the thermodynamic effects of anionic lipids on biomembrane stability.     

The bioenergetic correlation of COD to BOD

Summary A simple expression has been derived to demonstrate that the ratio of COD to BOD is not constant but a function of the thermodynamic efficiency of the growth process. An analysis of data reported in literature indicates that this ratio can assume a wide range of values for different substrates. The influence of a selected change in the environmental conditions, on the ratio, COD/BOD, has been evaluated experimentally, in a bacterial system.     

Models of active transport of ions in biomembranes of various types of cells

Nonequilibrium statistical models of the active transport of ions in biomembranes have been constructed. Differences of chemical potentials of the ATP-ADP reaction and the electrochemical potential of ions were taken as the thermodynamic forces responsible for the flow of ions through the membrane. The active transport of ions was viewed as a cross phenomenon arising from the chemical reaction of the ATP hydrolysis. These models provide independent calculations of the resting potential at the biomembrane and concentrations of ions in a cell on the assumption the free energy of the ATP-ADP reaction is fully (without the dissipation loss) converted to the free energy of transported ions. They take into account the presence of nonpenetrating ions in a cell. It was shown that different concentrations of nonpenetrating ions have a considerable effect on the resting potential. The proposed models were compared with experimental data obtained for different types of cells including neurons, muscular cells, bacteria, plants, and mitochondria. Calculated values of the membrane potential and ion concentrations were in good qualitative agreement with experimental data.     

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.     

Temperature dependence of thermodynamic properties for DNA/DNA and RNA/DNA duplex formation.

A clear difference in the enthalpy changes derived from spectroscopic and calorimetric measurements has recently been shown. The exact interpretation of this deviation varied from study to study, but it was generally attributed to the non-two-state transition and heat capacity change. Although the temperature-dependent thermodynamics of the duplex formation was often implied, systemic and extensive studies have been lacking in universally assigning the appropriate thermodynamic parameter sets. In the present study, the 24 DNA/DNA and 41 RNA/DNA oligonucleotide duplexes, designed to avoid the formation of hairpin or slipped duplex structures and to limit the base pair length less than 12 bp, were selected to evaluate the heat capacity changes and temperature-dependent thermodynamic properties of duplex formation. Direct comparison reveals that the temperature-independent thermodynamic parameters could provide a reasonable approximation only when the temperature of interest has a small deviation from the mean melting temperature over the experimental range. The heat capacity changes depend on the base composition and sequences and are generally limited in the range of -160 to approximately -40 cal.mol-1.K-1 per base pair. In contrast to the enthalpy and entropy changes, the free energy change and melting temperature are relatively insensitive to the heat capacity change. Finally, the 16 NN-model free energy parameters and one helix initiation at physiological temperature were extracted from the temperature-dependent thermodynamic data of the 41 RNA/DNA hybrids.     

Depletion effect and biomembrane budding

Depletion effects are well known to lead to phase separation in microsystems consisting of large and small particles with short-range repulsive interactions that act over macromolecular length scales. The equilibrium mechanics between an enveloped colloidal particle and a biomembrane caused by entropy is investigated by using a continuum model. We show that the favorable contact energy stems from entropy, which is sufficient to drive engulfment of the colloidal particle, and deformation of the biomembrane determines the resistance to the engulfment of the colloidal particle. The engulfment process depends on the ratio of the radii of the larger particle and smaller particles and the bending rigidity. The results show insights into the effects of depletion on biomembrane budding and nanoparticle transportation by a vesicle.     

Determination of the kinetics of guanine nucleotide exchange on EF-Tu and EF-Ts: continuing uncertainties

An analysis is made of the rate constants for the reactions involving the interactions of EF-Tu, EF-Ts, GDP, and GTP recently derived by Gromadski et al. [Biochemistry 41 (2002) 162]. Though their measured values appear to allow a reasonable rate of nucleotide exchange sufficient to support rates of protein synthesis in vivo, their data underestimate the thermodynamic barrier involved in nucleotide exchange and therefore cannot be considered definitive. A kinetic scheme consistent with the thermodynamic barrier can be achieved by modification of various rate constants, particularly of those involving the release of EF-Ts from EF-Tu.GTP.EF-Ts, but such constants are markedly different from what are experimentally observed. It thus remains impossible at present satisfactorily to model guanine nucleotide exchange on EF-Tu, catalysed by EF-Ts by a double displacement mechanism, with experimentally derived rate constants. Metabolic control analysis has been applied to determine the degree of flux control of the different steps in the pathway.     

The formation of polymeric model biomembranes from diacetylenic fatty acids and phospholipids

Diacetylenic fatty acid monolayers at the air/water interface and multilayers on suitable supports polymerise when exposed to ultraviolet radiation. It has been found that polymerisation still occurs when monolayers are diluted with cholesterol or gramicidin. The rigid, crystalline nature of the films formed makes them useful biomembrane models. Phospholipids made from the fatty acids were less reactive. Multilayers deposited on hydrophobic supports would polymerise but not monolayers on water.     

生物膜研究在生物物理研究所的兴起与发展

生物膜研究是现代生物学研究的前沿方向之一,本文对文革结束以后生物膜研究在中国科学院生物物理研究所(下称生物物理所)的兴起与发展进行了系统回顾.文革结束后,中国科学院领导了解到国外生物膜研究迅速发展的情况,迅即派遣以生物物理所杨福愉为组长,包括生物化学研究所、动物研究所、植物研究所、上海实验生物研究所等六人的代表团前往联邦德国进行考察.考察结束后院领导根据多学科交叉对生物膜研究的重要作用,组织了生物物理所、植物所、中国医学科学院、北京医科大学等单位联合申报国家自然科学基金委员会重大项目"膜脂-膜蛋白的相互作用及其在医学和农业上的应用",并获得立项.与此同时,院领导建议由中国生物物理学会、中国生化学会和中国细胞生物学会共同组织生物膜学术讨论会.首次会议于1979年3月在北京友谊宾馆举行,以后每3年召开1次,从未中断,有力地促进了生物膜研究的交流与发展.2003年举行的第200届香山会议专门组织讨论21世纪生物膜研究在中国的布局,进一步推动了生物膜研究的发展.本文还重点阐述了中国科学院生物物理研究所在生物膜研究方面所取得的代表性成果:a.金属离子通过膜脂-膜蛋白相互作用调控生物膜能量转换、物质运输及信号转导的分子机制;b.提出"克山病是一种心肌线粒体病"的重要观点;c.发现溶酶体内含有为量甚微、一般认为是消化酶的胰凝乳蛋白酶,并阐明了它通过溶酶体膜外泄后参与细胞凋亡的作用机制;d.确定了通过调控线粒体动态变化而干预肿瘤细胞迁移侵袭的新靶标.最后,特别值得一提的是,2004年,常文瑞与植物研究所匡廷云等在《自然》(Nature)发表了《菠菜中主要捕光色素复合体2.72魡分辨率的晶体结构分析》的研究论文,2005年饶子和与徐建兴等在《细胞》(Cell)发表了《猪心线粒体呼吸链复合体Ⅱ的晶体结构》的研究论文,充分标志着我国生物膜研究已在国际上占据一席之地.2004年,徐涛研究员因其在囊泡转运方面的丰厚学术积淀,作为首席科学家组织一批生物膜专家承担了国内首个生物膜973项目,这标志着国内生物膜研究开始进入一个新的发展时期.     

Bicontinuous microemulsions as a biomembrane mimetic system for melittin

Antimicrobial peptides effectively kill antibiotic-resistant bacteria by forming pores in prokaryotes' biomembranes via penetration into the biomembranes' interior. Bicontinuous microemulsions, consisting of interdispersed oil and water nanodomains separated by flexible surfactant monolayers, are potentially valuable for hosting membrane-associated peptides and proteins due to their thermodynamic stability, optical transparency, low viscosity, and high interfacial area. Here, we show that bicontinuous microemulsions formed by negatively-charged surfactants are a robust biomembrane mimetic system for the antimicrobial peptide melittin. When encapsulated in bicontinuous microemulsions formed using three-phase (Winsor-III) systems, melittin's helicity increases greatly due to penetration into the surfactant monolayers, mimicking its behavior in biomembranes. But, the threshold melittin concentration required to achieve these trends is lower for the microemulsions. The extent of penetration was decreased when the interfacial fluidity of the microemulsions was increased. These results suggest the utility of bicontinuous microemulsions for isolation, purification, delivery, and host systems for antimicrobial peptides.     

Relationship between chemiosmotic flows and thermodynamic forces in oxidative phosphorylation

A set of equations has been derived, describing quantitatively the relationships between flows and thermodynamic forces in the chemiosmotic model of oxidative phosphorylation.Experimental tests of these equations give information on the stoichiometric coupling constants between the different flows.     

Difference infrared spectroscopy of aqueous model and biological membranes using an infrared data station

A Perkin-Elmer infrared Data Station associated with a simple IR spectrometer (model 298) is shown to give excellent results with aqueous model and biomembrane systems. Examples are presented of difference spectra obtained with lipid--water systems, reconstituted lipid--protein systems and a natural biomembrane. The spectra of the lipid after water subtraction and of the intrinsic protein after lipid subtraction from a model reconstituted Ca2+-ATPase membrane system are shown. The potential for studying intrinsic protein conformations is emphasised.