On the role of anisotropy of membrane constituents in formation of a membrane neck during budding of a multicomponent membrane

The expression for the isotropic membrane bending energy was generalized for the case of a multicomponent membrane where the membrane constituents (single molecules or small complexes of molecules-membrane inclusions) were assumed to be anisotropic. Using this generalized expression for the membrane energy it was shown that the change of intrinsic shape of membrane components may induce first-order-like shape transitions leading to the formation of a membrane neck. The predicted discontinuous membrane shape transition and the concomitant lateral segregation of membrane components were applied to study membrane budding. Based on the results presented we conclude that the budding process might be driven by accumulation of anisotropic membrane components in the necks connecting the bud and the parent membrane, and by accumulation of isotropic (conical) membrane components on the bud. Both processes may strongly depend on the intrinsic shape of membrane components and on the direct interactions between them.     

紫膜LB膜的结构特性研究

研究了紫膜LB膜中的紫膜碎片的结构特性。扫描电子显微镜观察表明,紫膜LB膜中单个紫膜碎片的直径大约为0.3微米。表面轮廓测量仪(简称台阶仪)观察到紫膜LB膜中的紫膜碎片的厚度为40—50。在不同的表面压和不同紫膜含量时测量了紫膜碎片在紫膜LB膜中的形态学分布,当表面压为30mN/m或紫膜与大豆磷脂的重量比大于20:1时,紫膜碎片容易重叠或凝聚。     

Stress sensitive glucose oxidase-nylon membrane

Summary Glucose oxidase (GOD) was immobilized on a nylon membrane. No activity of the GOD-nylon membrane was observed under normal conditions but it appeared when the membrane was mechanically stretched. A linear relationship was observed between the stress strength and the GOD activity of the membrane. The appearance of the GOD activity of the membrane with stress was reproducible and the membrane could be stored for at least 2 months. Therefore, the GOD-nylon membrane can be called a stress sensitive membrane.     

膜生物反应器的研究进展

膜生物反应器是近年来发展的废水处理新技术,具有活性污泥浓度高、污泥龄长、占地面积小、投资省的特点。利用膜生物反应器进行污水处理不仅可以大大节约水资源,还可以大大节约能源,节省设备和运行费用,已成为二十一世纪研究热点。膜生物反应器是通过高效膜分离技术与活性污泥相结合,增大污泥中的特效菌来加快生化反应速率,提高废水处理效果。目前处理对象已从生活污水扩展到高浓度的有机废水和难降解的工业废水。本文综述了膜生物反应器在废水中的应用研究情况,并分析比较了各种膜材质的特点、适用范围以及膜的污染因素和清洗方法,展望了膜生物反应器的应用前景及进一步研究方向。     

Membrane topology of transmembrane proteins: determinants and experimental tools

Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.     

盐胁迫对玉米叶片叶肉细胞生物膜超微结构的影响

研究了NaCl胁迫对玉米叶肉细胞生物膜超微结构的影响. 结果表明：NaCl胁迫破坏了玉米叶片叶肉细胞生物膜的正常结构,50 mmol·L^-1 NaCl处理胁迫下,玉米叶肉细胞核膜,线粒体膜,细胞膜,叶绿体膜,液泡膜都受到不同程度的破坏,叶绿体基粒类囊体膨胀,间质片层空间增大,片层紊乱。100 mmol·L^-1 NaCl处理胁迫下,质膜,液泡膜,线粒体,叶绿体都受到严重的破坏。细胞质膜破坏,破损的叶绿体充斥在细胞间隙中;叶绿体外膜破坏,甚至解体消失,叶肉细胞中充满膜结构,基粒排列方向改变,垛叠层数减少,基粒和基质片层界限模糊不清,有的基粒解体消失,甚至叶绿体完全解体;核膜破坏、解体,核中的染色质高度凝缩;线粒体的数量增多,线粒体膜破坏,脊的数量减少,甚至整个线粒体破损解体;液泡膜破坏;由于各种生物膜的破坏,使细胞内充满许多囊状小泡、多泡体或斑层小体;叶肉细胞发生严重的质壁分离,严重时发生细胞壁断裂;甚至整个细胞溶解。     

Localization of proteolytic activity in the outer membrane of Escherichia coli.

An enzyme in the cytoplasmic membrane, nitrate reductase, can be solubilized by heating membranes to 60 degrees C for 10 min at alkaline pH. A protease in the cell envelope has been shown to be responsible for this solubilization. The localization of this protease in the outer membrane was demonstrated by separating the outer membrane from the cytoplasmic membrane, adding back various forms of outer membrane protein to the cytoplasmic membrane, and following the increase in nitrate reductase solubilization with increasing amounts of outer membrane proteins. This solubilization is accompanied by the cleavage of one of the subunits of nitrate reductase and is inhibited by the protease inhibitor p-aminobenzamidine. Analysis of membrane proteins synthesized by cells grown in the presence of various amounts of p-aminobenzamidine revealed that p-aminobenzamidine affects the synthesis of the major outer membrane proteins but has little effect on the synthesis of cytoplasmic membrane proteins. When outer membrane is reacted with the protease inhibitor [3H]diisopropylfluorophosphate, a single protein in the outer membrane is labeled. Since the interaction with diisopropylfluorophosphate is inhibited by p-aminobenzamidine, it is suggested that this single outer membrane protein is responsible for the in vitro solubilization of nitrate reductase and the in vivo processing of the major outer membrane proteins.     

Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains

A rat liver plasma membrane preparation was isolated and characterized both biochemically and morphologically. The isolation procedure was rapid, simple and effective in producing a membrane fraction with the following biochemical characteristics: approximately 40-fold enrichment in three plasma membrane markers, 5'-nucleotidase, alkaline phosphodiesterase I (both putative bile canalicular membrane enzymes), and the asialo-glycoprotein (ASGP) receptor (a membrane glycoprotein present along the sinusoidal front of hepatocytes); a yield of each of these plasma membrane markers that averaged approximately 16%; and minimal contamination by lysosomes, nuclei, and mitochondria, but persistent contamination by elements of the endoplasmic reticulum. Morphological analysis of the preparation revealed that all three major domains of the hepatocyte plasma membrane (sinusoidal, lateral, and bile canalicular) were present in substantial amounts. The identification of sinusoidal membrane was further confirmed when ASGP binding sites were localized predominantly to this membrane in the isolated PM using electron microscope autoradiography. By morphometry, the sinusoidal front membrane accounted for 47% of the total membrane in the preparation, whereas the lateral surface and bile canalicular membrane accounted for 6.8% and 23% respectively. This is the first report of such a large fraction of sinusoidal membrane in a liver plasma membrane preparation.     

Heat modifiability and detergent solubility of outer membrane proteins of Rhodopseudomonas sphaeroides

The outer membrane fraction from Rhodopseudomonas sphaeroides was isolated by isopycnic density centrifugation. The purity of this fraction was assayed by several methods. When the outer membrane fraction obtained after French press lysis of cells was compared with the outer membrane fragments released during spheroplast formation, the polypeptide profiles were identical. Detergent solubilization of membrane fractions showed that Triton X-100 nonselectively solubilizes both the cytoplasmic membrane and the outer membrane, whereas Deriphat 160 selectively solubilizes the cytoplasmic membrane. Several outer membrane polypeptides, including the major outer membrane protein, exhibited changes in electrophoretic mobility that depended upon the temperature of solubilization in sodium dodecyl sulfate. Solubilization at room temperature in the presence of ions reproduced the effect of thermal denaturation on the major outer membrane polypeptide.     

New membrane assembly in IgE receptor-mediated exocytosis

Summary The presence of excess membrane has been observed in the secretory granules of mast cells activated via the physiological mechanism of IgE receptor-mediated exocytosis. This excess membrane is the result of ade novo assembly from phospholipid, cholesterol, and other membrane components stored in the quiescent granule. Following receptor stimulation, membrane bilayer structures of varying size and shape can be seen in the subperigranular membrane space where the perigranular membrane has lifted away from the granule matrix. Vesicles as small as 25 nm in outer diameter are frequently found beneath the perigranular membrane at the site of granule fusion. Membrane in the form of elongated vesicles, tubes, or sheets has also been observed. The wide variation in size and shape of the newly assembled membrane may reflect the spontaneity of the entropy-driven membrane generation process and the fluid characteristic of the biological membrane in general. Fusion of the newly assembled membrane with the perigranular membrane enables the activated granule to enlarge. This rapid expansion process of the perigranular membrane may be the principal mechanism by which an activated granule can achieve contact with the plasma membrane in order to generate pore formation. The fact that new membrane assembly also occurs in the IgE receptor-mediated granule exocytosis, supports our observation thatde novo membrane generation is an inherent step in the mechanism of mast cell granule exocytosis. Whether new membrane assembly is a common step in the mechanism of secretory granule exocytosis in general, must await careful reinvestigation of other secretory systems.     

表位标签技术在膜蛋白研究中的应用

膜蛋白的研究包括埘膜蛋白在细胞内的运输和定位,膜蛋白的结构和功能,以及膜蛋白和其他蛋白质间的相互作用等方面的研究.在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的表位标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有表位标签的重组膜蛋白,不仅具有原有膜蛋白的功能活性,还能够被抗体特异性识别,并且结合相关的免疫荧光检测技术,将会极大地促进膜蛋白的结构和功能研究.本文就目前膜蛋白研究中所涉及的表位标签技术及其应用策略和所取得的进展作一简述.     

Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion complexes can result in membrane lysis. The convergence of these findings showing membrane integrity loss during biological membrane fusion suggests new mechanistic models for membrane fusion and the role of membrane fusion complexes.     

A comparison of receptive and non-receptive plasma membrane areas of photoreceptor cells in the leech,Hirudo medicinalis

Summary Microvillar (receptive) and external (non-receptive) portions of the plasmalemma of photoreceptor cells of Hirudo were compared electron microscopically in thin sections and freeze-fracture replicas. A morphometric approximation showed that the surface area of the microvillar membrane is about 19 times larger than that of the external membrane. The microvillar membrane most probably undergoes extensive membrane turnover. In both segments of the membrane the particles associated with the P- and the E-fracture faces are randomly distributed except at some specific sites. The particles adhere predominantly to the P-faces. The particle densities on the fracture faces of the microvillar membrane differ from those of the external membrane. The P-face particles of the external membrane appear to be larger than those of the microvillar membrane. It is suggested that the P-face particles of the microvillar membrane represent sites where the photopigment is incorporated into the membrane. The distinguishing structural features correspond to the functional differences postulated for both portions of the plasma membrane.     

The interaction of laminin and its membrane receptor on mouse macrophage membrane studied by STM and FRAP

The variation of membrane surface and lateral diffusion of membrane protein was studied after the interaction of laminin with its membrane receptor in mouse macrophages. A pattern of membrane surface which showed smaller and bigger peaks was obtained by scanning tunneling microscope(STM), looking like the domains of lipid groups and proteins in the model of fluid mosaic biomembrane. Some even more higher and wider peaks projected out from the membrane surface in STM image after the interacting of laminin with membrane receptor were, probably, the complexes of laminin and membrane receptor. Furthermore, the decreased lateral diffusion coefficient value (D) was obtained by fluorescence recovery after photobleaching (FRAP) after the laminin was reacted with membrane receptor. This phenomenon provides an evidence that the complexes of laminin and its membrane receptor were located on the membrane of macrophages. So we could consider that the laminin is combined with membrane receptor leading to the variation in the properties of membrane surface.     

Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in Ras

Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction.     

Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this change on plasma membrane properties were examined. The agents that were used were ionophore A23187 and dibucaine. Both agents activated calpain (the Ca2(+)-dependent protease), resulting in the hydrolysis of actin-binding protein and decreased association of actin with membrane glycoproteins. Disruption of actin-membrane interactions was accompanied by the shedding of procoagulant-rich microvesicles from the plasma membrane. The shedding of microvesicles correlated with the hydrolysis of actin-binding protein and the disruption of actin-membrane interactions. When the calpain-induced disruption of actin-membrane interactions was inhibited, the shedding of microvesicles was inhibited. These data are consistent with the hypothesis that association of the membrane skeleton with the plasma membrane maintains the integrity of the plasma membrane, preventing the shedding of procoagulant-rich microvesicles from the membrane of unstimulated platelets. They raise the possibility that the procoagulant-rich microvesicles that are released under a variety of physiological and pathological conditions may result from the dissociation of the platelet membrane skeleton from its membrane attachment sites.     

Comparison of membrane electroporation and protein denature in response to pulsed electric field with different durations

In this paper, we compared the minimum potential differences in the electroporation of membrane lipid bilayers and the denaturation of membrane proteins in response to an intensive pulsed electric field with various pulse durations. Single skeletal muscle fibers were exposed to a pulsed external electric field. The field‐induced changes in the membrane integrity (leakage current) and the Na channel currents were monitored to identify the minimum electric field needed to damage the membrane lipid bilayer and the membrane proteins, respectively. We found that in response to a relatively long pulsed electric shock (longer than the membrane intrinsic time constant), a lower membrane potential was needed to electroporate the cell membrane than for denaturing the membrane proteins, while for a short pulse a higher membrane potential was needed. In other words, phospholipid bilayers are more sensitive to the electric field than the membrane proteins for a long pulsed shock, while for a short pulse the proteins become more vulnerable. We can predict that for a short or ultrashort pulsed electric shock, the minimum membrane potential required to start to denature the protein functions in the cell plasma membrane is lower than that which starts to reduce the membrane integrity. Bioelectromagnetics 34:253–263, 2013. © 2012 Wiley Periodicals, Inc.     

Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions

Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains.     

Integrin-mediated membrane blebbing is dependent on sodium-proton exchanger 1 and sodium-calcium exchanger 1 activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.     

A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification

Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.