Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, and sphingolipid-enriched lipid rafts

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.     

The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: A synchrotron X-ray diffraction study

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20° to 36 °C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 °C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Quantitation of glycogen synthase kinase‐3 sensitive proteins in neuronal membrane rafts

We report a quantitative proteomic study to investigate the changes induced in membrane rafts by the inhibition of glycogen synthase kinase‐3. Sensitive quantitation of membrane raft proteins using isobaric tagging chemistries was enabled by a novel hybrid proteomic method to isolate low‐microgram (10–30 μg) membrane raft protein preparations as unresolved bands in a low‐density acrylamide gel. Samples were in‐gel digested, differentially tagged and combined for 2‐D LC and quantitative MS. Analysis of hippocampal membrane preparations using this approach resulted in a sixfold increase in sensitivity and a threefold increase in the number of quantifiable proteins compared with parallel processing using a traditional in‐solution method. Quantitative analysis of membrane raft preparations from a human neuronal cell line treated with glycogen synthase kinase‐3 inhibitors SB415286 or lithium chloride, that have been reported to modulate processing of the Alzheimer amyloid precursor protein, identified several protein changes. These included decreases in lamin B1 and lamin B receptor, as well as increases in several endosome regulating rab proteins, rab5, rab7 and rab11 that have been implicated in processing of the amyloid precursor protein in Alzheimer's disease.     

MBR运行初期对基因工程菌的截留特性研究

在膜-生物反应器(MBR)中实施基因工程菌生物强化时,运行初期基因工程菌流失是生态风险评价的重要内容.在一体式微滤膜-生物反应器中,考察了运行初期不同运行条件对基因工程菌流失密度和截留效率的影响,并对截留特性进行了探讨.结果表明,膜-生物反应器运行初期,不同运行条件对基因工程菌的截留效率影响不同:污泥浓度增加,截留效率提高;提高膜通量和曝气量,截留效率降低.基因工程菌接种密度为1.0×1010CFU/mL时,不同运行条件下的流失密度为1.0×102 CFU/mL～2.5×102 CFU/mL,最大截留效率大于8 lg.膜-生物反应器运行初期,膜组件截留、污泥吸附以及对悬浮细胞迁移阻碍是影响截留效率的主要因素,一定条件下其截留效率贡献率分别为82.3%、14.9%和2.8%.膜-生物反应器稳定运行过程中形成凝胶层,可以提高截留效率.一定条件下,膜组件、污泥和凝胶层对基因工程菌的截留贡献率分别为75.3%、10.7%和14.0%.     

MBR运行初期对基因工程菌的截留特性研究

在膜-生物反应器(MBR)中实施基因工程菌生物强化时, 运行初期基因工程菌流失是生态风险评价的重要内容。在一体式微滤膜-生物反应器中, 考察了运行初期不同运行条件对基因工程菌流失密度和截留效率的影响, 并对截留特性进行了探讨。结果表明, 膜-生物反应器运行初期, 不同运行条件对基因工程菌的截留效率影响不同：污泥浓度增加, 截留效率提高; 提高膜通量和曝气量, 截留效率降低。基因工程菌接种密度为1.0×1010 CFU/mL时, 不同运行条件下的流失密度为1.0×102 CFU/mL~2.5×102 CFU/mL, 最大截留效率大于8 lg。膜-生物反应器运行初期, 膜组件截留、污泥吸附以及对悬浮细胞迁移阻碍是影响截留效率的主要因素, 一定条件下其截留效率贡献率分别为82.3%、14.9%和2.8%。膜-生物反应器稳定运行过程中形成凝胶层, 可以提高截留效率。一定条件下, 膜组件、污泥和凝胶层对基因工程菌的截留贡献率分别为75.3%、10.7%和14.0%。     

Enzyme markers and isolation of the microvillar and perimicrovillar membranes of Dysdercus peruvianus (hemiptera: pyrrhocoridae) midgut cells

The midgut of Dysdercus peruvianus is divided into four sections (V₁-V₄). All the cells have microvilli ensheathed by a lipoprotein membrane (perimicrovillar membrane) extending toward the lumen as narrow tubes with dead ends. Subcellular fractionation of V₁ and V₂ tissue in isotonic and hypotonic conditions showed that -glucosidase is associated with membranous structures larger than those associated with β-glucosidase. The /β-glucosidase activity ratio is 34 ± 4 in V₁ tissue and 170 ± 10 in membranes recovered from the V₁ luminal contents. These membranes are resolved in sucrose gradients into low density (1.087 ± 0.001 g/cm³) -glucosidase-carrying membranes (/β-glucosidase activity ratio of 330±30) and high density (1.132 ± 0.002g/cm³) β-glucosidase-carrying-membranes. Low-density membranes have 1090 ± 60 μg lipid/mg protein and apparently are not contaminated by high-density ones (electron micrographs). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that membranes recovered from V₁ luminal contents are composed mainly of a-glucosidase-rich membranes. The data suggest that -glucosidase-rich membranes are perimicrovillar membranes which may be partly lost into luminal contents on dissection, with densities and lipid/protein ratios similar to that of myelin sheaths, in accordance with previous freeze-fracture data. β-Glucosidase-rich membranes are probably microvillar membranes with densities increased by the presence of associated portasomes.     

Binding protein for Escherichia coli heat-stable enterotoxin II in mouse intestinal membrane

Abstract The protein binding Escherichia coli heat-stable enterotoxin II (STII) was isolated from cell membranes of mouse intestine. The binding of ¹²⁵I-labeled STII to the proteins was inhibited by unlabeled STII, showing that it is specific. Proteins cross-linked with ¹²⁵I-STII were purified by column chromatography on hydroxyapatite and TSK gel. Analyses of the purified protein by SDS-polyacrylamide gel electrophorosis and gel filtration showed that the molecular mass was 25 kDa.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

Time-dependent elastic extensional RBC deformation by micropipette aspiration: redistribution of the spectrin network?

The time dependence of small elastic extensional RBC deformation by micropipette aspiration has been analyzed. This process shows two-phases which are characterized by time constants of the order of some tenths of seconds and about ten seconds, respectively. The equilibrium tongue length is reached after about 30 s. For the first, fast step we assume that the membrane model of immobilized boundaries holds, i.e., the skeleton is tightly associated with the lipid bilayer and no redistribution of the skeleton with respect to the lipid bilayer is allowed. This lipid-spectrin interaction or anchorage is characterized by some association force density. It has been shown that at a given tongue length the force generated owing to the membrane deformation and acting to redistribute the spectrin, overcomes (in some membrane area) the association force density and results in an additional increase of the sucked membrane length. Equations have been derived to describe this process. From the experimental conditions of an RBC aspiration and the determined tongue length corresponding to the second slow aspiration step, the association force density between the lipid bilayer and the spectrin network may be determined. From literature data and our own results a force density of between 40 and 50 Pa has been estimated. Offprint requests to: D. Lerche     

Differential effect of triiodothyronine and thyroxine on the liposomal membrane in liquid-crystalline and gel state

The effect of thyroid hormones on the degree of order or fluidity of dimyristoyl, dipalmitoyl or egg yolk phosphatidyl choline liposomes was evaluated by fluorescence spectroscopy methods. The freedom of molecular motion above the phase transition temperature was decreased, while below the transition, the mobility was actually increased by the incorporation of triiodothyronine to liposomes. While thyroxine decreases the fluidity in the liquid crystalline state, it cannot increase the fluidity in the gel state.A differential effect of triiodothyronine and thyroxine on the release of the liposomal content was found, depending on the liquid crystalline or gel state of the liposomes. These facts were correlated with the differential incorporation of the hormones to liposomes above and below the phase transition temperature of dimyristoyl and dipalmitoyl phospholipid choline. In gel state, a low incorporation of thyroxine compared with triiodothyronine was found.This work was supported by Grants PID 3-013800/89 from Consejo National de Investigaciones Científicas y Técnicas (CONICET), Fundación Antorchas A-12576/1-000065 and Consejo de Investigaciones de la Universidad National de Tucumán (CIUNT). We thank Dr. G. Rotillo for the space filling models.     

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.     

Munc18-1 Protein Molecules Move between Membrane Molecular Depots Distinct from Vesicle Docking Sites

Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.     

Novel, rapid purification of the membrane protein photosystem I by high-performance liquid chromatography on porous materials

New porous materials have been tested for their potential to speed up purification of membrane proteins. As an example the purification of photosystem I, a light-driven electron pump from the cyanobacterium Synechocystis PCC6803, was optimized. The combination of two HPLC steps (an anion-exchange chromatography followed by a hydrophobic interaction chromatography) yields homogeneous monomeric or trimeric photosystem I as determined by gel filtration and gel electrophoresis. In comparison to traditional purification schemes our method is at least three-times faster and allows for easy scale-up.     

Allosteric regulation of GRASP protein-dependent Golgi membrane tethering by mitotic phosphorylation

Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation.     

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrane and of the whole platelet components established in this system.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

低聚木糖分离纯化的研究进展

综述了低聚木糖分离纯化的研究进展。低聚木糖是一种非消化性寡糖 ,能选择性增殖肠道内双歧杆菌 ,可广泛应用于食品工业和饲料工业。低聚木糖的分离纯化技术主要包括层析分离技术 (包括凝胶过滤层析、离子交换层析和吸附层析 )和膜分离技术 (包括超滤、纳滤和反渗透 )。低聚木糖的提纯主要采用膜分离技术和层析分离技术 ,低聚木糖单一组分的分离主要采用凝胶过滤层析和吸附层析     

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by ¹²⁵I/lactoperoxidase or ³H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.