龙须菜叶绿素-蛋白复合物的分离及鉴定

以海洋经济海藻--龙须菜(Gracilarialemaneiformis)为材料,机械破碎与超声波相结合破碎这种含胶量非常多的细胞,蔗糖密度梯度超速离心纯化其类囊体膜,去垢剂Triton X-100增溶纯化的类囊体膜,再用蔗糖密度梯度超速离心方法分离其叶绿素-蛋白质复合物.P700差示光谱鉴定分离的光系统Ⅰ(PSⅠ)颗粒,并且检测到了具有DCIP光还原活性的光系统Ⅱ(PSⅡ)颗粒.结果表明,尽管海藻类囊体膜增溶困难,但只要条件合适,可以得到具有活性的光系统颗粒.     

龙须菜叶绿素- 蛋白复合物的分离及鉴定

以海洋经济海藻——龙须菜（Gracilaria lemaneiformis）为材料,机械破碎与超声波相结合破碎这种含胶量非常多的细胞,蔗糖密度梯度超速离心纯化其类囊体膜,去垢剂Triton X-100增溶纯化的类囊体膜,再用蔗糖密度梯度超速离心方法分离其叶绿素-蛋白质复合物。P700差示光谱鉴定分离的光系统Ⅰ（PSⅠ）颗粒,并且检测到了具有DCIP光还原活性的光系统Ⅱ（PSⅡ）颗粒。结果表明,尽管海藻类囊体膜增溶困难,但只要条件合适,可以得到具有活性的光系统颗粒。     

大鼠海马细胞质膜的分离及其蛋白质组学分析

运用差速离心和连续蔗糖密度梯度离心的方法分离纯化成年大鼠海马组织细胞质膜并用Western blotting对质膜样品进行检测.结果表明,在蔗糖密度梯度离心介质中,膜片主要集中于蔗糖浓度为32%~42%的区段(密度约1.13~1.18),其次是20%~25% (密度约1.08~1.10)的区段.39%(密度约1.17)层面上质膜浓度和纯度最高.一维SDS-PAGE分离和CapLC-MS/MS分析后,通过数据库搜寻从大鼠海马组织细胞质膜样品中共鉴定出135种蛋白质, 其中质膜蛋白和与膜相关的蛋白质共70种(占51.9%), 主要包括Na⁺/K⁺-ATPase、Glutamate/aspartate transporter、Lipophilin、GLAST 1a等. 为研究海马组织细胞的功能和大鼠海马组织细胞质膜蛋白质数据库的建立积累了有价值的资料.     

胡杨液泡膜微囊的纯化及其质子转运活性

 为进一步研究液泡膜及 H+ - ATP酶在胡杨抵御盐胁迫中所起的作用 ,比较了研磨、捣碎和超声破碎三种细胞破碎方法 ,从悬浮培养的胡杨细胞中制备液泡膜微囊的效果 ;并用差速离心和不连续蔗糖密度梯度离心纯化了胡杨液泡膜微囊 .通过测定 H+ - ATP酶对 NO-3 、VO3-4和 Na N3的敏感性 ,以及焦磷酸酶质子转运活性表明 ,液泡膜微囊主要分布在 0 %～ 2 5%的蔗糖界面上 .捣碎法破碎细胞结合差速离心和蔗糖密度梯度离心可获得正向微囊比例高、封闭性好和酶活性高的液泡膜微囊     

胡杨液泡膜H+-ATPase的部分纯化及其耐盐性研究

为了阐明液泡膜H^ -ATPase在盐胁迫下的作用和适应性机制,对悬浮培养的胡杨细胞在50mmol／L盐浓度下处理10d,结果表明液泡膜H^ -ATPase、焦磷酸酶的水解活性、质子泵活性增加。将通过差速离心和不连续蔗糖密度梯度离心富积的液泡膜微囊先由脱氧胆酸钠(DOC)和n-辛基-β-D-葡萄糖(OG)分步破膜抽提,经蔗糖密度梯度离心分离,部分纯化的酶含V型H^ -ATPase的主要亚基。     

奶牛Y精子膜蛋白的提取与分析

本实验旨在对奶牛Y精子膜蛋白的提取与SDS-PAGE分析,是分离纯化奶牛Y精子特异膜蛋白基础与关键。本实验采用超声波破碎法将精子膜与精子分离,分离后的精子膜粗品采用蔗糖密度梯度离心法进行纯化。纯化的精子膜经膜蛋白提取液(去污剂)提取,透析浓缩。经SDS-PAGE银盐染色,结果表明该方法成功地提取到膜蛋白,得到膜蛋白电泳图谱。BIO-RAD分析得知Y精子膜蛋白种类至少有10种,分子量范围为7.94kD～166.65kD,其中尤以15kD的蛋白含量最多。奶牛Y精子膜蛋白的提取与鉴定为深入研究Y精子特异膜蛋白在受精过程中的作用,从而获得一种更经济、更安全、更有效的奶牛性别控制方法提供了实验依据。     

从假根羽藻类囊体膜直接分离纯化主要捕光叶绿素a／b蛋白复合体

应用液相色谱层析技术从海生绿藻,即假根羽藻(Bryopsis corticulans Setch.)的类囊体膜直接分离纯化获得了主要捕光叶绿素a/b蛋白质复合体(LHC II)。类囊体膜提取物经3% n-Octyl-b-D-glucopyranoside去垢剂处理后,其中的LHC II蛋白质获得与Q型阴离子交换层析柱的特异亲和力,因此LHC II从类囊体膜中被高选择性分离出来。经过蔗糖密度梯度离心,获得了LHC II单体、三聚体和聚集体。多肽组分的SDS-PAGE分析证明纯化获得的LHC II单体、三聚体和寡聚体具有很高的纯度。该LHC II制备物的吸收光谱也说明了其结构的完整性。首次提出了通过少数简单步骤从类囊体膜直接分离、纯化LHC II是可以实现的。     

从假根羽藻类囊体膜直接分离纯化主要捕光叶绿素a/b蛋白复合体

应用液相色谱层析技术从海生绿藻,即假根羽藻(Bryopsis corticulans Setch.)的类囊体膜直接分离纯化获得了主要捕光叶绿素a/b蛋白质复合体(LHCⅡ).类囊体膜提取物经3％n-Octyl-b-D-glucopyranoside去垢剂处理后,其中的LHCⅡ蛋白质获得与Q型阴离子交换层析柱的特异亲和力,因此LHCⅡ从类囊体膜中被高选择性分离出来.经过蔗糖密度梯度离心,获得了LHCⅡ单体、三聚体和聚集体.多肽组分的SDS-PAGE分析证明纯化获得的LHCⅡ单体、三聚体和寡聚体具有很高的纯度.该LHCⅡ制备物的吸收光谱也说明了其结构的完整性.首次提出了通过少数简单步骤从类囊体膜直接分离、纯化LHCⅡ是可以实现的.     

一种适用于双向电泳分析的高效真菌线粒体提取及蛋白质制样方法

用液氮冷冻研磨法破碎真菌菌丝细胞,通过差速和蔗糖密度梯度离心分离纯化板栗疫病菌线粒体,所得线粒体产率(质量比)约为1/10~4。电子显微镜观察和蛋白印迹表明,所制备的线粒体完整性好,没有检测到其它细胞成分的污染。使用膜蛋白裂解液溶解线粒体制备蛋白样品,将蛋白样品200μg上样于pH3~10,24cm的非线性胶条进行等电聚焦,电聚焦后再进行第二向SDS-PAGE电泳分离,经银染获得重复性好、背景清晰、分辨率高(680±15个蛋白质点)的凝胶图谱。随机选择10个蛋白质点进行质谱分析,9个获得有效注释,均为线粒体特异性蛋白质,表明所制备的蛋白样品非常适合双向电泳分析及其后续的质谱鉴定。     

鼻咽癌5-8F细胞膜蛋白质组初步分析

细胞膜蛋白是细胞的重要组成部分,作为细胞的"门铃"与"门户",参与细胞内外物质交换、信息转换、细胞生长发育、细胞迁移以及免疫应答等重要生理活动.为鉴定鼻咽癌转移相关膜蛋白,运用差速离心联合双水相方法分离纯化鼻咽癌高转移细胞5-8F的细胞膜,SDS-PAGE分离膜蛋白,液相色谱/电喷雾串联质谱分析(LC-MS/MS)结合生物信息学分析鉴定出316种非冗余蛋白质,其中152种(48.5%)被注释为膜蛋白或膜相关蛋白.通过肿瘤差异蛋白质组数据库(dbDEPD)搜索,发现在114个膜蛋白中有49种膜蛋白与其它肿瘤的发生发展密切相关,其中21个膜蛋白与肿瘤转移相关.进一步分析发现膜蛋白CD104、VDAC2、CD298和SLC25A3与同属头颈部肿瘤的口腔癌转移相关,提示这4个膜蛋白也可能是鼻咽癌潜在的转移相关蛋白.研究结果提供了一个鼻咽癌细胞5-8F包含中高丰度膜蛋白的数据库,为进一步研究头颈部肿瘤鼻咽癌癌变分子机理积累了有价值的资料.     

Surface morphology of peritrophic membrane formation in the cabbage looper,Trichoplusia ni

Summary The development of the peritrophic membrane in the cabbage looper, Trichoplusia ni, was investigated by scanning electron microscopy. The development of this membrane is characterized by a series of events suggested by the observations to be (1) secretion of material among and above the microvilli of the midgut epithelial cells, (2) maturation of this material into a randomly cross-linked fibrous matrix, and (3) aggregation of amorphous materials in and within the matrix. The membrane, possessing small discontinuities, remains intact in the midgut, but shows gross damage by the time it is passed from the insect, surrounding the feces.     

Measurement of the transmembrane electrical potential of Dunaliella acidophila by microelectrodes

A method was developed to determine electrical potential differences across the plasma membrane of the microalga Dunaliella by means of potential-sensitive microelectrodes. Special emphasis was put on the measurement of the membrane potential in the acidophilic Dunaliella acidophila (optimal growth at pH 1.0), but neutrophilic, halotolerant Dunaliella species were used as reference systems. For Dunaliella acidophila positive membrane potentials (cytoplasma relative to the medium), ranging from +30 to +65mV were measured. Illumination caused a decrease of the positive potential by about 10 mV. The ATPase inhibitor omeprazole caused an increase of the positive membrane potential ranging from +60 to +100 mV, whereas the ionophore gramicidin caused a decrease of the MP to +10 to +30 mV. The salt tolerant, neutrophilic Dunaliella parva and Dunaliella bardawil exhibited negative membrane potentials in the order of -40 to -60mV, and light caused a hyperpolarization of about 10 mV. A negative membrane potential was measured also in D. acidophila cells transferred to pH 7.0. The physiological significance of a positive membrane potential for acidophilic algae is discussed.Abbreviations E _m membrane potential - PM plasma membrane - TPB^– tetraphenylborone anion - TPP⁺ tetraphenyl-phosphonium cation - SCN^– isothiocyanate     

Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles

Summary Membrane potential and resistance were recorded from parenchymal cells of oat (Avena) coleoptiles, using one and two intracellular electrodes. Membrane potential is largest (–100 mV) in impalements with low input resistance (2–4 M), and is less negative (–50 mV) in penetrations with high input resistance (> 20 m). The interpretation is that the electrode lodges in the vacuole which is positive to the cytoplasm (but still negative to the external solution), and that measurements of net membrane potential are compromised to varying degrees by leakage shunts introduced across the high resistance vacuolar membrane by the electrode. This conclusion is supported by several additional lines of evidence. (1) It is possible to convert large-R/small-V impalements into small-R/large-V penetrations by passing excess current through the electrode or by briefly ringing the capacitance neutralization circuit in the amplifier. The cells usually recover their resistance in a few minutes, with a concomitant decrease in the negativity of the membrane potential. (2) Changes in external [K] affect the measuree potential by an amount that is independent of the input resistance of the impalement. This is consistent with an effect of [K] _o on the potential of the plasma membrane and the occurrence of leakage shunts primarily at the tonoplast. (3) Quantitatively, the effects of a change in [K] _o on resistance indicate that nearly 90 percent of the input resistance of unshunted cells resides in the tonoplast. (4) The effects of metabolic inhibitors (DNP, CN^–) on potential are smaller in large-R than in small-R impalements. This observation suggests there are electrogenic pumps contributing to the membrane potential at both the plasmalemma and tonoplast. Finally, we conclude that with an electrode in the vacuole it is possible to record potentials that are dominated by the contribution of the plasma membrane, provided care is taken to select impalements combining both large, negative potential and low input resistance.     

The eggs of the freshwater fish Epiplatys dageti have tight plasma membranes without intramembranous particles

Summary The plasma membrane of fish (Epiplatys dageti) eggs, which are capable of developing in salt-free water despite intracellular osmolarity corresponding to a pressure gradient of 7 to 8 atmospheres, is almost devoid of intramembrane particles. The specific membrane resistance is at least 3 orders of magnitude larger than that of nerve or muscle cells of different species indicating that the membrane is tight. These findings support the view that intramembranous particles are involved in transmembrane transport of ions, and indicate that the ionic concentration gradient in this cell is maintained by a tight membrane rather than by active transport.     

Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.     

Formation de la membrane de fécondation de l'oeuf d'Artemia salina

Résumé L'oeuf vierge d'Artemia salina n'est pas entouré de membranes exocellulaires. Le plasme sous-cortical ne contient pas d'organites spéciaux. Dès la fécondation, une membrane est secrétée par l'oeuf. La substance membranogène, contenue dans le reticulum endoplasmique lisse, passe par les éléments golgiens, où elle semble modifiée, et est expulsée dans des vésicules qui se détachent du Golgi. Retenue par un enduit granuleux, qui couvre le plasmolemme, et qui peut être un glycocoat ou du suc du tractus génital, elle s'étale en une membrane de fécondation, qui se soulève pour constituer l'espace périvitellin. Le processus est progressif et dure environ une heure et demi.

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Formation of the fertilization membrane of the egg inArtemia salina

Summary The unfertilized egg ofArtemia salina is not covered with any extracellular structure. No special organelles are found in the sub-cortical plasma. From the moment of fertilization, a membrane is progressively secreted by the egg. The membranogenous substance is first seen as large granules in the smooth endoplasmic reticulum, presumably transformed within Golgi elements and extruded in vesicles liberated from the Golgi apparatus. Retained by a glycocoat or by contact with the fluid of the genital tract, it spreads out into a fertilization membrane, soon surrounding a perivitelline space. The process lasts till 1 1/2 h after fertilization.

     

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant

Summary We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485–510 kb) whereas sipC and sipD both map to 82 min (3850–3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.     

Evidence for two different nitrate-reducing activities at the plasma membrane in roots ofZea mays L.

Plasma-membrane (PM) vesicles isolated from 6-d-old corn roots by sucrose gradient centrifugation or two-phase partitioning showed an NADH-dependent nitrate reductase (NR) activity averaging at 40 nmol per milligram PM protein per hour. This membrane-associated NR activity could not be removed from two-phase-partitioned PM vesicles by salt washing, osmotic shock treatment, sonication, or freeze-thawing to reverse vesicle sidedness. Therefore, it could not be attributed to contamination of membrane vesicles by the soluble, cytosolic NR. Plasma-membrane vesicles reduced NO ₃ ^- in the presence of the electron donors NADH or NADPH at an activity ratio of 2.2. The NADH- and NADPH-dependent NR activities of outside-out oriented PM vesicles differed in their sensitivity toward the detergent Brij 58, leading to a latency of 65% or 29% using NADH or NADPH as electron donor, respectively. The activities of NO ₃ ^- reduction in the presence of saturating concentrations of NADH and NADPH were additive. Furthermore, both activities were characterized by a different pH dependence with a pH optimum of 7.5 for the NADH-dependent activity and of 6.8 for the NADPH-dependent activity. The membrane-associated NAD(P)H-dependent NR activities responded to different nitrogen nutrition of plants in a manner different from the soluble forms of the enzyme. The data confirm the existence of a corn PM NR and suggest that there may be two different NO ₃ ^- -reducing enzymes located at the PM of corn roots.Abbreviations PM Plasma membrane - NR nitrate reductase This research was supported by grants from the National Research Council of Italy (bilateral project between Italy and Germany to Z.V. and U.L.), by the Ministero dell' Università e Ricera Scientifice e Tecnologica (MURST 40%) and by the Deutsche Forschungsgemeinschaft.     

Activities of a Mechanosensitive Ion Channel in anE. Coli Mutant Lacking the Major Lipoprotein

Summary The activity of the mechanosensitive (MS) ion channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the IISP (the suction for ane-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S _p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in thelpp membranes. After lysolecithin addition, thelpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.