蟾毒灵与脂膜相互作用的研究

应用＾１３Ｃ－ＣＰ／ＭＡＳ和ＤＳＣ方法研究蟾毒灵与磷脂膜相互作用的执致相变特性及动力学特性。ＤＳＣ曲线表明蟾毒灵使磷脂膜相变温度降低,吸热峰变宽。＾１３Ｃ－ＣＰ／ＭＡＳ谱表明磷脂膜的ＮＭＲ信号峰化学位移随温度稍有变化,提示磷脂膜在液晶态脂肪烃链有不同程序的反－旁式异构化。含蟾毒灵的ＥＰＣ脂双层ＮＭＲ谱,随温度升高有蟾毒灵信号峰出现,ＥＰＣ脂双层分子内各部分的信号峰强度和峰形变化明显,说明脂双层分子     

马铃薯Y病毒普通系外壳蛋白基因在大肠杆菌中的表达

将马铃薯Ｙ病毒普通系（ＰＶＹ０）的外壳蛋白基因克隆到表达质粒ｐＭＡＬｃ２中,构建这一基因在大肠杆菌中的表达载体ｐＭＡＬｃ２ＰＶＹ０ＣＰ。ＳＤＳＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ检测结果表明,这一表达栽体在Ｅ．ｃｏｌｉＤＨ５α中经ＩＰＴＧ诱导可表达分子量为７１．８ｋＤａ的特异性融合蛋白。以ａｍｙｌｏｓｅｒｅｓｉｎ亲合柱层析纯化这一融合蛋白为抗原,免疫家兔制备了效价为１∶１０２４的特异性抗血清。用该抗血清可通过对流免疫电泳、免疫双扩散及Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ对ＰＶＹ进行检测     

大肠杆菌cai DE的基因克隆与表达

从Ｅ．ｃｏｌｉ ＭＣ４１００菌体的染色体ＤＮＡ中利用鸟枪法克隆含编码肉碱消旋酶及相关因子的ｃａｉＤＥ基因片段,并经序列分析验证,由重组质粒ｐｌＸ３９３亚克隆得到ｐＤＳＷ２重组表达质粒,后者转入Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ３）菌株中是丙基硫代半乳糖苷（ＩＰＴＧ）诱导,在聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）上分子量为３０ｋＤ和２４ｋＤ附近可见明显的表达蛋白带。     

膜脂的物理状态对G_s激活AC的影响

用生物膜的拆离与重建方法将从牛脑皮层膜中纯化的激活型ＧＴＰ结合蛋白（Ｇｓ）和腺苷酸环化酶（ＡＣ）在含有不同极性头部或不同脂肪酸侧链的磷脂组成的脂质体上重建形成脂酶体,测定脂酶体中ＡＣ的基础活力及Ｇｓ激活ＡＣ的活力。实验结果表明,磷脂影响ＡＣ的基础活力和Ｇｓ激活ＡＣ活力的顺序依次为：ＰＥ＞ＰＳ＞ＰＣ;含不同脂肪酸侧链的混合磷脂对Ｇｓ的激活活力的影响大于含单一脂肪酸侧链的纯磷脂,如ＰＥＤＰＰＥ,ＰＳＤＰＰＳ,ＰＣＤＰＰＣ。含不同脂肪酸侧链的磷脂影响Ｇｓ的活力的顺序为ＤＬＰＣ＞ＤＭＰＣ＞ＤＰＰＣ。用反映磷脂分子的堆积程度的荧光探剂ＭＣ５４０和脂双层的流动性变化的ＤＰＨ以及专一性标记蛋白质巯基（－ＳＨ）基团的荧光探剂ａｃｒｙｌｏｄａｎ的测定结果表明,不同磷脂影响Ｇｓ的活力的差异主要是由于脂质物理状态的不同所致。     

细胞周期蛋白（cyclin）D在鼻咽癌中的表达及功能初？…

探讨了细胞周期蛋白（ｃｙｃｌｉｎ）Ｄ在ＥＢ病毒（Ｅｐｓｔｅｉｎ－Ｂａｒｒ ｖｉｒｕｓ）潜伏膜蛋白１（ｌａｔｅｎｔ ｍｅｍｂｒａｎｅ ｐｒｏｔｅｉｎ,ＬＭＰ１）阳性和阴性表达的鼻咽癌细胞５系中的表达特征,利用流式细胞仪同时从ＤＮＡ及蛋白质水平初步分析了ｃｙｃｌｉｎ Ｄ１在鼻咽癌细胞系中的功能。Ｗｅｓｔｅｒｎ印迹的结果发现ｃｙｃｌｉｎｈ Ｄ１在ＣＮＥ－ＬＭＰ１中过表达,在ＨＮＥ１中表达较弱;Ｃｙｃｌｉ     

光系统II核心天线CP43的纯化及性质

菠菜放氧的ＰＳＩ核心复合物经０．８ｍｏｌ／ＬＴｒｉｓ－ＨＣｌ（ｐＨ８．０）洗涤后,用温和的非离子去垢剂ＤＭ和高浓度的ＬｉＣｌＯ４增溶,再经ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ－６５０Ｓ离子交换柱层析分离,可得到ＰＳＩＩ核心天线４３ｋＤ叶绿素ａ结合蛋白（ＣＰ４３）。ＳＤＳ－ＰＡＧＥ显示一条４３ｋＤ蛋白质带。根据Ａｒｎｏｎ法和Ｍａｒｋｗｅｌ法的结果表明,每个蛋白质分子结合有２０～２１个分子的叶绿素ａ。室温条件下,ＣＰ４３在红光区具有６７１ｎｍ的最大吸收峰和６８３ｎｍ的荧光发射峰,以及Ｗ型的圆二色信号,表明其处于较为天然的状态。文中还制备并鉴定了对ＣＰ４３特异的抗血清。     

光系统Ⅱ核心天线CP47的纯化及其抗血清的制备

将ＯＧ处理的ＰＳⅡ经高速离心和透析制得的ＯＥＣＣ，用ＤＭ增溶后，上ＤＥＡＥ－６５０５柱，ＬｉＣＩＯ４梯度洗脱可得ＰＳⅡ核心天线蛋白ＣＰ４７。根据Ａｍｏｎ法和Ｍａｒｋｗｅｌｌ法的结果表明，每个蛋白质分子结合１３～１４个分子的Ｃｈｌａ。室温条件下，ＣＰ４７在红光区具有６７４ｎｍ的最大吸收峰和６９３ｎｍ的荧光发射峰，以及Ｗ型的圆二色信号，表明其处于较为天然的状态。将制得的ＣＰ４７免疫家兔可制得相应的抗血     

PKC,PKA和TPK在血小板激活中的作用

利用＾３２Ｐ－ＮａＨ２ＰＯ４标记猪血小板,然后,以ＰＭＡ,凝血酶,ＰＧＥ１腺苷等处理,结果表明,随着ＰＭＡ激活ＰＫＣ,血小板发生聚集,３５μｍｏｌ／ＬＰＧＥ１或１ｍｍｏｌ／ＬｄｂｃＡＭＰ不能抑制５０ｎｍｏｌ／ＬＰＭＡ诱导的血小板聚集,腺苷却能抑制ＰＭＡ诱导的血小板聚集（ＥＣ５０＝０．１ｍｍｏｌ／Ｌ,ｄｂ－ｃＡＭＰ,腺苷都不能抑制１００ｎｍｏｌ／ＬＰＭＡ诱导的４０ｋＤ蛋白磷酸化,ＰＫＡ激活不能抑制Ｐ     

内皮素和bFGF对MC的促增殖作用及胰岛素的协同效应

采用３Ｈ－ＴｄＲ参入法,测定碱性成纤维细胞生长因子（ｂＦＧＦ）、胰岛素和内皮素－１（ＥＴ－１）对体外培养的大鼠肾小球系膜细胞（ＭＣ）增殖的影响,以及胰岛素与ｂＦＧＦ或ＥＴ－１促ＭＣ增殖的协同作用。结果表明,不同浓度的ｂＦＧＦ（５－２００ｎｇ／ｍｌ）和胰岛素（０．１－２．４Ｕ／ｍｌ）均显著升高ＭＣ的３Ｈ－ＴｄＲ参入值（ｃｐｍ值）（Ｐ＜０．０１）。ＥＴ－１对ＭＣ的ｃｐｍ值的影响依剂量不同呈现两种不同的效应,在１０－９－１０－７ｍｏｌ／Ｌ时,随着浓度的升高,ＭＣ的ｃｐｍ值明显升高（Ｐ＜０．０１）,并以１０－８ｍｏｌ／Ｌ作用最强;当升高到１０－６ｍｏｌ／Ｌ时,ＭＣ的ｃｐｍ值出现降低趋势。胰岛素与ｂＦＧＦ或低浓度ＥＴ－１（≤１０－８ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值明显高于二者单独作用之和（Ｐ＜０．０１）,与高浓度ＥＴ－１（＞１０－７ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值小于二者单独作用之和（Ｐ＞０．０５）。上述结果说明,胰岛素、ｂＦＧＦ和ＥＴ－１均能显著促进ＭＣ增殖;胰岛素与ｂＦＧＦ或低浓度的ＥＴ－１促ＭＣ增殖具有正协同作用,与高浓度ＥＴ－１呈现负协同作用。     

N~+离子注入对不同辐射敏感性微生物超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的影响

以Ｅ．ｃｏｌｉ和耐辐射生球菌（Ｄｅｉｎｏｃｏｃｃｕｓｒａｄｉｏｄｕｒａｎｓ）为试材,研究了Ｎ＾＋离子注入对其ＳＯＤ,ＣＡＴ和ＰＯＤ活性的影响及共对自由基的清除,结果表明：Ｄ．ｒａｄｉｏｄｕｒａｎｓ经Ｎ＾＋离子注入后ＳＯＤ和ＣＡＴ酶活高于Ｅ．ｃｏｌｉ的,而ＰＯＤ酶活不仅很低于Ｅ．ｃｏｌｉ的;随剂量的增大,两者ＳＯＤ和ＣＡＴ酶活均为增后减,只是其ＳＯＤ酶活的变化峰值对应剂量分别为６×１０＾１５Ｎ＾＋／     

Conformational changes of urea-denatured colicin E1 induced by phospholipid membranes.

The membrane insertion of urea-denatured colicin E1 was studied by using fluorescence spectroscopy, circular dichroism and monolayer techniques. The results showed that the denatured colicin E1 taking mainly the 'random coil' conformation may recover its orderliness to a certain extent under the induction of the phospholipid membrane and insert spontaneously into phospholipid membrane, indicating that unfolding of colicin E1 does not inhibit its membrane insertion. Among the four tryptophan residues of the membrane-bound colicin E1 molecules, at least two were accessible by the quenchers, i.e. not inserted into the membranes. Although urea-denatured colicin E1 interacted preferentially with negatively charged phospholipids, it seems less dependent on the negatively charged lipid than colicin A. The addition of urea increased the speed of the adsorption of colicin E1 to the membrane, but did not affect obviously its membrane insertion ability.     

Histidine 440 controls the opening of colicin E1 channels in a lipid-dependent manner

The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state.     

Acidic interaction of the colicin A pore-forming domain with model membranes of Escherichia coli lipids results in a large perturbation of acyl chain order and stabilization of the bilayer.

2H and 31P NMR techniques were used to study the effects on acyl chain order and lipid organization of the well-characterized pore-forming domain of colicin A (20-kDa thermolytic fragment of colicin A) upon insertion in model membrane systems derived from the Escherichia coli fatty acid auxotrophic strain K 1059, which was grown in the presence of [11,11-2H2]-labeled oleic acid. Addition of the protein to dispersions of the E. coli total lipid extract, in a 1/70 molar ratio of peptide to lipids, resulted in a large pH-dependent decrease in quadrupolar splitting of the 2H NMR spectra. The decrease of the quadrupolar splitting obtained at the various pH values was correlated with the pH dependence of the insertion of the protein in monolayer films using the same E. coli lipid extracts. The pK governing the perturbing effects on the order of the fatty acyl chains was around 5, in agreement with the values of the pH-dependent conformational changes of the pore-forming domain of colicin A required for membrane insertion as reported by van der Goot et al. [(1991) Nature 354, 408-410]. 31P NMR measurements show that the bilayer organization remains intact upon addition of the protein to dispersions of lipid extract. Surprisingly, 31P NMR measurements as a function of temperature indicate that the pore-forming domain of colicin A even stabilizes bilayer lipid structure at pH 4. Both the large effect of the protein on acyl chain order and its bilayer-stabilizing activity are indicative of a surface localization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected (13)C and (15)N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val C(alpha) signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the alpha-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by (2)H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.     

Conformational changes of colicin Ia channel-forming domain upon membrane binding: a solid-state NMR study

Channel-forming colicins are bactericidal proteins that spontaneously insert into hydrophobic lipid bilayers. We have used magic-angle spinning solid-state nuclear magnetic resonance spectroscopy to examine the conformational differences between the water-soluble and the membrane-bound states of colicin Ia channel domain, and to study the effect of bound colicin on lipid bilayer structure and dynamics. We detected ¹³C and ¹⁵N isotropic chemical shift differences between the two forms of the protein, which indicate structural changes of the protein due to membrane binding. The Val Cα signal, unambiguously assigned by double-quantum experiments, gave a 0.6 ppm downfield shift in the isotropic position and a 4 ppm reduction in the anisotropic chemical shift span after membrane binding. These suggest that the α-helices in the membrane-bound colicin adopt more ideal helical torsion angles as they spread onto the membrane. Colicin binding significantly reduced the lipid chain order, as manifested by ²H quadrupolar couplings. These results are consistent with the model that colicin Ia channel domain forms an extended helical array at the membrane-water interface upon membrane binding.     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

Insertion intermediates of pore-forming colicins in membrane two-dimensional space

The formation of integral membrane voltage-gated ion channels by the initially soluble C-terminal channel polypeptide (CP) of the pore-forming colicins is a fruitful area for studies on membrane protein import. The dependence of CP import on specific membrane parameters can be better understood using liposomes and planar membranes of defined lipid composition. The membrane surface and interfacial layer provide special conditions for the transition of a pore-forming colicin from the soluble to the integral membrane state. The colicin E1 CP is arranged in the membrane interfacial layer as a conformationally mobile helical array that is extended far more in the two dimensions parallel to the membrane surface than in the third dimension perpendicular to it. The alpha-helical content of CP(E1) increases by approximately 30% upon binding to the membrane. The sequence of kinetically distinguishable events in the CP(E1)-membrane interaction is binding, unfolding to a subtended area of 4200 A(2), helix extension, and insertion, the last three events overlapping in their time course ( approximately 10 s(-1)). The extension into two dimensions and the interaction with the membrane surface may explain the reversible denaturation and refolding of secondary structure that occurs after boiling of the CP-membrane complex. Although DSC showed the presence of helix-helix interactions in the membrane-bound state, the change in secondary structure and the extended surface area argue against a molten-globule intermediate in the CP-membrane interaction. However, the surface-bound state is mobile, as surface conformational mobility is a necessary prerequisite for insertion of CP trans-membrane helices into the bilayer. The requirement for this surface protein mobility, described by "thermal melting" FRET experiments, may provide the explanation for the precipitous decrease in the voltage-gated CP channel formation at high values of surface potential of planar bilayer membranes. Thus, the membrane interfacial layer, with the CP backbone situated near the acyl chain carbonyls, provides a favorable environment for the structure changes necessary for the transition from the soluble to the membrane-inserted state.     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

Isolation and Characterization of Mutants of Colicin Plasmids E1 and E2 After Mu Bacteriophage Infection

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.     

Lipid-destabilizing properties of the hydrophobic helices H8 and H9 from colicin E1

Colicins are toxic proteins produced by Escherichia coli that must cross the membrane to exert their activity. The lipid insertion of their pf domain is linked to a conformational change which enables the penetration of a hydrophobic hairpin. They provide useful models to more generally study insertion of proteins, channel formation and protein translocation in and across membranes. In this paper, we study the lipid-destabilizing properties of helices H8 and H9 forming the hydrophobic hairpin of colicin E1. Modelling analysis suggests that those fragments behave like tilted peptides. The latter are characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. Fluorescence techniques using labelled liposomes clearly show that H9, and H8 to a lesser extent, destabilize lipid particles, by inducing fusion and leakage. AFM assays clearly indicate that H8 and especially H9 induce membrane fragilization. Holes in the membrane are even observed in the presence of H9. This behaviour is close to what is seen with viral fusion peptides. Those results suggest that the peptides could be involved in the toroidal pore formation of colicin E1, notably by disturbing the lipids and facilitating the insertion of the other, more hydrophilic, helices that will form the pore. Since tilted, lipid-destabilizing fragments are also common to membrane proteins and to signal sequences, we suggest that tilted peptides should have an ubiquitous role in the mechanism of insertion of proteins into membranes.