尖吻蝮蛇毒磷脂酶A2基因的克隆与序列分析

从尖吻蝮蛇毒腺中抽提总ＲＮＡ,经ＲＴＰＣＲ扩增磷脂酶Ａ２的基因,以江浙蝮蛇的酸性磷脂酶Ａ２基因为探针杂交筛选克隆,分离得到４种磷脂酶Ａ２基因。经双向测序测定了这些磷脂酶Ａ２同功酶基因的全序列,并由此推导出编码的氨基酸序列。运用计算机软件推算了它们的等电点,按照等电点和结构特征将它们分别命名为尖吻蝮蛇毒酸性磷脂酶Ａ２Ｉ（Ａ．ａＡＰＬＡ２Ｉ）、尖吻蝮蛇毒酸性磷脂酶Ａ２ＩＩ（Ａ．ａＡＰＬＡ２ＩＩ）、尖吻蝮蛇毒碱性磷脂酶Ａ２（Ａ．ａＢＰＬＡ２）和尖吻蝮蛇毒Ｌｙｓ４９磷脂酶Ａ２（Ａ．ａＬｙｓ４９ＰＬＡ２）。其中Ａ．ａＡＰＬＡ２Ｉ的１～１０位氨基酸残基序列同以前分离得到的尖吻蝮蛇酸性磷脂酶Ａ２已测定的１～１０位氨基酸残基序列完全一致。Ａ．ａＬｙｓ４９ＰＬＡ２基因则由于其推导出的４９位氨基酸残基由Ｌｙｓ代替了Ａｓｐ而区别于以前克隆到的磷脂酶Ａ２基因。最后运用计算机软件比较了它们的同源性。这一组磷脂酶Ａ２基因的克隆,将为进一步研究磷脂酶Ａ２的结构与功能的关系提供更多的信息。     

哺乳动物细胞液磷脂酶A2

哺乳动物细胞液磷脂酶Ａ_２杨在清,甘莉（华中农业大学,武汉４３００７０）关键词细胞液磷脂酶Ａ_２细胞液磷脂酶Ａ２（ｃｙｔｏｓｏｌｉｃｐｈｏｓｐｈｏ－ｌｉｐａｓｅＡ２,ｃＰＬＡ２）是动物非胰腺磷脂酶Ａ２的一种,存在于哺乳动物的细胞液中,在花生四烯酸的代谢...     

磷脂酶A2的生理机能新说

磷脂酶Ａ２的生理机能新说杜晓燕周元聪（中国科学院上海生物化学研究所,上海２０００３１）关键词磷脂酶Ａ２生理机能磷脂酶Ａ２（ｐｈｏｓｐｈｏｌｉｐａｓｅＡ２,ＥＣ３．１．１．４）,简称ＰＬＡ２。它能水解甘油磷脂的第二位酯酰键,生成溶血磷脂和脂肪酸,如下式...     

江浙蝮蛇毒磷脂酶A2的结构模建与分析

我们用同源模建方法构建了江浙蝮蛇毒碱性－酸性杂合磷脂酶Ａ２－Ⅱ和中性磷脂酶Ａ２的三维结构,并对它们的结构特征做了比较分析。在此基础上,我们在三维结构水平上解释了磷脂酶Ａ２荧光光谱学研究的一些结果。我们还对四种江浙蝮蛇毒磷脂酶Ａ２的酶活性部位的静电势分布做了分析。我们认为：含钙的ＰＬＡ２酶活性部位周围的静电势有利于ＰＬＡ２与带负电荷的底物结合。     

竹叶青蛇毒磷脂酶A2的分离纯化和性质研究

竹叶青蛇毒磷脂酶Ａ_２的分离纯化和性质研究冯波,吴卫甲,钱嵘,王克夷,周元聪（中国科学院上海生物化学研究所,２０００３１）关键词竹叶青蛇毒,磷脂酶Ａ_２;血小板聚集磷脂酶Ａ。在哺乳动物胰脏和蛇毒毒液中含量较丰富,其中蛇毒磷脂酶Ａ。除能水解甘油磷脂的第二...     

磷脂酶A2阻断剂对失血性休克再灌注损伤的保护作用

磷脂酶Ａ２阻断剂对失血性休克再灌注损伤的保护作用颜光涛郝秀华王录焕江潮光１田亚平２王成彬２李振甲（解放军总医院临床医学研究所,１科训处,２生化科,北京１００８５３）磷脂酶Ａ２（ＰｈｏｓｐｈｏｌｉｐａｓｅＡ２,ＰＬＡ２）激活后水解释放花生四烯酸代谢产物...     

蝮蛇毒酸性磷脂酶A2高含钙量的晶体结构研究

钙离子是磷脂酶Ａ２（ＰＬＡ２）经必需的辅因子,以蝮蛇生磷脂酶Ａ２为材料,在晶体生长过程中加入ＣａＣｌ２培养了高含钙量的ＰＬＡ２晶体,Ｘ射互衍鉴定该晶体与天然酶同晶。民集了１．６埃分辩率的同步辐射衍射数据,经晶这修正所得高含钙酶的结构与天然酶结构要近,但钙结合部位与Ｃ端肽段有细微判别与钙配位的七个氧原子所形成的五角双锥构型比天然酶的完美。钙离了对磷脂酶Ａ２整个分子构象的影响较小,下离子的作用主要是通     

脱Ca~（2＋）与结合Ca~（2＋）蝮蛇毒酸性磷脂酶A_2晶体的培养和X射线衍射数据

钙离子是磷脂酶Ａ２催化必须的铺因子,以蝮蛇毒酸性磷脂酶Ａ２为材料,采用在晶体培养液中加入ＥＤＴＡ或ＥＧＴＡ螯合剂络合法除去ＰＬＡ２中可能有的Ｃａ＾２＋离子和加入ＣａＣｌ２以确保ＰＬＡ２能克分结合上钙离子的方法培养充分结合Ｃａ＾２＋和脱Ｃａ＾２＋的ＰＬＡ２晶体。加了螯合剂后长出的晶体为长棒状六方柱,加Ｃａ＾２＋离子后长出的晶体则为短粗的六方柱。经Ｘ射线鉴定两种晶体为同晶型,空间群为Ｐ６１,晶胞参数很     

磷脂酰胆碱特异性磷脂酶C的研究

随着人们对信号转导认识的逐步加深,各种磷脂酶在信号通路中的作用也日渐受到重视,并日趋明了。其中磷脂酶Ａ２（ＰＬＡ２）、磷脂酰胆碱特异性磷脂酶Ｄ（ＰＣ－ＰＬＤ）的基因已克隆,对磷脂酰肌醇特异性磷脂酶Ｃ（ＰＩ－ＰＬＣ）也有较深了解,而对磷脂酰胆碱特异性磷...     

尖吻蝮蛇毒酸性磷脂酶A_2I的表达及其生化特征

将尖吻蝮蛇毒酸性磷脂酶 Ａ２ Ｉ（ Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ） 的基因克隆至表达载体ｐ Ｂ Ｌ Ｍ Ｖ Ｌ２ , 在大肠杆菌 Ｒ Ｒ１ 中成功表达。表达产物 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ约占细菌蛋白质总量的３０ ％ , 以包含体的形式存在。纯化包含体后, 将产物变性、复性, 然后用 Ｆ Ｐ Ｌ Ｃ Ｓｕｐｅｒｏｓｅ Ｔ Ｍ１２ 纯化, 产物经过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 检测只有单一条带。对表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ进行了酶活性、抑制血小板聚集活性和溶血活性的测定。结果显示, 表达的 Ａ．ａ Ａ Ｐ Ｌ Ａ２ Ｉ的酶活性同变性后复性江浙蝮蛇酸性磷脂酶 Ａ２（ Ａ Ｐ Ｌ Ａ２） 的酶活性相近, 既具有抑制血小板聚集活性也具有溶血活性。最后对磷脂酶 Ａ２（ Ｐ Ｌ Ａ２） 的结构与这些活性的关系进行了讨论     

Isoelectric points of multi-domain proteins

Although the distribution of protein isoelectric points is multi-modal, large proteins show isoelectric points less variable than small proteins and their isoelectric points tend to converge to a unique value, close to the pH of the milieu in which the proteins are functional, as far as the protein dimension increases. This study demonstrates that large proteins, which contain more than a single domain, do have isoelectric points less variable than small proteins, which contains a single domain. However, the distribution of the isoelectric points of the single domains, contained in large proteins, resembles that of small proteins, which contain a single domain. Thus, large proteins can be soluble even if their pI is very close to the pH of the milieu, in which they perform their function, since they can contain several domains, the electrostatic properties of each of which mirror those of small proteins.     

Isoelectric focusing of interacting systems. I. Carrier ampholyte-induced macromolecular isomerization.

A phenomenological theory of isoelectric focusing is formulated for rapidly reversible, ampholyte-induced macromolecular isomerization. The calculations reveal that such interactions can give well resolved, bimodal transient and equilibrium isoelectric focusing patterns in which the two peaks correspond to different chemical equilibrium compositions and not to separated isomers. The kinetics of approach to the equilibrium pattern are characteristically biphasic: During the first phase, which is controlled by the rate of migration of the isomers in the electric field, two peaks are positioned in the region between the isoelectric points of the two isomers; one of the peaks then grows slowly at the expense of the other with a diffusion-dominated rate. The kinetics are dependent upon the initial distribution of macromolecule in the isoelectric focusing column, and in certain cases only a single peak is apparent during the first phase. These findings have practical implications for unambiguous interpretation of isoelectric focusing patterns, furnish explanations for hitherto puzzling experimental observations, and provide theoretical insights required for application of isoelectric focusing to the detection and characterization of macromolecular interactions in general.     

AMPHOTERIC COLLOIDS : II. VOLUMETRIC ANALYSIS OF ION-PROTEIN COMPOUNDS; THE SIGNIFICANCE OF THE ISOELECTRIC POINT FOR THE PURIFICATION OF AMPHOTERIC COLLOIDS.

1. It is shown by volumetric analysis that on the alkaline side from its isoelectric point gelatin combines with cations only, but not with anions; that on the more acid side from its isoelectric point it combines only with anions but not with cations; and that at the isoelectric point, pH = 4.7, it combines with neither anion nor cation. This confirms our statement made in a previous paper that gelatin can exist only as an anion on the alkaline side from its isoelectric point and only as a cation on the more acid side of its isoelectric point, and practically as neither anion nor cation at the isoelectric point. 2. Since at the isoelectric point gelatin (and probably amphoteric colloids generally) must give off any ion with which it was combined, the simplest method of obtaining amphoteric colloids approximately free from ionogenic impurities would seem to consist in bringing them to the hydrogen ion concentration characteristic of their isoelectric point (i.e., at which they migrate neither to the cathode nor anode of an electric field). 3. It is shown by volumetric analysis that when gelatin is in combination with a monovalent ion (Ag, Br, CNS), the curve representing the amount of ion-gelatin formed is approximately parallel to the curve for swelling, osmotic pressure, and viscosity. This fact proves that the influence of ions upon these properties is determined by the chemical or stoichiometrical and not by the "colloidal" condition of gelatin. 4. The sharp drop of these curves at the isoelectric point finds its explanation in an equal drop of the water solubility of pure gelatin, which is proved by the formation of a precipitate. It is not yet possible to state whether this drop of the solubility is merely due to lack of ionization of the gelatin or also to the formation of an insoluble tautomeric or polymeric compound of gelatin at the isoelectric point. 5. On account of this sudden drop slight changes in the hydrogen ion concentration have a considerably greater chemical and physical effect in the region of the isoelectric point than at some distance from this point. This fact may be of biological significance since a number of amphoteric colloids in the body seem to have their isoelectric point inside the range of the normal variation of the hydrogen ion concentration of blood, lymph, or cell sap. 6. Our experiments show that while a slight change in the hydrogen ion concentration increases the water solubility of gelatin near the isoelectric point, no increase in the solubility can be produced by treating gelatin at the isoelectric point with any other kind of monovalent or polyvalent ion; a fact apparently not in harmony with the adsorption theory of colloids, but in harmony with a chemical conception of proteins.     

全柱成像毛细管等电聚焦电泳测定艾塞那肽等电点

目的:采用新一代全柱成像毛细管等电聚焦电泳技术(CIEF-WCID)测定艾塞那肽等电点。方法:采用互补性金属氧化物半导体成像技术对样品等电聚焦过程进行实时记录,根据适宜的marker计算得到艾塞那肽的等电点,并对方法的准确度与重复性进行考察。结果:测得艾塞那肽等电点为5.46,与凝胶电泳结果基本一致,相对标准偏差为0.11%。CIEF-WCID方法快速准确,相对误差小于2.5%,重复性良好。结论:CIEF-WCID作为一种新的技术手段可用于艾塞那肽等电点的分析,方法快速、准确、重复性好,可为多肽的质量控制提供一种可靠的分析方法。     

Transient state isoelectric focusing. Digital measurement of zone position, zone area, segmental pH gradient, and isoelectric point as a function of time

Transient state isoelectric focusing (TRANSIF) is a kinetic method which offers quantitative information about relevant parameters pertaining both to methodological aspects and to the physical characterization of amphoteric molecules. TRANSIF data are obtained with an improved scanning isoelectric focusing assembly coupled to an on-line digital data acquisition and processing system which is used to continuously record changes in peak position ($\text{x}$), peak area, segmental pH gradient ($\text{Δ(pH)}\text{Δx}$), and isoelectric point (pI) of proteins during focusing. This improved apparatus has made it feasible to follow the temporal stability of an isoelectric focusing system in a quantitative fashion.     

Fractionation,Purification and Some Properties of Proteolytic Enzymes from Stem Bromelain

The heterogeneity of the proteolytic enzymes in the stem bromelain was investigated by the isoelectric focusing with carrier ampholytes. The isoelectric focusing of the stem bromelain demonstrated the presence of two types of proteolytic enzymes which were distinguishable from each other by their isoelectric points. One of these was a basic protein having an isoelectric point of 9.45. This basic enzyme comprised almost all of basic protein which are found in stem bromelain. The other was an acidic protein having an isoelectric point near pH 4.7. This was a minor compooent. The purification of the two enzymes was carried out by use of chromatographies on CM-Sephadex, DEAE-Sephadex and Sephadex at pH 7.0.     

Steroid receptors in human endometrium. Comparison of data obtained by three different methods

To find a method for steroid receptor measurement in small endometrial tissue samples (less than 100 mg), an isoelectric focusing assay has been compared with a dextran-coated charcoal assay for oestradiol receptor. The results correlated well (r = 0.85) and this indicates that isoelectric focusing is a good technique for oestradiol receptor determination. Te isoelectric focusing of progesterone receptor has been compared with a dextran-coated charcoal assay and sucrose density gradient centrifugation. Isoelectric focusing gave recoveries of 0-26% compared to receptor values obtained with the two other methods, which correlated well (r = 0.97). The low recovery implies that the isoelectric focusing assay is not suitable for progesterone receptor determination.     

牛凝血酶等电点的初步测定

作为一种新型的速效局部止血药和工具酶,凝血酶在临床和生物学研究中的应用十分广泛,牛血浆是其重要的来源之一。等电点沉淀是提取牛凝血酶首要和关键的步骤,测定其等电点后,再用此法时将得到更纯的凝血酶粗制品。本实验的目的是采用载体两性电解质pH梯度等电聚焦电泳的方法,结合SDS-PAGE测定牛凝血酶的等电点。经双向电泳后,SDS聚丙烯酰胺凝胶中出现了4个清晰的斑点,分别测定它们的分子量和等电点, 其中一个斑点与牛凝血酶B链的分子量一致为32kDa,其等电点为5．19．     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

High resolution two-dimensional electrophoresis of basic as well as acidic proteins.

A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.