一种改进的蛋白质超薄凝胶电泳方法

介绍了一种将聚丙烯酰胺凝胶固定在电泳夹板上的蛋白质电泳方法.通过此方法蛋白质电泳可以在0.4 mm厚的聚丙烯酰胺凝胶上进行.实验证明,经此方法处理的玻板结合凝胶非常牢固,在电泳后的所有处理步骤中都不会发生凝胶脱落现象.     

滇金丝猴和菲氏叶猴血红蛋白及几种同功酶的比较研究

用聚丙烯酰胺凝胶电泳技术对滇金丝猴和菲氏叶猴血红蛋白和几种同功酶在相同电泳条件下进行了分析和比较。 电泳:采用聚丙烯酰胺凝胶圆盘电泳(8.9×0.5cm),T=6.5％;聚丙烯酰胺凝胶板电泳(14×13cm),T=7.2％,pH8.3的Tris—Glycine缓冲液;北京六一仪器厂DYY—Ⅲ型电泳仪;电流3mA/管、2mA/cm~2;电流时间3h(圆盘),5h(板)。染色参考Davidson et al.(1965)、Tashion et al.(1971)、Mikio Kuboki(1978)、牛满江、童弟周(1978)等的方法并略加修改。     

小儿副轮状病毒的检出

用聚丙烯酰胺凝胶电泳法,从婴幼儿急性腹泻粪便中检出轮状病毒RNA电泳阳性293份,发现一株副轮状病毒(青-27株),此株病毒经电泳观察,呈典型的轮状病毒形态,但易破碎。ELISA检测表明不具有一般轮状病毒的特异性群抗原,病毒RNA基因组由11个片段组成,但电泳图型特殊,吴4:3:2:2排列模式,本文证实,此一小儿副轮状病毒与国外报道的,散发罕见的小儿副轮状病毒RNA电泳图型相同,提示此病毒的重要意义。     

电泳用水平聚丙烯酰胺干胶研制成功

聚丙烯酰胺凝胶电泳是快速低廉的生化分析技术。由于凝胶具有三维网状结构,电泳时兼具有分子筛效应和电荷效应,从而比其他电泳的分辨力大为提高,并在此基础上又发展了ＳＤＳ聚丙烯酰胺电泳、梯度胶电泳和等电聚焦聚丙烯酰胺电泳等技术。除大量用于各种蛋白质分析、分型...     

我国不同地区东亚钳蝎(Buthus martensii Karsch)毒在三种类型电泳中的比较研究

用SDS—不连续聚丙烯酰胺凝胶板电泳、pH4.5—不连续聚丙烯酰胺凝胶板电泳和聚丙烯酰胺凝胶圆盘等电聚焦电泳,分离我国不同地区东亚钳蝎毒。其图谱表明:不同产地的东亚钳蝎毒蛋白的种类及含量存在一定的差异,地区相近,差异较小,反之亦然。     

我国不同地区东亚钳蝎（Buthus martensii Karsch）毒在三种…

用ＳＤＳ－不连续聚丙烯酰胺凝胶板电泳、ｐＨ４．５－不连续聚丙烯酰胺凝胶板电泳和聚丙烯酰胺凝胶圆盘等电聚焦电泳,分离我国不同地区东亚钳蝎毒。其图谱表明：不同产地的东亚钳蝎毒蛋白的种类及含量存在一定的差异,地区相近,差异较小,反之亦然。     

一种提取小鼠表皮总RNA的新方法

目的:建立一种从小鼠表皮组织提取高质量RNA的方法。方法:用热击法分离小鼠表皮,用TRIzol法提取RNA,用紫外分光光度计测定RNA的产率和纯度,用琼脂糖电泳和RT-PCR检测RNA的质量和完整性。结果:采用新方法提取的小鼠表皮总RNA,其D260nm/D280nm值为1.8～2.0,大于1.5,且RNA产率高于100μg/g;琼脂糖电泳出现5S、18S和28S等3条清晰的rRNA条带,而且28SrRNA条带的亮度约为18S的2倍;用新方法制备的总RNA可成功地用于RT-PCR实验。结论:采用热击法分离表皮并结合TRIzol法可提取到高质量、完整性好的小鼠表皮总RNA,并能用于相关的分子生物学实验。     

牦牛血超氧化物歧化酶电泳多谱带现象研究

采用聚丙烯酰胺凝胶电泳、ＳＤＳ－聚丙烯酰胺凝胶电泳、线性梯度聚丙烯酰胺凝胶电泳、制备电泳、等电聚焦电泳以及不同试剂处理等多种方法对牦牛血铜锌超氧化物歧化酶电泳多谱带现象进行了研究。结果表明在不同的实验条件下,各带会发生相互转化。该多谱带现象是由聚合和构象异构体两种原因造成。     

植物组织中蛋白质及同功酶的聚丙烯酰胺凝胶盘状电泳

聚丙烯酰胺凝胶电泳是鉴定蛋白质、酶的有力工具,是生物化学研究上不可缺少的实验技术。聚丙烯酰胺凝胶是一种合成的凝胶,是由丙烯酰胺单体和交联剂甲义丙烯酰胺在催化剂的作用下聚合而成的大分子。不同的甲义丙烯酰胺在和丙烯酰胺浓度可制成孔径不同大小的电泳基质。电泳时蛋白质混     

Molecular forms of acetylcholinesterase in developing Torpedo embryos

We have studied the evolution of acetylcholinesterase molecular forms during the embryonic development of Torpedo marmorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, ‘myogenic phase’ of electric organ development), globular forms of acetylcholinesterase (G₄ and G₂) are abundant in both tissues and the collagen-tailed form A₁₂ is already present. In the electric organs, this form accumulates rapidly after the 55–60 mm stage (‘electrogenic phase’), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCl₂, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form A₈ shows a transient increase which coincides approximately with the maximal accumulation of A₁₂, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G₂, which eventually becomes predominent in the adult electric organs, lags behind that of A₁₂. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of A₁₂. In the electric lobes, the tetrameric form (G₄) is abundant during development, as well as G₂ and G₁ at certain stages, but the A₁₂ form is predominant in the adult.     

超嗜热古菌Thermococcus eurythermalis A501编码B家族DNA聚合酶的生化特性及PCR应用

DNA聚合酶广泛应用于PCR技术,在生命科学研究及相关领域发挥重要作用。但目前商业化DNA聚合酶仍不能完全满足科研需要,有必要寻求高性能DNA聚合酶。文中克隆表达了超嗜热古菌（Thermococcus eurythermalis）A501来源的B家族DNA聚合酶基因（NCBI数据库基因登录号为TEU＿RS04875）、表征该重组蛋白的生化特性、评价了其PCR应用。将删除intein蛋白序列的DNA聚合酶（Teu-PolB）进行体外重组表达,经亲和层析和离子交换层析纯化获得Teu-PolB蛋白;利用5′端带荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳鉴定Teu-PolB的生化特性;以噬菌体λDNA基因组为模板,探究Teu-PolB的PCR应用。结果显示,Teu-PolB具有DNA聚合酶活性和3′→5′核酸外切酶活性,该酶在98℃下的半衰期约为2 h,热稳定性高。使用Teu-PolB进行PCR扩增,最适PCR缓冲液为50 mmol/L Tris-HCl pH 8.0,2.5 mmol/L MgCl₂,60 mmol/L KCl,10 mmol/L （NH<...     

Bioconversion of phospholipids by immobilized phospholipase A2

Phospholipase A₂ selectively hydrolyses the ester linkage at the sn-2 position of phospholipids forming lysocompounds. This bioconversion has importance in biotechnology since lysophospholipids are strong bioemulsifiers. The aim of the present work was to study the kinetic behaviour and properties of immobilized phospholipase A₂ from bee venom adsorbed into an ion exchange support. The enzyme had high affinity for CM-Sephadex^® support and the non-covalent interaction was optimum at pH 8. The activity of immobilized phospholipase A₂ was comparatively evaluated with the soluble enzyme using a phospholipid/Triton X-100 mixed micelle as assay system. The immobilized enzyme showed high retention activity and excellent stability under storage. The activity of the immobilized system remained almost constant after several cycles of hydrolysis. Immobilized phospholipase A₂ was less sensitive to pH changes compared to soluble form. The kinetic parameters obtained (V_max 883.4 μmol mg⁻¹ min⁻¹ and a K_m 12.9 mM for soluble form and V_max = 306 μmol mg⁻¹ min⁻¹ and a K_m = 3.9 for immobilized phospholipase A₂) were in agreement with the immobilization effect. The results obtained with CM-Sephadex^®-phospholipase A₂ system give a good framework for the development of a continuous phospholipid bioconversion process.     

Extracellular exo-polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Exo-polygalacturonase (exo-PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular exo-PGase was purified to electrophoretic homogeneity using DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G-200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)-PAGE after treatment with SDS and 2-mercaptoethanol. The isoelectric point was at pH 6.2. The K_m and V_max values for polygalacturonate (degree of polymerization: 52) were 14.4 μ M and 25.6 μmol (mg protein)⁻¹ h⁻¹, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba²⁺, Cu²⁺, Mn²⁺ and Hg²⁺. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

构建噬菌体展示抗体库过程中电穿孔法的条件优化

目的：确定稳定且高效的电转化实验方案以达到构建高库容噬菌体展示抗体库的目的。方法：探索电压、脉冲时间、噬菌粒DNA质量与浓度、TG1大肠杆菌的生长周期、重悬洗涤缓冲液及培养基优化等方面对TG1大肠杆菌电转化效率的影响。结果：当电转杯电极间距为2mm时,设定电转仪参数为3kV、25μF、5ms、200Ω;外源DNA经纯化后加入感受态菌液中使终浓度为1ng/μl;培养基中加入20mmol/L的MgCl₂,并将TG1的生长阶段调控在OD₆₀₀=0.8,用无菌超纯水重悬及洗涤细胞,将感受态细胞浓度调整为4×10¹⁰个/ml。在上述条件下,电转化效率可达到4.9×10⁹CFU/μg DNA。结论：通过多种条件优化,提高了电转化效率,为构建高库容噬菌体展示抗体库建立了基础。     

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: Characterization by radioligand binding and photoaffinity labelling

A₁ adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[³H]dipropyl-xanthine ([³H]DPCPX) gave a K_d value of 0.7 nM and a B_max value of 82 ± 13 fmol/mg protein. For the highly A₁-selective agonist 2-chloro-N⁶-[³H]cyclopentyladenosine ([³H]CCPA) a K_d value of 1.7 nM and a B_max value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [³H]DPCPX binding gave a pharmacological profile with R-N⁶-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5′-N-ethylcarboxamido-adenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N⁶-¹²⁵I-p-hydroxyphenylisopropyladenosine ([¹²⁵I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A₁ receptors labelled in brain membranes. Therefore, coated vesicles contain A₁ adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A₁ receptors in these vesicles are not a processed receptor form. These results confirm that A₁ receptors in coated vesicles are coupled to a G-protein, and it appears that the A₁ receptor systems in coated vesicles and in plasma membranes are identical.     

UV effects on photosynthesis and phycobiliprotein composition in the flagellate Cyanophora paradoxa

Abstract The effects of solar and artificial ultraviolet radiation on photosynthetic oxygen production and phycobiliprotein composition were investigated in the freshwater flagellate, Cyanophora paradoxa . The phycobiliproteins of the cell acting as photosynthetic accessory pigments were found to be readily affected by even short exposure to ultraviolet radiation, while the membrane-bound chlorophyll protein complexes were hardly impaired as demonstrated by SDS polyacrylamide gel electrophoresis, isoelectric focussing and fast protein liquid chromatography (FPLC). The phycobilisomes are dissembled from the outside inwards and go from higher molecular weight components to hexamers (αβ)₆, trimers (αβ)₃ and finally monomers (αβ). Fluorescence spectra indicated that the energy transfer from accessory pigments to the photosystems was impaired by ultraviolet radiation. Photosynthetic oxygen production was affected on a much faster timescale than changes in the absorption spectra or at the protein level.     

Phospholipase A2 in human placental serum

Intervillous blood was collected from term placentae at delivery, and sera were tested for phospholipase A₂ under various experimental conditions. Enzyme activity was found to develop upon extended storage in the cold or at 37°C. The enzyme is reversibly inhibited by dithiothreitol, requires Ca⁺⁺ ions for activity, and tolerates various detergents. The apparent molecular weight is 42 kDa. In all these parameters the serum enzyme behaves similar to the 42 kDa phospholipase A₂ which we recently purified to homogeneity from thoroughly washed placental tissue. Serum phospholipase A₂ appears to be generated by proteolytic processing from a slightly larger inactive precursor which was detected immunochemically. Most likely this protein originates from fetal cells and may be released by membrane damage. We conclude that both placental serum and tissue harbour a novel type of phospholipase A₂ which is distinct from cytosolic and secretory phospholipases A₂. Preference for arachidonate containing substrate suggests a role in eicosanoid production within gestational tissues.