运动后尿液蛋白质分子量与等电点的变化特征

通过对９名男性受试者在分别完成１００－２００ｍ,４００－８００ｍ和１ ５００－３ ０００ｍ跑步间歇训练后尿蛋白分子量和等电点的测定发现：①运动时尿液高、低分子量蛋白质排泄率均较运动前明显增加,但以高分子量蛋白质排泄为主;②运动时尿液高、低分子量蛋白质排泄率均以４００－８００ｍ间歇训练时最高,１００－２００ｍ间歇训练时次之,１ ５００－３ ０００ｍ间歇训练时最低;③运动时尿液排出的蛋白质以负离子为主     

五龄家蚕若干组织器官蛋白质数据库构建

为构建家蚕蛋白质数据库,达到从蛋白质水平研究基因的表达及表达产物的加工修饰的目的,采用五龄家蚕休体壁、中肠和脂肪为材料获取蛋白质样品,用蛋白质双向电泳技术分离、纯化蛋白质,对其中的40个蛋白质斑点进行了氨基酸序列分析及同源性检索,发现其中58.5％的蛋白质与果蝇的有关蛋白质具有较高同源性,36.5％与其他生物的蛋白质具有较高同源性,只有5％与家蚕已知蛋白拷贝产高同源性,研究发现有27个蛋白质的N－末端氨基酸序列在家蚕中是第一次被检测到,并通过互联网向SWISS－PROT数据库进行了登录。研究证明利用蛋白质数据库的结果,可以得到基因表达及其产物修饰的信息。     

蛋白质组学技术及其在肺脏疾病研究中的应用

蛋白质组学是旨在研究蛋白质表达谱和蛋白质与蛋白质之间相互作用的新领域,其研究必须依赖高通量、高自动化的技术.简要介绍了蛋白质组分离技术(双向电泳、色谱),蛋白质组分析技术(质谱分析、氨基酸组成分析、蛋白质芯片,Edman降解法测N端序列),蛋白质相互作用技术(酵母双杂交系统、表面等离子共振)以及生物信息学.并从寻找差异表达的蛋白质,寻找用于诊断的疾病相关的标记分子,研究疾病的发病机制三方面介绍了蛋白质组技术在肺脏疾病研究中的应用.     

N-乙酰氨基葡萄糖化在信号转导中的作用

蛋白质磷酸化在生命活动以及信号转导过程中的重要作用已经被研究证实,但不少研究发现在大多数核,胞液蛋白质上不仅存在磷酸化动态修饰,还存在广泛的动态N－乙酰氨基葡萄糖修饰,N－乙酰氨基葡萄糖基转移酶和N－乙酰氨基葡萄糖基酶以类似于蛋白质激酶和磷酸酶的方式调节蛋白质是否发生N－乙酰氨基葡萄糖化。N－乙酰氨基葡萄糖化蛋白质主要分布在细胞核与胞液,其生理功能涉及细胞基本生命活动和调节信号传递。N－乙酰氨基葡萄糖的作用基础与阻断或影响蛋白质的磷酸化有关。     

光合作用系统II外周蛋白——P23k去折叠的热力学和动力学

利用荧光光谱学等方法结合高压力技术研究了光合作用系统II中的一个外周蛋白——— 2 3kD(以P2 3k表示 )蛋白的去折叠。热力学研究表明 ,在 2 0℃、180MPa(1MPa =10 .0大气压 )可使该蛋白质完全去折叠 ,而在3℃ ,16 0MPa即可使该蛋白质完全去折叠 ,这是迄今为止有关研究中最易被高压力去折叠的一个蛋白质。在2 0℃ ,该蛋白质在常压下去折叠反应的标准自由能与标准体积变化分别为 2 3.4 5kJ mol和 - 15 0 .3ml mol;动力学研究揭示该蛋白质的折叠反应的活化体积ΔV f 为正值 (84 .1ml mol) ,而去折叠反应的活化体积ΔV u 为负值(- 6 6 .2ml mol)。在常压下 ,折叠和去折叠反应的速度常数 (K0f,K0u)分别为 1.87s- 1 和 1.3× 10 - 4s- 1 ,这些结果为解释该蛋白质易被压力去折叠提供了线索     

蛋白质组学在木薯育种中的应用

蛋白质组学通过研究整体蛋白质活动来揭示生命运动规律,是解析功能基因组表达的必要手段。目前蛋白质组学为木薯选育种研究提供了重要手段和新思路。介绍了蛋白质组学研究平台并对其在选育高光效高淀粉积累、高蛋白质、高类胡萝卜素及抗逆木薯种质方面的应用进行了综述。     

核酸－蛋白质互作的生物化学研究方法

研究核酸－蛋白质的互作是揭示生命活动机理的基础, 文章简要综述了用于研究核酸－蛋白质互作的各种生物化学方法。从体内、体外两个研究角度, 针对核酸、蛋白以及复合物3个研究水平, 概述了硝化纤维膜过滤实验、足迹法、EMSA、Southwestern杂交等经典分析方法的原理、发展和运用。还着重介绍了最近在表观遗传学领域中广泛运用的nChIP、xChIP等基本染色质免疫沉淀(ChIP)技术及其衍生出的ChIP-on-chip等方法。     

基因敲除技术对5－HT受体研究的新贡献

基因敲除技术对５－ＨＴ受体研究的新贡献随着５－ＨＴ受体家族的扩大,寻找高选择性的受体激动剂及阻断剂日趋困难。基因药理的研究对象不是特定的受体蛋白质,而是编码蛋白质的基因或ｍＲＮＡ．通过基因敲除技术,可以繁殖出变异鼠,使之缺少某种特定基因,从而使之不能...     

以麦芽根为原料发酵生产单细胞蛋白研究初报

以麦芽根为原料发酵生产单细胞蛋白研究初报刘海滨（烟台大学生化系．烟台）单细胞蛋白（Ｓｉｎｇｌｅ－ｃｅｌｌｐｒｏｔｅｎ,ＳｃＰ）是近年来生物技术方面研究比较活跃的领域之一。与传统的蛋白质原料相比,单细胞蛋白具有蛋白质含量高、氨基酸组成齐全、富含维生素并...     

新型表达载体 pEC34 试用于人 γ 干扰素基因的高表达

构建了一个新的双顺反子表达载体ｐＥＣ３４,它的第一个顺反子是已高表达的ｅｒａ基因的部分序列,并带有原核翻译增强子。当检测ｐＥＣ３４的可用性时,获得了人γ－干扰素的极高表达。目的蛋白占菌体总蛋白的８５．５％,诱导后经ＳＤＳ－ＰＡＧＥ分析,出现分子量为１５．５ｋＤａ左右的蛋白表达带。Ｗｅｓｔｅｒｎ ｂｌｏｔ结果证实为人γ－干扰素。无压力传代３０次表达质粒仍然稳定,表明ｐＥＣ３４可能用作真核蛋白质高表达     

DNA及其蛋白质复合体的电镜观察及研究应用

对生物大分子形状的正确了解有助于对其功能的解析。事实上,借助于电子显微镜观察法,使我们在对构成细胞各组分功能的探讨和研究方面收益匪浅。自从生物学家们认识到DNA在细胞行使其功能中的重要性以来,电子显微镜便成为一种最重要的技术手段而被用于分析DNA的结构,研究DNA的复制、重组和转录机制。近年来,这一领域的研究技术又有了非常大的改进,使人们已经能够借助于电子显微镜,观察介入DNA复制、重组和转录等过程中的DNA蛋白质复合体的活体状态。今后,在对生物大分子的活体行为进行探讨时,电子显微镜技术将发挥越来越重要的作用。     

beta-lactoglobulin under high pressure studied by small-angle neutron scattering

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.     

Dynamic light scattering: a practical guide and applications in biomedical sciences

Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a very powerful tool for studying the diffusion behaviour of macromolecules in solution. The diffusion coefficient, and hence the hydrodynamic radii calculated from it, depends on the size and shape of macromolecules. In this review, we provide evidence of the usefulness of DLS to study the homogeneity of proteins, nucleic acids, and complexes of protein–protein or protein–nucleic acid preparations, as well as to study protein–small molecule interactions. Further, we provide examples of DLS’s application both as a complementary method to analytical ultracentrifugation studies and as a screening tool to validate solution scattering models using determined hydrodynamic radii.     

Denatured State Structural Property Determines Protein Stabilization by Macromolecular Crowding: A Thermodynamic and Structural Approach

Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total macromolecular concentration never surpasses 10 g l⁻¹. However, biological macromolecules are known to evolve and function under crowded intracellular environments that comprises of proteins, nucleic acids, ribosomes and carbohydrates etc. Crowded environment is known to result in altered biological properties including thermodynamic, structural and functional aspect of macromolecules as compared to the macromolecules present in our commonly used experimental dilute buffers (for example, Tris HCl or phosphate buffer). In this study, we have investigated the thermodynamic and structural consequences of synthetic crowding agent (Ficoll 70) on three different proteins (Ribonuclease-A, lysozyme and holo α-lactalbumin) at different pH values. We report here that the effect of crowding is protein dependent in terms of protein thermal stability and structure. We also observed that the structural characteristics of the denatured state determines if crowding will have an effect or not on the protein stability.     

Characterization of dynamic behavior of side groups of globular proteins by spin-label, NMR and luminescence polarization methods

In this work we suggest a quantitative estimation of a complicated motion of side groups of globular proteins. In the general case, three basic parameters determine the motion: (a) rotational correlation time of a side unit under study, or covalently bound spin label, or dye, (b) parameter S that reflects sterical restrictions for re-orientation of the given unit (these two parameters depending on the side-chain structure and its conformational change within the immediate dynamic protein surrounding whereas correlation times of side units on microviscosity in addition), (c) rotational correlation time of protein globule. These parameters can be measured by spin-label, NMR and fluorescence polarization techniques. An attempt to describe a complicated dynamic behaviour of side units of protein macromolecules with a single dynamics parameter--rotational correlation time--not only leads to a loss of part of information about the local structural dynamics of macromolecules but also can diminish the tau value.     

Turnover of cell-surface macromolecules in cultured dog tracheal epithelial cells

We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.     

DNA-binding and enzymatic domains of the bifunctional biotin operon repressor (BirA) of Escherichia coli

The negative regulation of the biotin biosynthetic (bio) operon in Escherichia coli is mediated by the bifunctional birA gene product, which serves as the bio repressor and biotin-activating enzyme. Nucleotide sequence analysis of 18 mutations in the birA gene was employed to study the DNA-binding and enzymatic functions of the BirA protein. The results indicate that a predicted helix-turn-helix structure, from amino acid (aa) positions 18 to 39 within the 321-aa BirA protein, may be responsible for sequence-specific DNA binding, whereas the temperature-sensitive mutations affecting biotin activation are found in two regions from aa positions 83-119 and 189-235.     

High pressure simulations of biomolecules

Pressure is a thermodynamic variable which is particularly suitable for exploration of the properties of biological macromolecules. For proteins, in particular, denaturation induced by pressure is different from that induced by temperature or denaturants. The response of proteins to pressure changes can provide information on properties of their native and non-native states. This review focuses on molecular dynamics studies of the effect of pressure on detailed atomic models of proteins. It also reports on other theoretical approaches, such as Monte Carlo simulations, which have been used to study simplified models. Another purpose of this review is to try to point out potential future studies that may be both interesting and feasible, with constantly increasing computing power.     

SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

The Arabidopsis SUC5 protein represents a classical sucrose/H⁺ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co‐factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis‐defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild‐type plants. Although suc5 mutants were like the wild‐type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5.