Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.     

磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定 ,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程 ,在细胞中磷蛋白含量低 ,磷酸化位点可变 ,且磷酸肽的质谱信号常常会受到抑制 ,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前 ,质谱仍然是核心的鉴定技术 ,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定 ,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展 ,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

蛋白质组双向电泳实验中一些常见失误的分析

从以固相pH梯度等电聚焦为第一向,SDS-PAGE电泳为第二向进行的蛋白质组双向电泳实验中挑选了一些常见的硝酸银染色2-DE失误图谱,将它们进行分类,初步分析造成各类失误的可能原因,探讨相应的解决方法和总结实验操作中应该注意的事项．     

大肠癌双向电泳分离条件的建立及其初步分析

采用固相pH梯度等电聚焦为第一向,SDS-PAGE垂直电泳为第二向建立了大肠癌双向电泳分离技术实验条件,对样品的处理、水化、等电聚焦、凝胶平衡等步骤进行了优化,成功地获得了大肠癌分辨率高、重复性好的双向电泳图谱.癌组织样品经3次重复实验共获得蛋白质斑点数1 186±46个,蛋白质斑点位置在IEF方向平均偏差为1.67±0.29 mm,在SDS-PAGE方向为1.41±0.16 mm,蛋白质表达量的相对标准差为6.67 %±2.25 %.经ImageMaster 2D Elite软件初步分析后发现了一些差异表达的蛋白质.     

糖组学研究技术及其进展

多细胞生物机体内,蛋白质糖基化是一个重要后修饰事件 . 蛋白质的糖链不仅仅是区别细胞种类的标志,且与众多的生物现象有关,如细胞发育、分化、形态、肿瘤转移、微生物感染等 . 糖组学的内容主要涉及单个个体的全部糖蛋白结构分析,确定编码糖蛋白的基因和蛋白质糖基化的机制 . 综述了糖组学的分离和结构鉴定技术及其最新进展 .     

Functional Compartmentalization of the Translation Apparatus and Channeling of tRNA/Aminoacyl-tRNA in Cells of Higher Eukaryotes

Literature data and authors' results on the structural and functional organization of the translation apparatus in higher eukaryotes are considered. Proofs are presented of the channeling of tRNA/aminoacyl-tRNA in the course of eukaryotic protein synthesis. The concept of the shuttle role of eEF1A is grounded; the factor, being in a GTP-bound form, delivers aminoacyl-tRNA to the ribosome and then, in the having undergone to a GDP -form after hydrolysis of GTP on the ribosome, forms a complex with the deacylated tRNA and delivers it to the aminoacyl-tRNA synthetase. The notion of a translational compartment is defined.     

Ternary complex between elongation factor Tu•GTP and Phe-tRNA

The effect of aminoacylation and ternary complex formation with elongation factor Tu•GTP on the tertiary structure of yeast tRNA^Phe was examined by ¹H-NMR spectroscopy. Esterification of phenylalanine to tRNA^Phe does not lead to changes with respect to the secondary and tertiary base pair interactions of tRNA. Complex formation of Phe-tRNA^Phe with elongation factor Tu•GTP results in a broadening of all imino proton resonances of the tRNA. The chemical shifts of several NH proton resonances are slightly changed as compared to free tRNA, indicating a minor conformational rearrangement of Phe-tRNA^Phe upon binding to elongation factor Tu•GTP. All NH proton resonances corresponding to the secondary and tertiary base pairs of tRNA, except those arising from the first three base pairs in the aminoacyl stem, are detectable in the Phe-tRNA^Phe•elongation factor Tu•GTP ternary complex. Thus, although the interactions between elongation factor Tu and tRNA accelerate the rate of NH proton exchange in the aminoacyl stem-region, the Phe-tRNA^Phe preserves its typical L-shaped tertiary structure in the complex. At high (> 10⁻⁴ M) ligand concentrations a complex between tRNA^Phe and elongation factor Tu•GDP can be detected on the NMR time-scale. Formation of this complex is inhibited by the presence of any RNA not related to the tRNA structure. Using the known tertiary structures of yeast tRNA^Phe and Thermus thermophilus elongation factor Tu in its active, GTP form, a model of the ternary complex was constructed.     

双向凝胶电泳缺陷胶及其原因

双向凝胶电泳是蛋白质组学研究中的关键技术之一,涉及较多的实验步骤和试剂,常出现缺陷胶而导致实验失败。从数千张电泳胶图中选取了21张典型的缺陷胶图,对它们进行了归类和总结,分析和讨论了形成缺陷胶的多种原因,提出了实验改进的方法和注意事项。     

用于双向凝胶电泳分析的奶牛乳清蛋白样品制备方法的比较

为建立适用于双向凝胶电泳分析的奶牛乳清蛋白的制备方法,分别比较了直接裂解法、三氯乙酸-丙酮法,Trizol法和2-D clean up kit法对奶牛乳清蛋白提取效率和双向凝胶电泳图谱的影响.用2-D Quant Kit试剂盒测定蛋白浓度,分别用十二烷基磺酸钠 聚丙烯酰胺凝胶电泳和双向凝胶电泳进行奶牛乳清蛋白的分离.蛋白定量结果表明,2-D clean up kit法产率最高,直接裂解法、三氯乙酸-丙酮法次之,trizol法产率最低;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳结果表明,2- D clean up kit法提取的蛋白质量最高;双向电泳图谱分析表明,2-D clean up kit法得到的蛋白图谱与另外3种方法相比,检测到的蛋白点最多,图谱背景清晰,分辨率最高.结果提示,2-D clean-up法相对最适合于双向凝胶电泳分析奶牛乳清蛋白样品的制备,尤其对一些低丰度高分子量蛋白的分离效果较为明显.     

成年和老年小鼠脑蛋白质组双向电泳图谱比较

使用双向电泳(2-DE)比较成年和老年小鼠脑蛋白质差异,从分子水平初步探索老年脑蛋白整体变化规律.以固相pH梯度等电聚焦为第一向,SDS-聚丙烯酰胺凝胶水平电泳(PAGE)为第二向进行2-DE.图象分析软件Imagemaster^® 2D Elite分析电泳图谱.重复性实验结果显示,同组样品在三次不同实验中所得蛋白质斑点数目的相对标准差（变异系数）为4.43%±0.25%;同一蛋白质斑点在三次实验中等电点、分子质量和蛋白质量的相对标准差分别为8.76%±5.14%, 13.00%±4.22%和10.84%±9.16%.成年和老年小鼠脑组织2-DE图谱分别获得996和1256个蛋白质斑点,其中8个蛋白质在老年脑组织中含量降低,20个蛋白质斑点含量增加.另至少有4个蛋白质斑点在老年脑组织中缺失,14个蛋白质点为老年脑特有. 以上差异点的发现为研究脑老化和退行性疾病机理提供了有益的线索.     

蛋白质组分析中蛋白质分步提取方法的建立

利用细胞裂解液充分溶解细胞蛋白质是成功进行蛋白质组分析的先决条件.尝试利用三步提取法,即以三种溶解性能不同的裂解液分步提取细胞中的蛋白质组,并分别进行二维聚丙烯酰胺凝胶电泳（two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE）分离.通过对2-D PAGE蛋白质图谱的比较,发现其与常规方法相比,具有蛋白质提取率高、双向电泳(two-dimensional electrophoresis,2-DE)分辨率高等优点.     

The nitrate/nitrite ABC transporter of Phormidium laminosum: Phosphorylation state of NrtA is not involved in its substrate binding activity

Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr²⁰³, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.     

Comparative effects of hepatocyte growth factor and epidermal growth factor on motility,morphology, mitogenesis,and signal transduction of primary rat hepatocytes

Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) are major hepatacyte mitogens, but HGF, also known as scatter factor (SF), has also been shown as a potent motogen for epithelial and endothelial cells. The mechanisms by which HGF is a stronger motogen compared to other mitogens are not understood. Here we report a comparative study of the effect of the two growth factors on cultured primary rat hepatocytes regarding their differential effects on morphology, mitogenicity, and motility as well as the phosphorylation of cytoskeletal-associated proteins. Using three different motility assays, both HGF and EGF increased the motility of hepatocytes, but HGF consistently elicited a significantly greater motility response than EGF. Additionally, HGF induced a more flattened, highly spread morphology compared to EGF. To examine if HGF and EGF phosphorylated different cytoskeletal elements as signal transduction targets in view of the observed variation in morphology and motility, primary cultures of ³²P-loaded rat hepatocytes were stimulated by either HGF or EGF for up to 60 min. Both mitogens rapidly stimulated four isoforms of MAP kinase with similar kinetics and also rapidly facilitated the phosphorylation of cytoskeletal-associated F-actin. Two cytoskeletal-associated proteins, however, were observed to undergo rapid phosphorylation by HGF and not EGF during the time points described. One protein of 28 kDa was observed to become phosphorylated fivefold over controls, while the EGF-stimulated cells showed only a slight increase in the phosphorylation of this protein. Another protein with an apparent mwt of 42 kDa was phosphorylated 20-fold at 1 min and remained phosphorylated over 50-fold over control up to the 60 min time point. This protein was observed to become phosphorylated by EGF only after 10 min, and to a lesser extent (20-fold). Taken together, the data suggest that HGF and EGF stimulate divergent as well as redundant signal transduction pathways in the hepatocyte cytoskeleton, and this may result in unique HGF- or EGF-specific motility, morphology, and mitogenicity in hepatocytes. © 1994 Wiley-Liss, Inc.     

定量蛋白质组学分析方法及应用

蛋白质组学经历了近10年的发展,现在已经初具规模。但是由于它是动态地观察生物体不断变化的所有蛋白质,所以技术难度非常之大。为使研究简化并更具针对性,人们着重进行比较蛋白质组学的研究。为了具体量化这些蛋白质的变化产生了定量蛋白质组学,近几年各种标记技术的进步使得该学科得以迅猛发展。     

蛋白质组学在细菌应激反应研究中的应用

当外部生存环境发生变化时,细菌会在短时间内发生应激反应。利用双向凝胶电泳技术结合生物质谱鉴定的方法对细菌蛋白表达谱变化进行研究,是细菌转录谱变化研究的深入和扩展,是细菌应激反应研究中的新热点。综述了蛋白质组学在细菌应激反应研究中的应用现状和存在问题。     

Concepts and approaches towards understanding the cellular redox proteome

The physiological activity of a significant subset of cell proteins is modified by the redox state of regulatory thiols. The cellular redox homeostasis depends on the balance between oxidation of thiols through oxygen and reactive oxygen species and reduction by thiol-disulfide transfer reactions. Novel and improved methodology has been designed during recent years to address the level of thiol/disulfide regulation on a genome-wide scale. The approaches are either based on gel electrophoresis or on chromatographic techniques coupled to high end mass spectrometry. The review addresses diagonal 2D-SDS-PAGE, targeted identification of specific redox-interactions, affinity chromatography with thioredoxins and glutaredoxins, gel-based and non-gel based labelling techniques with fluorophores (such as Cy3, Cy5, ICy), radioisotopes, or with isotope-coded affinity tags (ICAT), differential gel electrophoresis (DIGE) and combined fractional diagonal chromatography (COFRADIC). The extended methodological repertoire promises fast and new insight into the intricate regulation network of the redox proteome of animals, bacteria, and plants.     

Analysis of leaf proteins in late flowering mutants of Arabidopsis thaliana

Late flowering monogenic mutants of Arabidopsis thaliana (L.) Heynh. at the loci co, gi, fca, fve, fwa, fha, fpa, fy and their corresponding wild type, Landsberg erecta , were analysed by two-dimensional gel electrophoresis. All plants were grown under continuous light and proteins were extracted from leaves of the same age (20-day-old). The polypeptide patterns of the mutants at the loci co, gi, fca, fve, fwa, fha, fpa , and Landsberg erecta were identical. The mutant at the fy locus showed a qualitative difference with Landsberg erecta . Crosses were made between this line and the wild type Landsberg erecta . F2 plants, resulting from autopollination of the hybrid, were analysed and showed no cosegregation between the observed protein and the flowering phenotype, indicating that these two lines differ by more than a single mutation.     

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-termin vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extendedin vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.     

采用亲和层析结合制备凝胶电泳获得高纯度的重组抗原蛋白用于制备蛋白质芯片

为了得到制备抗原芯片所需的高纯度重组抗原蛋白,需要建立一套适合于多种重组抗原表达和纯化的技术路线．采用了亲和层析结合制备胶电泳的方法,对16种用于构建蛋白质芯片的食管癌相关抗原基因进行了克隆重组并在大肠杆菌中进行了表达．对高表达的重组蛋白首先制备包涵体,然后采用Ni-Sepharose亲和层析得到初步纯化的蛋白质,最后使用SDS-PAGE制备胶电泳作进一步纯化．经过透析复性后,用于制备蛋白质芯片．采用亲和层析纯化重组蛋白,得率为71% ,纯度约为70%;在SDS-PAGE制备胶进一步纯化后,得率为32%,纯度为95%,经过透析和复性后,最终得率为21%,纯度为95%．得到的重组蛋白RPS4在ELISA检测中可以和血清中识别RPS4 的自身抗体起反应,并且,采用精纯抗原制备的蛋白质芯片,在检测抗原与抗体这一对反应中也具有较高的敏感性和特异性,适合大规模血清抗体的检测．研究表明,采用亲和层析结合制备凝胶电泳纯化抗原蛋白,是一条简便快捷,适合需要量不大,但对纯度要求比较高的蛋白质芯片制备的技术路线．