Ribosomal proteins in halobacteria

The amino acid sequences of 16 ribosomal proteins from archaebacterium Halobacterium marismortui have been determined by a direct protein chemical method. In addition, amino acid sequences of three proteins, S11, S18, and L25, have been established by DNA sequencing of their genes as well as by protein sequencing. Comparison of their sequences with those of ribosomal proteins from other organisms revealed that proteins S14, S16, S19, and L25 are related to both eukaryotic and eubacterial ribosomal proteins, being more homologous to eukaryotic than eubacterial counterparts, and proteins S12, S15, and L16 are related to only eukaryotic ribosomal proteins. Furthermore, some proteins are found to be similar to only eubacterial proteins, whereas other proteins show no homology to any other known ribosomal proteins. Comparisons of amino acid compositions between halophilic and nonhalophilic ribosomal proteins revealed that halophilic proteins gain aspartic and glutamic acid residues and significantly lose lysine and arginine residues. In addition, halophilic proteins seem to lose isoleucine as compared with Escherichia coli ribosomal proteins.     

Isolation and characterization of the major proteins of ram seminal plasma

Mammalian seminal plasma contains among others, two major families of proteins, namely spermadhesins and those proteins that contain fibronectin type II domains. Spermadhesins are the major proteins of boar and stallion seminal plasma and homologous proteins have been identified in the bull. These proteins appear to be involved in capacitation and sperm-egg interaction. In bovine seminal plasma, proteins containing fibronectin type II domains are the major proteins and are designated BSP proteins. These proteins play a role in sperm capacitation. In this study, we present the isolation and characterization of the major proteins of ram seminal plasma. Precipitated proteins from Suffolk ram seminal plasma were loaded onto a gelatin-Agarose column. The unadsorbed (fraction A) and retarded proteins (fraction B) were removed by washing the column with phosphate buffered-saline and the adsorbed proteins (fraction C) were eluted with 5 M urea. SDS-PAGE of fraction B indicated the presence of a 15.5 kDa protein, which is the major protein of ram seminal plasma (approximately 45% of total protein by weight) and was identified as a spermadhesin by N-terminal sequencing. SDS-PAGE analysis of fraction C revealed the presence of four proteins, which represented approximately 20% of total ram seminal plasma proteins by weight, and were identified as proteins of the BSP family and named RSP proteins. These RSP proteins were designated RSP-15 kDa, RSP-16 kDa, RSP-22 kDa, and RSP-24 kDa. Only RSP-15 kDa and -16 kDa proteins cross-reacted with antibodies against BSP proteins. Ram spermadhesin and RSP proteins interact with heparin but only RSP proteins bind to hen's egg yolk low-density lipoprotein. In conclusion, spermadhesin is the major protein of ram seminal plasma and other major proteins belong to the BSP protein family.     

表位标签技术在膜蛋白研究中的应用

膜蛋白的研究包括埘膜蛋白在细胞内的运输和定位,膜蛋白的结构和功能,以及膜蛋白和其他蛋白质间的相互作用等方面的研究.在研究过程中,如果能够基于膜蛋白的拓扑学结构预测,选择合适的表位标签,利用基因融合技术在基因水平上对膜蛋白进行改造,可以产生含有表位标签的重组膜蛋白,不仅具有原有膜蛋白的功能活性,还能够被抗体特异性识别,并且结合相关的免疫荧光检测技术,将会极大地促进膜蛋白的结构和功能研究.本文就目前膜蛋白研究中所涉及的表位标签技术及其应用策略和所取得的进展作一简述.     

抗真菌蛋白研究进展

对几种抗真菌蛋白 :病程相关蛋白、防卫素、核糖体失活蛋白、几丁质连接蛋白、蛋白酶抑制剂等的类型、特征、抗菌菌机理进行了阐述 ,着重讨论了病程相关蛋白和核糖体失活蛋白的最新研究进展 ,并且讨论了抗真菌蛋白在植物真菌病害综合防治中的应用前景。     

Characterization of the proteins of guinea-pig hair and hair-follicle tissue

This work forms a part of a study of the mechanism and control of protein synthesis in the hair follicle and concerns the characterization of the proteins of hair-follicle tissue and for comparative reasons those of the hair itself. 1. Five different groups of reduced carboxymethylated proteins were delineated from both tissues; these were: group 1A proteins, which appeared to be aggregates of the group 2 proteins; group 1B proteins, soluble at pH4.4, which were thought to originate from the medulla and inner-rootsheath layers; group 2 proteins, which were defined as the main low-sulphur keratin proteins insoluble at pH4.4; group 3 proteins, the precise origin of which is not known; and the group 4 proteins, which were defined as the main high-sulphur keratin proteins soluble at pH4.4. 2. With the single exception of the group 1B proteins, the types and properties of all hair and hair-follicle proteins were identical as far as could be determined by use of such criteria as multiplicity of components, molecular charge, molecular weight and amino acid composition. 3. Two significant quantitative differences were noted: in follicle extracts there were more group 2 proteins but less group 3 and group 4 proteins than in hair extracts; and secondly, in the follicle group 4 proteins, there were more proteins of lowest molecular weight and S-carboxymethylcysteine content, but fewer proteins of the highest molecular weight and S-carboxymethylcysteine conent than in the hair group 4 proteins. 4. These quantitative differences are discussed in terms of the mechanism of synthesis of the keratin proteins. 5. Follicle group 1B proteins are postulated to have arisen from the trichohyalin droplets of the developing medulla and inner-root-sheath layers of the follicle and may be precursors of the proteins of the mature medulla and inner root sheath.     

The centrosome is a polyfunctional multiprotein cell complex

Contemporary knowledge about centrosome proteins and their ensembles, which can be divided into several functional groups--microtubule-nucleating proteins, microtubule-anchoring proteins, centriole-duplication proteins, cell cycle control proteins, primary cilia growth regulation proteins, and proteins of regulation of cytokinesis--is reviewed. Structural-temporal classification of centrosomal proteins and the scheme of interconnection between the different centrosomal protein complexes are presented.     

~(60)Co-γ射线诱发皖麦50 Glu-1A亚基等位基因突变体差异蛋白质组学研究

研究了皖麦50及~(60)Co-γ射线诱发的Glu-1A突变体的比较蛋白质组学,结果表明,突变体蛋白质含量增加,2-D电泳图上有11个蛋白质斑点表达差异显著,其中3个蛋白点上调,8个蛋白点下调。经MALDI-TOF/TOF质谱鉴定,差异蛋白点涉及44个蛋白质。通过GO分析,参与生物过程的差异蛋白中,18个蛋白质参与代谢过程,18个蛋白质参与细胞过程,8个蛋白质参与刺激响应。参与分子功能差异蛋白中,18个蛋白质参与催化活性,10个蛋白质参与链结,7个肽段为分子功能调解剂,9个肽段为未知蛋白。参与细胞成分的差异蛋白中,12个蛋白质为胞外区蛋白,12个蛋白质为细胞,9个蛋白质为细胞器,2个蛋白质为膜,9个蛋白质为未知蛋白。KEGG通路注解表明,差异蛋白质中,4个蛋白质参与淀粉和蔗糖代谢,3个蛋白质参与氨基糖和核苷酸糖代谢,1个参与丙酮循环,1个参与半胱氨酸和甲硫氨酸代谢,1个参与丙酮酸代谢,1个参与乙醛酸和二羧酸代谢,1个参与光合生物碳固定,1个参与碳代谢信息通路。     

An Electrophoretic Analysis of the Seed Protein Body Proteins from Pinus Nigra

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.     

The human erythrocyte proteome: analysis by ion trap mass spectrometry

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.     

2-DE技术中疏水性和碱性蛋白质的研究进展

双向凝胶电泳(2-DE)具有高分辨率、高通量等特点,已被广泛地用于蛋白质组的分离．但是它在分离疏水性蛋白质和碱性蛋白质时却遇到了极大的挑战．然而,疏水性与碱性蛋白质在全蛋白质中占相当大的比例,且具有很重要的生物学意义．因而,近年来,越来越多的研究者将目标瞄准这些蛋白质,并且取得了一些令人鼓舞的进展：用亚细胞预分离技术,顺序提取法等方法来富集疏水性蛋白质,用一些新的有效的增溶剂如硫脲,ASB一14等来改善疏水性蛋白质的溶解,应用这些技术2一DE可分辨出总平均疏水值达O．80的蛋白质;在碱性蛋白质分离方面,通过等电聚焦预处理,使用窄pH梯度胶条等大大地改善了碱性蛋白质在2-DE中的分离,能分辨出等电点达11．7的蛋白质．现对2-DE技术中疏水性和碱性蛋白质分离的研究进展进行综述．     

白桦叶绿体RNA结合蛋白的蛋白质组学分析

在高等植物叶绿体中,RNA结合蛋白在转录后RNA处理、运输以及mRNA的稳定等方面发挥重要作用.本项研究使用多聚腺苷酸(polyA）吸附柱或单链DNA(ssDNA)吸附柱富集白桦叶绿体的polyA结合蛋白或RNA结合蛋白,并通过MALDI-TOF-MS以及ESI MS/MS进行鉴定,13个叶绿体蛋白质得到了鉴定.按照Swiss Prot数据库的注释,这些蛋白质的功能主要包括4个相关种类,分别为NAD结合蛋白、RNA结合蛋白、DNA结合蛋白和ATP结合蛋白.使用这些方法还鉴定出包括转录因子的4个高丰度蛋白.这些结果加深了对树木中叶绿体RNA结合蛋白的全面了解,可以将其应用于其他树木叶绿体中RNA 蛋白质的相互作用的研究.     

全基因组预测目标基因的新方法及其应用

运用隐马尔可夫模型, 利用Perl编程, 以几种模式生物的蛋白质数据库为基础, 构建了目标基因的全基因组预测的新方法。该方法具有高通量, 准确度高且操作简易等优点, 特别在多结构域蛋白家族预测上更显优势。应用该方法对几种模式生物的全基因组PPR和TPR蛋白家族进行了预测, 其中粳稻日本晴中含有536个PPR蛋白、199个TPR蛋白; 籼稻9311中含有519个PPR蛋白、177个TPR蛋白; 拟南芥中含有735个PPR蛋白、292个TPR蛋白; 红藻中6个PPR蛋白、32个TPR蛋白; 蓝细菌以及古细菌中没有PPR蛋白, 但蓝细菌含有10个TPR蛋白, 古细菌有4个TPR蛋白, 并对所得结果进行了进一步生物信息学分析。     

羊布鲁氏菌的分泌蛋白质组分析

分泌蛋白是指那些分泌到细胞外的蛋白质。布鲁氏菌的分泌蛋白可能介导了病原与宿主之间的相互作用, 在布鲁氏菌的毒力方面发挥一定的作用, 但是研究方法的局限性限制了分泌蛋白的研究。本文报道了利用蛋白质组的方法来寻找羊布鲁氏菌的分泌蛋白。首先用TCA-丙酮法提取布鲁氏菌培养上清中的分泌蛋白, 双向电泳进行分离, 然后用质谱来鉴定这些蛋白, 最终鉴定到40种蛋白。通过生物信息学分析, 发现这些蛋白主要是ABC转运系统的底物结合蛋白、外膜蛋白和热休克蛋白。这些蛋白的识别不仅有助于对布鲁氏菌致病机制的理解, 而且也可为     

LC-MS/MS analysis of apical and basolateral plasma membranes of rat renal collecting duct cells

We used biotinylation and streptavidin affinity chromatography to label and enrich proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts (IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-reactive biotinylation reagents at 2 degrees C using a double barreled pipette. The perfusion-biotinylated proteins and proteins bound to them were isolated with CaptAvidin-agarose beads, separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein identification (LTQ, Thermo Electron Corp.). 17 integral and glycosylphosphatidylinositol (GPI)-linked membrane proteins and 44 non-integral membrane proteins were identified. Immunofluorescence confocal microscopy confirmed ACVRL1, H(+)/K(+)-ATPase alpha1, NHE2, and TauT expression in the IMCDs. Basement membrane and basolateral membrane proteins were biotinylated via incubation of IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral membrane proteins preferentially identified in the perfusion-biotinylated and not in the incubation-biotinylated IMCDs revealed protein kinases, scaffold proteins, SNARE proteins, motor proteins, small GTP-binding proteins, and related proteins that may be involved in vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A World Wide Web-accessible database was constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior studies.     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

StaRProtein,A Web Server for Prediction of the Stability of Repeat Proteins

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.     

Partial purification and characterization of GDP dissociation stimulator (GDS) for the rho proteins from bovine brain cytosol

A novel type of regulatory proteins for the rho proteins (rhoA p21 and rhoB p20), ras p21-like small GTP-binding proteins (G proteins), are partially purified from bovine brain cytosol. These regulatory proteins, named rho GDP dissociation stimulator (GDS) 1 and -2, stimulate the dissociation of GDP from rhoA p21 and rhoB p20. rho GDS1 and -2 are inactive for other ras p21/ras p21-like small G proteins including c-Ha-ras p21, smg p21B, and smg p25A. Since we have previously shown that the rate limiting step for the GDP/GTP exchange reaction of the rho proteins is the dissociation of GDP from these proteins, the present results suggest that rho GDS1 and -2 stimulate the GDP/GTP exchange reaction of the rho proteins. rho GDS1 and -2 are distinct from the GAP- and GDI-types of regulatory proteins for the rho proteins previously purified from bovine brain cytosol. rho GAP stimulates the GTPase activity of the rho proteins and rho GDI inhibits the GDP/GTP exchange reaction of the rho proteins. The present results together with these earlier observations indicate that the rho proteins are regulated by at least three different types of regulatory proteins, GDS, GDI, and GAP.     

Characterization of the major hnRNP proteins from Drosophila melanogaster

To better understand the role(s) of hnRNP proteins in the process of mRNA formation, we have identified and characterized the major nuclear proteins that interact with hnRNAs in Drosophila melanogaster. cDNA clones of several D. melanogaster hnRNP proteins have been isolated and sequenced, and the genes encoding these proteins have been mapped cytologically on polytene chromosomes. These include the hnRNP proteins hrp36, hrp40, and hrp48, which together account for the major proteins of hnRNP complexes in D. melanogaster (Matunis et al., 1992, accompanying paper). All of the proteins described here contain two amino-terminal RNP consensus sequence RNA-binding domains and a carboxyl-terminal glycine-rich domain. We refer to this configuration, which is also found in the hnRNP A/B proteins of vertebrates, as 2 x RBD-Gly. The sequences of the D. melanogaster hnRNP proteins help define both highly conserved and variable amino acids within each RBD and support the suggestion that each RBD in multiple RBD-containing proteins has been conserved independently and has a different function. Although 2 x RBD-Gly proteins from evolutionarily distant organisms are conserved in their general structure, we find a surprising diversity among the members of this family of proteins. A mAb to the hrp40 proteins crossreacts with the human A/B and G hnRNP proteins and detects immunologically related proteins in divergent organisms from yeast to man. These data establish 2 x RBD-Gly as a prevalent hnRNP protein structure across eukaryotes. This information about the composition of hnRNP complexes and about the structure of hnRNA-binding proteins will facilitate studies of the functions of these proteins.