Two-dimensional electrophoresis of soluble leaf proteins, isolated from two wheat species (Triticum durum and Triticum aestivum) differing in sensitivity towards NaCl

Plants of two wheat species (Triticum aestivum cv. Tanit and T. durum cv. Ben Bachir), differing in their sensitivity to NaCl were cultivated in the presence or absence of 100 mM NaCl for 21 days. Soluble proteins extracted from leaves were analyzed by two-dimensional electrophoresis in order to detect NaCl-induced changes in the polypeptide patterns. In all, 500 spots were detected. Results showed species-dependent differences. The greatest alterations in the polypeptide profiles following salt stress were found in the most sensitive cultivar: among the 12 spots (molecular mass, 15-31 kDa) specifically considered in the acidic region of the gel, 11 declined, even disappeared in the NaCl-sensitive leaf profiles, while in the tolerant species only five spots were affected by the salt treatment and five remained untouched; moreover in the latter, two new polypeptides were shown to be induced by NaCl.     

Tuning the conformation of synthetic co‐polypeptides of serine and glutamic acid through control over polymer composition

Ring opening polymerization (ROP) of N‐carboxy anhydride (NCA) amino acids presents a rapid way to synthesize high molecular weight polypeptides with different amino acid compositions. The compositional and functional versatility of polypeptides make these materials an attractive choice for biomaterials. The functional performance of polypeptide materials is equally linked to their conformation which is determined by the amino acid sequence in the polymer chains. Here, the interplay between composition and conformation of synthetic polypeptides obtained by NCA polymerization was explored. Various copolypeptides from Glu(Bzl) and Ser(Bzl) were prepared to investigate how polypeptide composition affected the conformation of the resulting copolymer. Polymerization kinetics indicated that the copolymerization of Glu(Bzl) and Ser(Bzl) preferentially yielded alternating copolymers. Both the polydispersity and the conformation of the polypeptides were dependent on the Ser(Bzl) content in the polymer, demonstrating that polypeptide functionalities could be tuned directly by altering the relative amounts of amino acids in the chain. This work presents the first step toward an improved understanding and control over polypeptide conformation through modulating the amino acid composition of the material. Understanding this sequence–functionality relationship is essential to advancing the use of ROP as a technique to design smart polypeptide based materials with specific functions. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2331–2336     

Mineral uptake and biochemical changes in Helianthus annuus under treatment with different sodium salts

Experiments were conducted to study the effects of different sodium salts viz., sodium chloride (NaCl), sodium sulphate (Na(2)SO(4)) and sodium carbonate (Na(2)CO(3)) on growth, dry matter production, mineral contents, biochemical constituent and enzyme activities of sunflower (Helianthus annuus L.). The germinating sunflower seeds were treated with 10, 20 and 50mM NaCl and Na(2)SO(4) and 5, 10 and 15 mM Na(2)CO(3). The seedling growth, minerals, chlorophyll content and biochemicals like protein and free amino acid contents with enzyme activities like ATPase and protease were analysed on 8 DAS. The seedlings were separated into root, stem, leaf and cotyledon on 8 DAS. All the treatments decreased the germination percentage; shoot length, root length, leaf area and dry weight, chlorophyll and protein contents significantly. Potassium, sodium and free amino acid contents; activities of ATPase and protease were increased when compared to control. This effect was very high in the Na(2)CO(3) treated seedlings this was followed by Na(2)SO(4) and NaCl treated seedlings. From the results of this investigation, it is clear that, the sunflower seedlings were affected significantly in the Na(2)CO(3) treatments, and followed by Na(2)SO(4) and NaCl treatments.     

梅花鹿茸中活性多肽的纯化、测序及功能研究

利用凝胶过滤、离子交换层析及C18反相柱层析等方法, 从梅花鹿茸中分离得到了一个多肽CNT14, SDS-PAGE电泳显示其为一条带, HPLC图谱为单峰, 激光解析电离飞行时间质谱测定其分子量为1479.9028. 氨基酸组成分析结果表明, 此多肽主要含缬氨酸、亮氨酸、谷氨酸及脯氨酸, 用电喷雾串联质谱法测序结果为E-P-T-V-L-D-E-V-C-L-A-H-G-P. 活性检测结果表明, 该多肽能够明显促进小鼠海马HT22细胞增殖. 同时, 采用固相合成法对该多肽进行了合成, 与所分离的天然多肽进行了结构与性质的比较.     

Sequence Analysis of Polypeptides and Proteins by Combined Gas Chromatography-Mass Spectrometry

A new method for determining the amino acid sequence of polypeptides consists in initial partial hydrolysis to yield a complex mixture of oligopeptides. After derivatization to enhance its volatility, the mixture is analyzed by combined gas chromatography and mass spectrometry. The sequence of the polypeptide is established by a computer from the identified oligopeptides. So far polypeptides having up to 40 amino acids have been analyzed by this method. The advantages and disadvantages of the new method compared with the stepwise procedure of the Edman degradation are considered. Since the two methods are based on fundamentally different principles they may prove to be complementary.     

Contribution of blotting techniques to the study of rapeseeds (Brassica napus L.) lipases

A recent advance in the study of plant lipases involving immunological techniques is presented. In an attempt to characterize lipases of cotyledons from germinating rapeseed seedlings and to investigate an eventual cross-reactivity with animal lipases, we have prepared anti-porcine pancreatic lipase antibodies raised in rabbit. It is shown by enzyme-linked immunosorbent assay and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Preincubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. We demonstrate immunochemical cross-reactivity between rapeseed and porcine pancreatic lipase. Using the immunoblotting procedure, it is found that antibodies bind specifically to a single polypeptide with a molecular mass of about 55 kDa. Rapeseed lipase activity decreased after immunoprecipitation suggesting that antibodies were bound to some catalytic site residues. We conclude from the data obtained in this study that the two different lipase species present close similarities in amino acid sequence and antigen characteristics.     

Translation of genetic information on the ribosome

The information for protein structure that is contained in the base sequence of the nucleic acids is translated on the ribosome into the amino acid sequence. This translation can be divided into chain initiation, chain growth, and chain termination. Several specific protein factors and nucleic acids are involved in each section.—For chain initiation, start complexes are formed from the initiating amino acyl-tRNA, mRNA carrying the start signal, and the small and large subunits of a ribosome. GTP and the initiating factors are also involved in this process.—In chain elongation, one amino acid at a time is transferred, in a reaction cycle, from the linkage with tRNA into a linkage with the polypeptide chain. The amino acid to be incorporated is initially bound to the ribosome as amino acyl-tRNA, a process for which GTP and protein factors are necessary. The subsequent formation of a peptide linkage is catalyzed by the peptidyl transferase of the large ribosomal subunit. The peptidyl-tRNA with its newly added amino acid residue is then transferred from the amino acyl-tRNA acceptor site A to the peptidyl donor site P of the ribosome. This requires another protein factor and cleavage of GTP into GDP and phosphate.–Ghain termination begins as soon as one of the three terminator triplets UAA, UAG, or UGA in the mRNA reaches the ribosome. The mRNA is moving in relation to the ribosome from the 5′ end to the 3′ end. Release of the completed polypeptide chain from the ribosome is dependent on release factors. Before initiation of a new polypeptide chain, the ribosomes dissociate into their subunits.     

Evaluation of temperature,excipients impact on the primary structure of Ganirelix in an injectable formulation,and comparison with Orgalutran® using MALDI‐TOF/TOF‐MS

Ganirelix is a linear polypeptide consisting of covalently bonded 10 amino acid residues. The amino acid sequence in a peptide determines the properties of the molecule. The slightest change in the primary structure (amino acid sequence) of therapeutic peptides can significantly impact its safety, efficacy, and immunogenicity. Hence, the primary structure analysis of therapeutic peptides is regarded as a critical quality attribute (CQA). A vast array of analytical techniques can be used to capture the primary structure of the peptide. In this study, we applied matrix‐assisted laser desorption ionization (MALDI)/tandem time of flight mass spectroscopic (TOF/TOF MS) method to demonstrate the primary structure of Ganirelix in an injectable formulation. The apparent monoisotopic molecular mass of Ganirelix is 1,568.9 Da. The attained primary amino acid sequence of Ganirelix in temperature‐stressed generic product matched with the theoretical sequence and showed homology with those of the reference listed drug (RLD).     

Polypeptide changes induced by salt stress, water deficit, and osmotic stress in barley roots: a comparison using two-dimensional gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis was used to analyze and compare the effects of short term treatments (24 h) of salt stress, water deficit (desiccation), and osmotic stress (polyethylene glycol and mannitol) on protein synthesis in roots of barley seedlings (Hordeum vulgare L. cv. CM 72). These comparisons were made to determine if the polypeptides of Mr 26,000 and 27,000 and pI of 6.3 and 6.5 that were observed previously to increase significantly with salt stress (Plant Physiol. 1987, 83 517-524) also increased with water deficit and osmotic stress. The polypeptide patterns for control- and stress-treated plants were qualitatively similar, but the net synthesis of a number of polypeptides was quantitatively altered by each of the stress treatments. Of the polypeptide changes induced by the stress treatments, many were unique to a specific stress. Other polypeptide changes were common between two or more of the stress treatments. Only one polypeptide change, a decrease, was common to all of the stress treatments. An important finding was that polypeptides that increased significantly in response to salt stress did not increase in response to water deficit or osmotic stress.     

Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with L- and D-amino acid substitutions

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.     

孔石莼质体蓝素的柱色谱纯化及其N-端氨基酸序列的分析测定

通过DEAE-Sepharose Fast Flow阴离子交换柱和Sephadex G-75凝胶过滤柱分离纯化得到了孔石莼(Ulva pertusa)的质体蓝素。其步骤为:将孔石莼样品以0.02 mol/L磷酸盐缓冲液(pH 7.2)进行匀浆,然后离心去除沉淀,将上清液用硫酸铵分级盐析获得饱和度为40%~80%的盐析蛋白;通过DEAE-Sepharose 柱色谱,在含有0~1.0 mol/L NaCl 的0.01 mol/L磷酸盐缓冲液线性梯度洗脱下,盐析蛋白有3个主要的洗脱峰,然后在Sephadex G-75凝胶过滤色谱柱中进一步纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示,该蛋白质被纯化为单一条带。根据蛋白质电泳迁移率,纯化蛋白质的相对分子质量约为10000。该蛋白质不含糖。纯化的蛋白质经电转移至聚偏二氟乙烯（PVDF）膜后,以Edman降解法进行N-端氨基酸序列测定,前20个氨基酸残基序列为AAIVKLGPDDGSLAFVPSKI。通过对相关蛋白质数据库的检索,发现该序列与3种已报道的海藻的质体蓝素具有较高的序列同源性,其同源性分别为85%,85%和90%。据此,认为孔石莼的质体蓝素已获得纯化,其N-端20个氨基酸残基与已报道的海藻质体蓝素的氨基酸残基有较大的同源性,也存在着一定的变异。     

Metabolites of the pathogenic fungusVerticillium dahliae. XI. Primary structure of the polypeptide moiety of the phytotoxic pigment PKZh-1

The sequence of the 27 amino acid residues composing the polypeptide moiety of the phytotoxic metabolite PKZh-1 of the fungusV. dahliae has been determined on the basis of the structures of tryptic and chymotryptic peptides. It has been established that the linkage of the polypeptide with 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which is the chromophoric moiety of PKZh-1, is effected through an ester bond formed by the carboxy group of the C-terminal amino acid and one of the hydroxy groups of flaviolin.     

梅花鹿茸多肽的化学结构及生物活性

在马鹿茸活性多肽结构与功能研究基础上, 从新鲜梅花鹿茸中分离纯化了活性单体多肽, 确定了其化学结构, 并与马鹿茸多肽进行结构与活性比较. 利用离子交换层析、 凝胶过滤层析及反相高效液相色谱层析等生物化学技术, 从梅花鹿茸中分离得到1个新多肽, SDS-PAGE电泳显示为一条带, HPLC图谱为单一峰, MALDI-TOF MS给出该多肽的精确分子量为3263.4, 其等电点pI=8.15. 一级结构研究表明, 该多肽是由32个氨基酸残基组成的直链多肽, 不含半胱氨酸, 富含缬氨酸、 赖氨酸、 亮氨酸和甘氨酸, 氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. 生物活性检测结果表明, 该多肽可促进原代培养的表皮细胞和软骨细胞增殖, 也能刺激NIH3T3成纤维细胞株的分裂. 梅花鹿茸多肽与马鹿茸多肽在结构上均为32个氨基酸残基组成的直链多肽, 但第5, 8, 11和30位氨基酸残基不同. 2种多肽结构上的变化并未影响其促细胞增殖生物活性.     

A Novel Polypeptide from Cervus elaphus Linnaeus

A novel polypeptide having stimulant effect on some cell proliferation was isolated from the velvet antler (Cervus elaphus Linnaeus). The velvet antler polypeptide consists of a single chain of 32 amino acid residues. Amino acid sequence of the polypeptide was identified as:VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM.     

Synthesis,conformation and cytotoxicity of new,branched polymeric polypeptides containing hydrophobic amino acid or arginine moiety

In this paper we report on the synthesis and solution conformation of a new set of structurally related polycationic branched chain polypeptides (poly[Lys(X _i -dl-Ala _m )]) with hydrophobic (Ile, Nle, Val) or cationic (Arg) amino acids at the N-terminal end of the side chains as well as their cytotoxic effect on murine bone marrow derived macrophages. Solution conformation of the polypeptides was studied with circular dichroism spectroscopy under different conditions (pH, ionic strength). The results of these comparative studies indicate that a) polypeptides could adopt an ordered (mainly helical) conformation at physiological pH and salt concentration (pH 7.4, 0.2 M NaCl); b) the nature of side chain terminal amino acid (X) could determine under which conditions the ordered structure was formed. Thus, the solution conformation of branched polypeptides could be modulated by the selection of amino acid X under physiological conditions. All polypeptides with hydrophobic amino acid at the terminal position were essentially non-toxic on macrophages, whereas the polypeptide with terminal Arg proved to be markedly cytotoxic.

     

Probing Protein Surface with a Solvent Mimetic Carbene Coupled to Detection by Mass Spectrometry

Much knowledge into protein folding, ligand binding, and complex formation can be derived from the examination of the nature and size of the accessible surface area (SASA) of the polypeptide chain, a key parameter in protein science not directly measurable in an experimental fashion. To this end, an ideal chemical approach should aim at exerting solvent mimicry and achieving minimal selectivity to probe the protein surface regardless of its chemical nature. The choice of the photoreagent diazirine to fulfill these goals arises from its size comparable to water and from being a convenient source of the extremely reactive methylene carbene (:CH₂). The ensuing methylation depends primarily on the solvent accessibility of the polypeptide chain, turning it into a valuable signal to address experimentally the measurement of SASA in proteins. The superb sensitivity and high resolution of modern mass spectrometry techniques allows us to derive a quantitative signal proportional to the extent of modification (EM) of the sample. Thus, diazirine labeling coupled to electrospray mass spectrometry (ESI-MS) detection can shed light on conformational features of the native as well as non-native states, not easily addressable by other methods. Enzymatic fragmentation of the polypeptide chain at the level of small peptides allows us to locate the covalent tag along the amino acid sequence, therefore enabling the construction of a map of solvent accessibility. Moreover, by subsequent MS/MS analysis of peptides, we demonstrate here the feasibility of attaining amino acid resolution in defining the target sites.     

The Thermostability of Two Kinds of Recombinant ∆6-Fatty Acid Desaturase with Different N-Terminal Sequence Lengths in Low Temperature

Two recombinant Rhizopus stolonifer ?6-fatty acid desaturase enzymes with different-length N-termini were cloned and expressed in Saccharomyces cerevisiae strain INVScl: LRsD6D begins with the sequence of the N-terminal of the R. stolonifer ?6-fatty acid desaturase native, encoding a deduced polypeptide of 459 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A-M-K-F), whereas SRsD6D begins with the amino acid sequence of the predicted ORF, encoding a deduced polypeptide of 430 amino acids (M-K-F) and LRsD6D is longer than SRsD6D by 29 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A). Bioinformatic analysis characterized the two recombinant ?6-fatty acid desaturase enzymes with different-length N-termini, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5-like domain in the N-terminus. When the coding sequence was expressed in S. cerevisiae strain INVScl, the coding produced ?6-fatty acid desaturase activity exhibited by RsD6D, leading to a novel peak corresponding to γ-linolenic acid methyl ester standards, which was detected with the same retention time. The residual activity of LRsD6D was 74 % at 15 °C for 4 h and that of SRsD6D was 43 %. Purified recombinant LRsD6D was more stable than SRsD6D, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant LRsD6D.     

REACTIVITY OF SINGLET OXYGEN TOWARD LARGE PEPTIDES

Abstract— The reactions of singlet oxygen, ¹O₂, with amino acids and their derivatives have been studied previously. It was found that only five amino acid residues interact readily with ¹O₂. Here we describe its reactions with the large peptides melittin, neuropeptide Y (NPY) and insulin in their native and in their denatured forms. The singlet oxygen quenching by a polypeptide was compared with that of a solution at the same concentration as those of its constituent amino acids, which are known to react efficiently with ¹O₂. It was found that the quenching rate by such a mixture exceeded that of the polypeptides in their native form. The ratio of the rate constants for NPY to that of the corresponding amino acid mixture in solution was 0.75. For melittin in its monomeric form it was 0.83 and for a tetramer of melittin (at high ionic strength) it was 0.70. For native insulin the ratio of the rate constants was 0.55. For oxidized insulin with its -S-S- bridges opened the figure became 0.80. However, the quenching by all the polypeptides in their fully denatured form (in the presence of 6 M urea) equalled that of the corresponding amino acid mixtures. Although polypeptides are generally supposed not to possess a stable secondary structure in solution the effects are explained by shielding of some of the reactive amino acid residues in the chain by temporary folding or incipient secondary structures of the native polypeptide.
It is shown that the kinetics for a homogeneous solution of quenchers applies also to measurements in a polypeptide solution where the quenchers are localized along the polypeptide backbone and thus form clusters in solution.     

Properties of grafted amphiphilic chains. A computer simulation study

The model of a heteropolymer film formed by polypeptide chains was used for theoretical considerations. The linear chains consisting of amino acid residues were approximated by alpha carbon chains. Each chain was constructed on a very flexible [310] lattice. The inter- and intramolecular interactions consisted of the long-range contact potential between residues. The chains were built of hydrophilic and hydrophobic residues. Chains were terminally attached to an impenetrable surface with lateral motions possible. The Monte Carlo simulations of this model were carried out by using the Metropolis algorithm. The influence of the grafting density, the sequence of the amino acid residues, and the temperature on the static properties of the formed layer were studied and discussed. It was shown that homopolymer chains collapsed at higher temperature than the heteropolymers. The size of the polymers forming brush was smaller for homopolymers than for heteropolymers. The structure of the resulting polymer film and of its external surface was determined. The block copolymers formed well defined hydrophobic and hydrophilic layers, while for the amphiphilic case the composition of the brush layers changed continuously at high temperature. It was observed that the latter effect vanished at the collapsed amphiphilic copolymer.     

The desalination of a mixed solution of an amino acid and an inorganic salt by means of electrodialysis with charge-mosaic membranes

A new electrodialysis with charge-mosaic membranes was proposed to achieve efficient desalination of a mixed solution of an amino acid and an inorganic salt. For such a mixed solution, the conventional electrodialytic desalination with both cation-and anion-exchange membranes had resulted in a considerable loss of the amino acid through the membranes. In this method, however, the amino acid in the desalination channel of the electrodialyzer migrates away from the membranes so that the permeation loss of the amino acid through the membrane can be prevented.

Batchwise desalination experiments by this method were carried out with a glutamic acid or arginine solution including NaCl under the condition of constant electric current density. Similar experiments by the conventional method were also carried out. As a result of comparing both methods, the amino acid loss in this method became much smaller than that in the conventional one. It was confirmed that this method was very useful for the desalination of an amino acid solution. The effects of operating conditions on the desalination process are also discussed.