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Amino Acid Sequencing (amino + acid_sequencing)
Kinds of Amino Acid Sequencing Selected AbstractsIdentification of a 250 kDa putative microtubule-associated protein as bovine ferritinFEBS JOURNAL, Issue 3 2005Evidence for a ferritin, microtubule interaction We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072,1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances. [source] Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosaFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2-3 2003Linda D. Thomas Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa 13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine. [source] Involvement of RNase G in in vivo mRNA metabolism in Escherichia coliGENES TO CELLS, Issue 5 2001Genryou Umitsuki Background Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5, end-processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism. Results We found that rng::cat mutant strains overproduced a protein of about 100 kDa. N-terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng::cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin-chase experiment revealed that the half-life of adhE mRNA was 2.5-fold longer in the rng::cat disruptant than in the wild-type. Conclusion These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli. [source] Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybeanJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2005T. Zendo Abstract Aims:, Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. Methods and Results:, The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca2+ (CaCO3 or CaCl2). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6·0, whereas the highest cell growth was obtained at pH 7·0. Conclusions:,Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca2+ and pH) influenced the bacteriocin production. Significance and Impact of the Study:, A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains. [source] Penicillium chrysogenum glucose oxidase , a study on its antifungal effectsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2004É. Leiter Abstract Aims:, Purification and characterization of the high molecular mass Candida albicans -killing protein secreted by Penicillium chrysogenum. Methods and Results:, The protein was purified by a combination of ultrafiltration, chromatofocusing and gel filtration. Enzymological characteristics [relative molecular mass (Mr) = 155 000, subunit structure ,2 with Mr,, = 76 000, isoelectric point (pI) = 5·4] were determined using SDS-PAGE and 2D-electrophoresis. N-terminal amino acid sequencing and homology search demonstrated that the antifungal protein was the glucose oxidase (GOX) of the fungus. The enzyme was cytotoxic for a series of bacteria, yeasts and filamentous fungi. Vitamin C (1·0 mg ml,1) prevented oxidative cell injuries triggered by 0·004 U GOX in Emericella nidulans cultures but bovine liver catalase was ineffective even at a GOX : catalase activity ratio of 0·004 : 200 U. A secondary inhibition of growth in E. nidulans cultures by the oxygen-depleting GOX,catalase system was likely to replace the primary inhibition exerted by H2O2. Conclusions:,Penicillium chrysogenum GOX possesses similar enzymological features to those described earlier for other Penicillium GOXs. Its cytotoxicity was dependent on the inherent antioxidant potential of the test micro-organisms. Significance and Impact of the Study:,Penicillium chrysogenum GOX may find future applications in glucose biosensor production, the disinfection of medical implants or in the food industry as an antimicrobial and/or preservative agent. [source] Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLCJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 12 2001Fionnuala T. Lundy Abstract Background: The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromotography quadropole mass spectrometry. Aims: To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease. Methods: GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms. Results: Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied. Conclusions: The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis. Zusammenfassung Grundlagen: Der Proteingehalt des GCF, abgetrennt werden mit einer reversphasigen Microbore-HPLC auf einer C18-Säule mit Detektion auf der Basis der Absorption von 214 nm. Ein einziges symmetrisches Protein-Hauptpeak-Eluat, der mit einer Retentionszeit von 26 Minuten eluiert wurde (50% Acetonnitril) war in gingivalen Sulkusfluid (GCF) von Parodontitispatienten deutlich sichtbar, jedoch nicht im GCF von Gesunden. Dieses Protein wurde mitels N-terminaler Aminosären-Sequenzierung und Flüssigkeits-Chromatographie mit Quadropol-Massenspektrometrie als humanes MRP-8 identifiziert. Ziele: Quantifizierung der Menge an MRP-8, die im GCF von gesunden Personen, Patienten mit Gingivitis und mit Parodontitis nachweisbar ist und Studieren der eventuell möglichen Beziehungen zwischen den Titern dieses Reaktionsproteins und parodontaler Gesundheit bzw. Erkrankung. Methoden: Bei Parodontitispatienten (n=15) wurde das GCF von gesunden Bereichen, sowie von Stellen mit Gingivitis und Parodontitis gewonnen (30 Sek.) sowie bei Kontrollpersonen (n=5) mit klinisch gesunder Gingiva und ohne Parodontitis. Das gereinigte MRP-8 wurde mittels Edman-Degradierung Sequenziert und der Phenyl-Thiohydantoin (PTH) Aminosäure-Yield bestimmt (durch Vergleich der Peakbereiche mit externen PTH Aminosäurestandards). Darauffolgend wurde dieser Wert verwendet, um die relative Menge des Proteins im Peak-Eluat mit einer Retentionszeit von 26.0 Min. (MRP-8) in den individuellen Chromatogrammen zu berechnen. Ergebnisse: An entzündeten Stellen wurden höhere Titer von MRP-8 nachgewiesen: Parodontitis 457.0 (281.0) ng; Gingivitis 413.5 (394.5) ng verglichen mit parodontal gesunden Stellen bei erkrankten Patienten 14.6 (14.3) ng und den Kontrollen 18.6 (18.5) ng, p=0.003. An den entzündeten Stellen gab es im Vergleich mit den gesunden Stellen wenigstens 20 mal mehr MRP-8. Schlußfolgerungen: Die vorläufigen Daten zeigen, daß MRP-8 im GCF vorhanden ist und an erkrankten Stellen signifikant höhere Mengen vorhanden sind, als an gesunden Stellen. Eine systematische Studie der Beziehung dieses Proteins zur Parodondalerkrankung könnte sich als nützlich erweisen, um des weiteren zu klären, ob MRP-8 eine verläßlicher Biomarker für Gingivitis und Parodontitis ist. Résumé Origine: Les composants proéiques du fluide gingival peuvent être séparés par HPLC microbore en phase inverse sur une colonne C18 avec une détection sur une base d'absorption de 214 nm. Un unique pic majeur symétrique ayant un temps de rétention de 26 min (50% acetonitrile) était manifeste dans le fluide gingival (GCF) des patients atteints de parodontite, mais pas chez les patients sans. Cette protéine fut identifiée comme étant l'MRP-8 humaine après séquençage de l'acide aminé N terminal et spectrométrie de masse quadropole par chromatographie liquide. But: L'objectif est de quantifier la quantité de MRP-8 détectable dans le GCF de site sains, atteints de gingivite ou de parodontite et d'étudier, s'il y en a, la relation entre les niveaux de cette réponse protéique et la santé et la maladie parodontale. Méthodes: Le GCF frut prélevé (30 s) dans des sites sains, atteints de gingivite ou de parodontite, chez des sujets atteints de parodontite (n=15) ou chez des contrôles (n=5), ayant une gencive cliniquement saine sans parodontite. Le MRP-8 purifié fut séquencé par dégradation d'Edmann et le débit d'acide aminé phenylthiohydantoine (PTH) déterminé (par comparaison avec la surface de pic avec des standards d'acide aminé PTH externe). Cette valeur fut ensuite utilisée pour calculer la quantité relative de protéine dans le pic avec un temps de rétention de 26.0 mn (MRP-8) sur des chromatogrammes individuels de GCF. Résultats: De plus hauts niveaux de MRP-8 étaints détectés dans les sites inflammatoires: Parodontite 457.0 (281.0) ng; gingivite 413.5 (394.5) ng par rapport aux sites sains des sujets malades 14.6 (14.3) ng et des sujets contrôles 18.6 (18.5) ng, p=0.003. Il y avait au moins 20× plus de MRP-8 dans les sites inflammatoires par rapport au sites sains. Conclusions: Les données préliminaires indiquent que la MRP-8 est présente dans le GCF, en quantité significativement plus importante dans les sites malades. Une étude systèmatique de la relation entre cette protéine et la maladie parodontale pourrait se révéler utile pour encore plus expliciter si MRP-8 pourrait être un biomarqueur fiable du GCF des gingivites et des parodontites. [source] Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysisPHYSIOLOGIA PLANTARUM, Issue 4 2002Han-Min Chen Starch phosphorylase (SP) is an enzyme used for the reversible phosphorolysis of the ,-glucan in plant cells. When compared to its isoform in an animal cell, glycogen phosphorylase, a peptide containing 78 amino acids (L78) is inserted in the centre of the low-affinity type starch phosphorylase (L-SP). We found that the amino acid sequence of L78 had several interesting features including the presence of a PEST region, which serves as a signal for rapid degradation. Indeed, most L-SP molecules isolated from mature sweet potato roots were nicked in the middle of a molecule, but still retained their tertiary or quaternary structures, as well as full catalytic activity. The nicking sites on the L78 were identified by amino acid sequencing of these peptides, which also enabled us to propose a proteolytic process for L-SP. Enzyme kinetic studies of L-SP in the direction of starch synthesis indicated that the Km decreased during the proteolytic process when starch was used as the limiting substrate, but the Km for the other substrate (Glc-1-P) increased. On the other hand, the maximum velocities (Vmax) increased for both substrates. Mobility of the nicked L-SP was retarded on a native polyacrylamide gel containing soluble starch, indicating the increased affinity for starch. Results in this study suggested that L78 and its proteolytic modifications might play a regulatory role on the catalytic behaviour of L-SP in starch biosynthesis. [source] Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451TPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Sebastian Hörmann Abstract High hydrostatic pressure (HHP) exerts diverse effects on microorganisms, leading to stress response and cell death. While inactivation of microorganisms by lethal HHP is well investigated in the context of food preservation and the hygienic safety of minimal food processes, sublethal HHP stress response and its effect on adaptation and cross-protection is less understood. In this study, the HHP stress response of Lactobacillus sanfranciscensis was characterized and compared with cold, heat, salt, acid and starvation stress at the proteome level by using 2-DE so as to provide insight into general versus specific stress responses. Sixteen proteins were found to be affected by HHP and were identified by using N-terminal amino acid sequencing and MS. Only one slightly increased protein was specific to the HHP response and showed homology to a clp protease. The other proteins were influenced by most of the investigated stresses in a similar way as HHP. The highest similarity in the HHP proteome was found to be with cold- and NaCl-stressed cells, with 11,overlapping proteins. At the proteome level, L.,sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses. Our data suggest that a specific pressure response does not exist in this bacteria. [source] Proteins of calcified endoskeleton: II Partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactylaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2005M. Azizur Rahman Abstract Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N -termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N -terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process. [source] Two homologous parasitism-specific proteins encoded in Cotesia plutellae bracovirus and their expression profiles in parasitized Plutella xylostellaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2008Sunyoung Lee Abstract A wasp, Cotesia plutellae, parasitizes the diamondback moth, Plutella xylostella, and interrupts host physiology for wasp survival and development. Identification of parasitism-specific factors would be helpful to understand the host,parasitoid interaction. This study focused on identification of a 15-kDa protein found only in plasma of the parasitized P. xylostella. Degenerate primers were designed after N-terminal amino acid sequencing of the parasitism-specific protein and used to clone the corresponding gene from the parasitized P. xylostella by a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Two homologous genes were cloned and identified as "CpBV15," and "CpBV15,," respectively, due to the identical size (158 amino acid residues) of the predicted open reading frames, in which they shared amino acid sequences in both terminal regions, but varied in internal sequences. Southern hybridization analysis indicated that both genes were located on C. plutellae bracovirus genome. Real-time quantitative RT-PCR revealed that both genes were mostly expressed at the late parasitization period, which was further confirmed by an immunoblotting assay using CpBV15 antibody. A recombinant CpBV15 protein was produced from Sf9 cells via a baculovirus expression system. The purified CpBV15 protein could enter hemocytes of P. xylostella and were localized in the cytosol. Along with the sequence similarities of CpBV15s with eukaryotic initiation factors, their putative biological role has been discussed in terms of the host translation inhibitory factor. Arch. Insect Biochem. Physiol. 67:157,171, 2008. © 2008 Wiley-Liss, Inc. [source] Characterization of the major allergens of Pachycondyla chinensis in ant sting anaphylaxis patientsCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009E. K. Lee Summary Background The ant species Pachycondyla chinensis, which has spread from Far Eastern Asia to New Zealand and North America, induces anaphylactic reactions in human with its sting. However, the major allergens of P. chinensis have not yet been characterized. Methods We selected seven patients with histories of anaphylaxis induced by P. chinensis. Two-dimensional electrophoresis (2-DE) was used to identify the major allergens. We subsequently performed Western blots for P. chinensis -specific IgEs, N-terminal amino acid sequencing, ESI-MS/MS, and RT-PCR using primers based on the N-terminal sequence. Results Six of the anaphylactic subjects had an IgE specific to a 23 kDa allergen of P. chinensis. Two candidates for major allergens, 23 kDa (pI 8.7) and 25 kDa (pI 6.2), were revealed by 2-DE using P. chinensis -specific IgE immunoblotting. In N-terminal sequencing and ESI-MS/MS analysis, 23 kDa (pI 8.7) and 25 kDa (pI 6.2) allergens, belonging to the protein families of antigen 5, were identified and share marked amino acid sequence similarity. The 23 kDa allergen is 206 amino acids in length and homology searches showed 54.0% and 50.0% homology with Sol i 3 and Ves v 5, respectively. Conclusion The major allergens of P. chinensis are 23 kDa (pI 8.7) and 25 kDa (pI 6.2) proteins that belong to the antigen 5 family of proteins. [source] Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE-binding capacitiesCLINICAL & EXPERIMENTAL ALLERGY, Issue 10 2000A. DÍaz-Perales Background Lipid-transfer proteins (LTPs), but not Bet v 1 homologues, have been identified as major allergens of apple and peach in the Rosaceae fruit-allergic population in the Mediterranean area. Many of these patients show cosensitization to mugwort pollen. LTPs have an ubiquitous distribution in tissues of many plant species, and have been proposed as a novel type of plant panallergens. Objective We sought to isolate LTPs from Artemisia pollen and from a plant food not belonging to the Rosaceae family, such as chestnut nut, and to compare their amino acid sequences and IgE-binding capacities with those of apple and peach LTPs. Methods Allergens (LTPs) were isolated by different chromatographic methods (gel-filtration, ion exchange and/or reverse-phase HPLC), and characterized by N-terminal amino acid sequencing and MALDI analysis. Specific IgE-quantification and immunodetection, as well as immunoblot and ELISA inhibition assays, were carried out using sera from patients allergic to both apple and peach. Results Purified LTPs from Artemisia pollen and from chestnut seed showed molecular masses about 9 700d, and 43,50% sequence identity with the equivalent allergens of apple and peach in the first 30 N-terminal residues, which comprise about one third of the total amino acid sequence. A similar degree of sequence identity (50%) was found between the Artemisia and chestnut proteins. Both isolated LTPs bound specific IgE of sera from Rosaceae fruits allergic patients. However, substantially lower values of specific IgE-binding and maximum ELISA inhibition percentages were obtained for Artemisia and chestnut LTPs when compared to those from apple and peach. Conclusion LTPs from Artemisia pollen and chestnut crossreact with allergens (LTPs) of Rosaceae fruits, but significant differences in specific IgE-binding capacities were observed among members of the plant LTP family. Thus, further studies are needed to evaluate the clinical significance of the observed cross-reactivities of plant LTPs. [source] | ||||||||||||||||