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1.
Evidence for the presence of a CmuA methyltransferase pathway in novel marine methyl halide-oxidizing bacteria 总被引:3,自引:0,他引:3
Marine bacteria that oxidized methyl bromide and methyl chloride were enriched and isolated from seawater samples. Six methyl halide-oxidizing enrichments were established from which 13 isolates that grew on methyl bromide and methyl chloride as sole sources of carbon and energy were isolated and maintained. All isolates belonged to three different clades in the Roseobacter group of the alpha subdivision of the Proteobacteria and were distinct from Leisingera methylohalidivorans, the only other identified marine bacterium that grows on methyl bromide as sole source of carbon and energy. Genes encoding the methyltransferase/corrinoid-binding protein CmuA, which is responsible for the initial step of methyl chloride oxidation in terrestrial methyl halide-oxidizing bacteria, were detected in enrichments and some of the novel marine strains. Gene clusters containing cmuA and other genes implicated in the metabolism of methyl halides were cloned from two of the isolates. Expression of CmuA during growth on methyl halides was demonstrated by analysis of polypeptides expressed during growth on methyl halides by SDS-PAGE and mass spectrometry in two isolates representing two of the three clades. These findings indicate that certain marine methyl halide degrading bacteria from the Roseobacter group contain a methyltransferase pathway for oxidation of methyl bromide that may be similar to that responsible for methyl chloride oxidation in Methylobacterium chloromethanicum. This pathway therefore potentially contributes to cycling of methyl halides in both terrestrial and marine environments. 相似文献
2.
Strohalm M Kodícek M Pechar M 《Biochemical and biophysical research communications》2003,312(3):811-816
The reaction with 2-hydroxy-5-nitrobenzyl bromide (HNB) is a common covalent modification of tryptophan. It results in several products which have been described by classical physico-chemical methods. To improve the understanding of the HNB-modified tryptophan structure, we synthesized a model peptide containing one tryptophan only, modified it by HNB, and analyzed the product by MALDI-TOF mass spectrometry. Surprisingly, several multi-modified products (up to 5 HNB moieties per one tryptophan) were identified. the influence of HNB concentration and pH on the degree of modification was also analyzed. In addition, a splitting of modified tryptophan peaks in MALDI-TOF spectrum was described; most probably, this effect is a common MALDI artifact of nitro-aromatic compounds which facilitates the identification of HNB-modified tryptophan by MALDI-TOF MS significantly. 相似文献
3.
Gordon A. Morris Zdenka Hromdkov Anna Ebringerov Anna Malovíkov Juraj Alfldi Stephen E. Harding 《Carbohydrate polymers》2002,48(4):351-359
Citrus pectin with a low degree of methyl esterification (LMP) and its deesterified form, potassium pectate (KP), were modified with a low amount of UV-absorbing substituents. For this purpose, two different substitution reactions were used (a) alkylation of hydroxyl groups with p-carboxybenzyl bromide in aqueous alkali and (b) alkylation of the carboxylate group with benzyl bromide in the DMSO/TBAI/catalyst system. Chemical and spectroscopic methods reveal a low degree of substitution (DS<0.1) for the derivatives. The hydrodynamic properties were assessed by analytical ultracentrifugion, viscometry, and HPGPC. The results indicate that the introduction of small amounts of p-carboxybenzyl ether groups practically had no effect on the hydrodynamic properties in the case of KP, whereas, it was accompanied with a decrease of the molecular mass for LMP. The degradation was more pronounced during the benzyl esterification of LMP. The results confirmed that LMP is susceptible to chain cleavage due to β-elimination during both modification reactions. However, KP seems to be more tolerant of the reaction conditions. 相似文献
4.
Summary Periodate oxidation of free and protein-bound Amadori compounds formed by the condensation of reducing sugars with primary amino groups generates, on acid hydrolysis, N-carboxymethyl derivatives of amino acids. The analysis of these modified amino acids may be used to estimate both the extent and the site of protein glycosylation. The present study describes the use of gas chromatography-mass spectrometry (GC/MS) and gas chromatographytandem mass spectrometry (GC/MS/MS) for the identification of the various N-carboxymethylamino acids. Application of this approach to the quantitation of N-carboxymethylvaline and N
-carboxymethyllysine resulting from the oxidation of glycosylated haemoglobin is presented. 相似文献
5.
Determination of the sugar sequences and the glycosidic-bond arrangements of immunogenic heteroglycans 总被引:1,自引:0,他引:1
The complete sugar sequences and glycosidic-bond arrangements have been determined by a combined analytical scheme for a diheteroglycan of D-glucose and D-galactose and a tetraheteroglycan of 6-deoxy-L-talose, L-rhamnose, D-galactose, and D-glucuronic acid. The analytical scheme included methylation analysis by g.l.c. and mass spectrometry, periodate oxidation followed by borohydride reduction and identification of the residual fragments, enzymic hydrolysis followed by characterization of the modified glycan, and chemical degradation followed by characterization of the resulting fragments. The diheteroglycan and the tetraheteroglycan are immunogenic substances on the cell surface of the organisms, and are the group specific carbohydrates in the cell walls of Streptococcus faecalis, strain N, and Streptococcus bovis, strain C3. The combined analytical scheme should be of general applicability for the structural analysis of heteroglycans for which selective-degradation procedures can be devised. 相似文献
6.
Evaluation of the extent of heterogeneity in the Glycera dibranchiata monomer haemoglobin fraction by the use of n.m.r. and ion-exchange chromatography.
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The coelomic haemoglobin of Glycera dibranchiata is known to be separable into monomeric and higher-Mr fractions. Although exhibiting homogeneity with respect to Mr, the extent of haemoglobin heterogeneity for the monomer fraction has never been adequately assayed. In the present paper we demonstrate that there exists in the monomer haemoglobin fraction reproducibly detectable heterogeneity regardless of the presence or absence of proteinase inhibitors during the isolations. These results show that, considered on the same time scale as previous preparations used for amino acid sequencing, crystallography and kinetics, the monomer haemoglobin fraction is highly heterogeneous. Application of ion-exchange chromatography and ion-filtration methods resulted in the isolation of four resolvable haem protein components from the Glycera monomer haemoglobin fraction. Three of these components were isolated in sufficient quantity to employ proton n.m.r. as a successful analytical tool for discriminating the individual haemoglobins. These results are not surprising. Several previous studies indicated less extensive heterogeneity in the monomer fraction. Moreover, the ability of the Glycera monomer haemoglobin to bind oxygen at even quite low partial pressures has been attributed to functional diversity originating in multiple haemoglobin components. The present work reveals the extent of the haemoglobin heterogeneity. The results show that it is more extensive than previously believed. Examination of this monomer fraction is particularly important, since crystallography indicates that one of the components of the monomer fraction lacks the E-7 (distal) histidine residue. As a consequence, the identification of such extensive heterogeneity is important to many previously published ligand-binding studies. 相似文献
7.
(1) The antagonistic effect of atropine methyl bromide entrapped in liposomes on contraction of Xenopus intestine in vitro induced by acetylcholine was studied. The results provided some insight into cell-liposome interaction. (2) Acetylcholine (0.1 mM) was added to the medium in the bath (serosal solution), while liposomes containing atropine methyl bromide in their internal and external phases were added on the mucosal side of the intestine. Large multilamellar liposomes were prepared from egg lecithin (phosphatidylcholine, PC) and cholesterol in various molar ratios. Atropine methyl bromide had most effect in liposomes composed of PC and cholesterol in a ratio of 7:3, less in those with a ratio of 4:5, and none in those with a ratio of 9:1. These effects were parallel with the sizes of these liposomes, determined by quasi-elastic light-scattering; that is, the larger the liposomes, the greater was their effect. Addition (to the liposomes) of phosphatidic acid, the negative charge of which increases the distance between the lamellar layers, increased the effect, indicating that atropine methyl bromide in the space between lamellar layers was effective. Another type of liposomes in which atropine methyl bromide was present only in the external phase of liposomes was as effective as liposomes in which atropine methyl bromide was present in both the internal and external phases. (3) From these results the following new model for liposome-mediated stimulation of transport of atropine methyl bromide is proposed. Large multilamellar liposomes have structural defects in their external lamellae through which atropine methyl bromide in the mucosal solution can penetrate into the space between the external lamellar layers and move into intestinal cells through regions of fusion between the outermost layers of the liposomes and the cell membrane. 相似文献
8.
Top-down mass spectrometry strategies allow identification and characterization of proteins and protein networks by direct fragmentation. These analytical processes involve a panel of fragmentation mechanisms, some of which preserve protein post-translational modifications. Thus top-down is of special interest in clinical biochemistry to probe modified proteins as potential disease biomarkers. This review describes separating methods, mass spectrometry instrumentation, bioinformatics, and theoretical aspects of fragmentation mechanisms used for top-down analysis. The biological interest of this strategy is extensively reported regarding the characterization of post-translational modifications in biochemical pathways and the discovery of biomarkers. One has to bear in mind that quantitative aspects that are beyond the focus of this review are also of critical important for biomarker discovery. The constant evolution of technologies makes top-down strategies crucial players in clinical and basic proteomics. 相似文献
9.
Mass-spectrometric structure elucidation of dog bile azopigments as the acyl glycosides of glucopyranose and xylopyranose 总被引:13,自引:10,他引:3
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F. Compernolle G. P. Van Hees J. Fevery K. P. M. Heirwegh 《The Biochemical journal》1971,125(3):811-819
1. The structures of the alpha(2)- and alpha(3)-azopigments, prepared by diazotization of dog bile with ethyl anthranilate, were shown by mass spectrometry and g.l.c. to correspond to azobilirubin beta-d-xylopyranoside and azobilirubin beta-d-glucopyranoside respectively. 2. Both azopigments consist of a mixture of two methyl vinyl isomers having structures (IIIa) and (IIIb) for the alpha(2)-azopigment and structures (IVa) and (IVb) for the alpha(3)-azopigment. Separation of methyl vinyl isomers was obtained by t.l.c. or column chromatography performed on the acetylated azopigments. Hydrolysis of the less polar acetates derived from components (IIIa) and (IVa) gave rise to the azopigment (Ia), whereas hydrolysis of the more polar acetates derived from components (IIIb) and (IVb) gave rise to the azopigment acid (Ib). The positions of methyl and vinyl substituents in compounds (Ia) and (Ib) were assigned on the basis of their n.m.r. spectra. 3. Molecular ions in the mass spectra of the trimethylsilyl and acetyl derivatives of the azopigments indicated the presence of a pentose and a hexose conjugating sugar. 4. The ester functions linking the sugars to the propionic acid side chain of azobilirubin were demonstrated by ammonolysis and identification of the amide of azobilirubin as the aglycone derivative. 5. The sugar moieties were shown to occur as xylopyranose (alpha(2)) and glucopyranose (alpha(3)), bound at C-1, by application of a sequence of reactions performed on a micro-scale. The sugar hydroxyl groups were acetylated and the 1-acyl aglycone removed selectively by treatment with hydrogen bromide in acetic acid. Hydrolysis of the 1-bromo sugar acetates followed by acetylation afforded the alpha- and beta-xylopyranose tetra-acetates and alpha- and beta-glucopyranose penta-acetates, identified by a combination of g.l.c. and mass spectrometry. 6. The validity of this degradation scheme was confirmed (a) by g.l.c.-mass spectrometry identification of the alpha- and beta-1-propionyl derivatives of glucopyranose tetra-acetate, obtained from the alpha(3)-azopigment after final reaction with propionic anhydride; (b) by subjecting the acetates of alphabeta-glucopyranose, alphabeta-xylofuranose and alphabeta-glucofuranose to the same sequence of reactions. 相似文献
10.
The identification and characterization of biotinylated lysyl residues in a polypeptide chain by automated sequence analysis is described. An in depth analytical study was conducted for the delineation of N epsilon modification of lysyl residues with N-hydroxysuccinimide esters of biotin and 6-aminohexanoic biotin. Confirmation of the structure of the phenylthiohydantoin derivatives of N epsilon biotinylated lysine was achieved by mass spectrometry. The analytical study focused on the identification of biotinylated lysine-4 of neuropeptide Y which served as a model peptide for the analytical procedures detailed. 相似文献
11.
Stroma-free solutions of human haemoglobin modified with pyridoxal-5-phosphate, glutaraldehyde, borohydride and serum albumin were injected into the artery of an isolated rat heart perfused with Krebs-Henseleit solution under hypoxic conditions. About 70% of the oxygen transported by the modified haemoglobin was found to be utilized for a marked increase in the force of heart contraction. The results were in general correlation to the analysis of oxygenation curves of haemoglobin samples under study and confirmed the oxygen offloading ability. 相似文献
12.
Margaret M. Krahn Donald W. Brown Tracky K. Collier Andrew J. Firedman Rand G. Jenkins Donald C. Malins 《Journal of biochemical and biophysical methods》1980,2(4):233-246
A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system. 相似文献
13.
The basic fragmentation mechanism of methyl(methyl 4-deoxy-2,3-di-O-methyl-β-l-threo-hex-4-enopyranosid)uronate has been deduced by deuteromethylation analysis, metastable transition measurements, and by interpreting the spectra of weakly excited foregoing molecules. The differences in fragmentation of partially methylated derivatives of methyl 4-deoxy-β-l-threo-hex-4-enopyranosiduronic acid compared to that of the fully methylated substance are discussed in detail and the criteria are proposed for identification of the compounds concerned by mass spectrometry. 相似文献
14.
This paper describes the identification of a human metabolite of propetamphos ((E-O-2-isopropylcarbonyl-1-methylvinyl-O-methylethylphosphoramidothioate), formed by the hydrolytic cleavage of the enol-vinyl-phosphate bond, and the development of an analytical method suitable for biological monitoring of propetamphos exposure. The metabolite has been detected in the urine of exposed workers but not in that of control subjects. The analytical method involves azeotropic distillation of the urine with acetonitrile, followed by derivatization with pentafluorobenzyl bromide and analysis using gas chromatography with flame photometric detection. 相似文献
15.
Leppänen AS Niittymäki O Parikka K Tenkanen M Eklund P Sjöholm R Willför S 《Carbohydrate research》2010,345(18):2610-2615
The C-6 unit of methyl α-d-galactopyranoside was selectively modified by combining enzymatic oxidation with an indium-mediated allylation reaction. The Barbier-Grignard type reaction, where a carbonyl group reacts with an allyl halide, proceeds in aqueous solution, even with water as the only solvent; thus carbohydrates can be modified without the need for drying or protection-deprotection steps. The corresponding homoallyl alcohols are produced in high yields of >90% in the reactions with allyl bromide and cinnamyl chloride. The main products were isolated and characterized by GC-MS and NMR spectroscopy. 相似文献
16.
James M. Grace Timothy L. MacDonald Robert J. Roberts Michael Kinter 《Free radical research》1996,25(1):23-29
Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1, 4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1, 4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions. 相似文献
17.
Geoffrey W. Zehnder Changbin Yao Gang Wei Joseph W. Kloepper 《Biocontrol Science and Technology》2000,10(5):687-693
Field experiments were conducted to evaluate growth promotion and induced systemic disease resistance (ISR) in cucumber mediated by plant growth-promoting rhizobacteria (PGPR) with and without methyl bromide soil fumigation. In both fumigated and nonfumigated plots, numbers of cucumber beetles, Acalymma vittata (F.), and the incidence of bacterial wilt disease, caused by the beetle-transmitted pathogen Erwinia tracheiphila , were significantly lower with PGPR treatment compared with the nonbacterized control. However, in PGPR-treated plots, the incidence of bacterial wilt was more than 2-fold lower in the nonfumigated treatments compared with fumigated treatments, indicating that the level of PGPR-mediated ISR was greater without methyl bromide fumigation than with methyl bromide. Cucumber plant growth at 21 days after planting was greater in fumigated plots than in nonfumigated plots; however, plant height values in the nonfumigated, PGPR treatments and the fumigated, PGPR treatments were equivalent. This suggests that PGPR treatment compensated for delayed plant growth that often occurs in nonfumigated soil. These results indicate that, in cucumber production systems, withdrawal of methyl bromide will not negatively impact PGPRmediated ISR, and also that PGPR may have potential as an alternative to methyl bromide fumigation. 相似文献
18.
Krull RE Chen P Novak J Kirk M Barnes S Baker J Krishna NR Caufield PW 《The Journal of biological chemistry》2000,275(21):15845-15850
Mutacin II is a post-translationally modified lantibiotic peptide secreted by Streptococcus mutans T8, which inhibits the energy metabolism of sensitive cells. The deduced amino acid sequence of promutacin II is NRWWQGVVPTVSYECRMNSWQHVFTCC, which is capable of forming three thioether bridges. It was not obvious, however, how the three thioether bridges are organized. To examine the bridging, the cyanogen bromide cleavage products of mutacin II and its variants generated by protein engineering, C15A, C26A, and C15A/C26A, were analyzed by mass spectrometry. Analysis of the wild type molecule and the C15A variant excluded several possibilities and also indicated a high fidelity of formation of the thioether bridges. This allowed us to further resolve the structure by analysis (mass spectrometry and tandem mass spectrometry) of the cyanogen bromide cleavage fragments of the C26A and C15A/C26A mutants. Nuclear magnetic resonance analysis established the presence of one and two dehydrobutyrine residues in mutacin II and the C15A variant, respectively, thus yielding the final structure. The results of this investigation showed that the C-terminal part contains three thioether bridges connecting Cys residues 15, 26, and 27 to Ser/Thr residues 10, 12 and 19, respectively, with Thr(25) being modified to dehydrobutyrine. 相似文献
19.
An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry. 相似文献
20.
Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component 总被引:45,自引:0,他引:45
R J Anderegg R Betz S A Carr J W Crabb W Duntze 《The Journal of biological chemistry》1988,263(34):18236-18240
Mating type a cells of the yeast Saccharomyces cerevisiae produce a mating hormone, the a-factor, that we have previously characterized as a very hydrophobic, modified dodecapeptide (Betz, R., Crabb, J. W., Meyer, H. E., Wittig, R., and Duntze, W. (1987) J. Biol. Chem. 262, 546-548). We have investigated the molecular structure in detail using mass spectrometry and proton NMR spectrometry of the intact hormone and authentic component molecules. Tandem mass spectrometry confirms the previously determined peptide sequence of the hormone and shows that it contains additional structural components with masses of 205 and 15 daltons. These were identified by proton NMR and mass spectrometry as a farnesyl (C15H25) residue and a terminal methyl ester group. The farnesyl moiety is attached to the sulfur atom of the carboxyl-terminal cysteine residue, as revealed by NMR of synthetic S-farnesyl cysteine methyl ester. The stereochemical configuration of the farnesyl moiety was determined to be trans,trans by comparison of gas chromatography retention times, mass spectra, and NMR spectra with those of standards. These results define the structure of a-factor as: (Sequence: see text). Replacement of the farnesyl by a methyl group leads to a partial reduction in specific biological activity of the a-factor, whereas hydrolysis of the carboxyl-terminal methyl ester causes a complete loss of activity. 相似文献