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221.
Quentmeier A Hellwig P Bardischewsky F Grelle G Kraft R Friedrich CG 《Biochemical and biophysical research communications》2003,312(4):1011-1018
The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, formed complexes with subunits associated and covalently bound. In denaturing SDS-polyacrylamide gel electrophoresis (PAGE) SoxY migrated at 12 and SoxZ at 16kDa. SDS-PAGE of homogeneous SoxYZ without reductant separated dimeric complexes of 25, 29, and 32kDa identified by the N-terminal amino acid sequences as SoxY-Y, SoxY-Z, and SoxZ-Z, and subunit cleavage by reduction suggested their linkage via protein disulfide bonds. SoxYZ was reversibly redox active between -0.25 and 0.2V, as monitored by a combined electrochemical and FTIR spectroscopic approach. The dimanganese SoxB protein (58.611Da) converted the covalently linked heterodimer SoxY-Z to SoxYZ with associated subunits which in turn aggregated to the heterotetramer Sox(YZ)(2). This reaction depended on time and the SoxB concentration, and demonstrated the interaction of these two Sox proteins. 相似文献
222.
Gallego-Nicasio J López-Rodríguez G Martínez R Tarancón MJ Fraile MV Carmona P 《Biopolymers》2003,72(6):514-520
The compositional and structural changes of lipids and apolipoproteins during in vitro oxidation of low density lipoprotein (LDL) are investigated in this study by IR spectroscopy. For comparison, LDL samples containing either copper or glucose at physiological or pathological concentrations are considered in order to know the separate affects of these chemical factors on LDL oxidation. The results show that the initial steps of lipid oxidation proceed through hydrogen atom loss from methylene groups, as well as loss of cholesteryl ester molecules, and later a recovering of carbonyl compounds resulting from aldehyde formation that generally occurs in autooxidation processes. Lipid oxidation is induced by copper ions, and glucose enhances metal ion induced LDL oxidation as determined by conjugated diene formation. As to the protein conformational changes, IR spectroscopy reveals for the first time that LDL oxidation involves formation of beta-sheet structures, these being more abundant in LDL samples with pathological concentrations of glucose or copper. Consequently, the LDL structural changes may contribute to the recognition of oxidized LDL particles by scavenger receptors. 相似文献
223.
Person MD Brown KC Mahrus S Craik CS Burlingame AL 《Protein science : a publication of the Protein Society》2001,10(8):1549-1562
In the presence of a suitable oxidizing agent, the Ni(II) complex of glycyl-glycyl-histidine (GGH) mediates efficient and specific oxidative protein cross-linking. The fusion of GGH to the N terminus of a protein allows for the cross-linking reagent to be delivered in a site-specific fashion, making this system extremely useful for analyzing protein-protein contacts in complicated mixtures of biomolecules. Tyrosine residues have been postulated to be the primary amino acid target of this reaction, and using the dimeric serine protease inhibitor ecotin, we previously demonstrated that engineering a tyrosine at the protein interface of a dimer dramatically increased cross-linking efficiency. Cross-linking increased four-fold for GGH-ecotin D137Y in comparison to wild-type GGH-ecotin, presumably through bityrosine formation at the dimer interface. Here we report the first complete structural analysis of the cross-linked GGH-ecotin D137Y dimer. Using a combination of mass spectrometric and chemical derivatization methods, a sole novel cross-link between the N-terminal glycine residues and the engineered tyrosine at position 137 has been characterized. The dimer cross-link is localized to a single site without other protein modifications, but different reaction pathways produce structurally related products. We propose a mechanism that involves covalent bond formation between the protein backbone and a dopaquinone moiety derived from a specific tyrosine residue. This finding establishes that it is not necessary to have two tyrosine residues within close proximity in the protein interface to obtain high protein cross-linking yields, and suggests that the cross-linking reagent may be of more general utility than previously thought. 相似文献
224.
Differential in vitro and in vivo behavior of mouse ascorbate/Fe3+ and diamide oxidized erythrocytes 总被引:1,自引:0,他引:1
Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe3+ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe3+ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe3+ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used. 相似文献
225.
226.
The oxidation mechanism of caffeic acid (CAF) has been studied by means of cyclic voltammetry with the plastic formed carbon or glassy carbon electrode. CAF gives a well-developed two-electron reversible wave in acidic media, whereas it shows an irreversible behavior, i.e., a decrease of the rereduction peak, in less acidic media, suggesting that the oxidation of CAF follows an irreversible chemical reaction(s). Digital simulation analyses based on different oxidation mechanisms have been performed for the voltammograms obtained with the GC electrode in 1:1 (v/v) water:ethanol solutions. The results clearly show that the seeming two-electron oxidation of CAF occurs stepwise via one-electron processes, each of which follows an irreversible chemical reaction. It has also been suggested that the semiquinone radical as an intermediate of the one-electron oxidation should play an important role in the oxidation reaction. Evaluations of the rate constants for the chemical reactions have further suggested that the chemical reactions are dimerization reactions. 相似文献
227.
By monitoring R(pip)/R(Fpg), i.e. the relative sensitivity to hot piperidine and to formamidopyrimidine DNA glycosylase (Fpg protein) of the guanine lesions induced in DNA exposed to UV laser irradiation, we have previously observed that the formation of the two major types of one-electron oxidative guanine modifications, oxazolone and 7,8-dihydro-8-oxoguanine (8-oxodG), depends on DNA conformational features. While oxazolone is largely predominant at each site of single-stranded DNA (R(pip)>R(Fpg)), 8-oxodG is the major lesion at most of the sites of double-stranded DNA (R(pip)R(Fpg) at 20 degrees C and the ratio R(pip)/R(Fpg) does not vary significantly during the melting process. Interestingly, these guanine residues display a high sensitivity to dimethyl sulfoxide methylation while the opposite cytosine residues are unsensitive, suggesting that the prevalence of R(pip) over R(Fpg) is related not to base-pairing disruption but rather to the local helical alteration of the B-DNA stacking geometry. This leads us to propose that the slight variations in the ratios R(pip)/R(Fpg) observed, at individual sites, at temperatures below the helix-coil transition reflect local small-scale breathing motions, unstacking single dinucleotide steps prior to opening. Our results thus support the view that the temperature dependence of the ratio of R(pip)/R(Fpg) at sites of B-DNA provides a sensitive probe of the DNA internal local thermal stability and are discussed in relation with the mechanisms proposed for the intramolecular rearrangement of the guanyl radical. 相似文献
228.
Takahashi M Maraboeuf F Sakai Y Yakushiji H Mishima M Shirakawa M Iwai S Hayakawa H Sekiguchi M Nakabeppu Y 《Journal of molecular biology》2002,319(1):129-139
The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd. 相似文献
229.
Thies MJ Talamo F Mayer M Bell S Ruoppolo M Marino G Buchner J 《Journal of molecular biology》2002,319(5):1267-1277
The non-covalent homodimer formed by the C-terminal domains of the IgG1 heavy chains (C(H)3) is the simplest naturally occurring model system for studying immunoglobulin folding and assembly. In the native state, the intrachain disulfide bridge, which connects a three-stranded and a four-stranded beta-sheet is buried in the hydrophobic core of the protein. Here, we show that the disulfide bridge is not required for folding and association, since the reduced C(H)3 domain folds to a dimer with defined secondary and tertiary structure. However, the thermodynamic stability of the reduced C(H)3 dimer is much lower than that of the oxidized state. This allows the formation of disulfide bonds either concomitant with folding (starting from the reduced, denatured state) or after folding (starting from the reduced dimer). The analysis of the two processes revealed that, under all conditions investigated, one of the cysteine residues, Cys 86, reacts preferentially with oxidized glutathione to a mixed disulfide that subsequently interacts with the less-reactive second thiol group of the intra-molecular disulfide bond. For folded C(H)3, the second step in the oxidation process is slow. In contrast, starting from the unfolded and reduced protein, the oxidation reaction is faster. However, the overall folding reaction of C(H)3 during oxidative folding is a slow process. Especially, dimerization is slow, compared to the association starting from the denatured oxidized state. This deceleration may be due to misfolded conformations trapped by the disulfide bridge. 相似文献
230.
Castegna A Aksenov M Thongboonkerd V Klein JB Pierce WM Booze R Markesbery WR Butterfield DA 《Journal of neurochemistry》2002,82(6):1524-1532
Alzheimer's disease (AD) is a neurodegenerative disorder in which oxidative stress has been implicated as an important event in the progression of the pathology. In particular, it has been shown that protein modification by reactive oxygen species (ROS) occurs to a greater extent in AD than in control brain, suggesting a possible role for oxidation-related decrease in protein function in the process of neurodegeneration. Oxidative damage to proteins, assessed by measuring the protein carbonyl content, is involved in several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, neuronal death. The present investigation represents a further step in understanding the relationship between oxidative modification of protein and neuronal death in AD. Previously, we used our proteomics approach, which successfully substitutes for labor-intensive immunochemical analysis, to detect proteins and identified creatine kinase, glutamine synthase and ubiquitin carboxy-terminal hydrolase L-1 as specifically oxidized proteins in AD brain. In this report we again applied our proteomics approach to identify new targets of protein oxidation in AD inferior parietal lobe (IPL). The dihydropyrimidinase related protein 2 (DRP-2), which is involved in the axonal growth and guidance, showed significantly increased level in protein carbonyls in AD brain, suggesting a role for impaired mechanism of neural network formation in AD. Additionally, the cytosolic enzyme alpha-enolase was identified as a target of protein oxidation and is involved the glycolytic pathway in the pathological events of AD. Finally, the heat shock cognate 71 (HSC-71) revealed increased, but not significant, oxidation in AD brain. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. 相似文献