共查询到20条相似文献,搜索用时 8 毫秒
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The modification of deoxyribonucleohistone by trypsin and chymotrypsin 总被引:11,自引:0,他引:11
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Xuhui Tong William Lopez Jayalakshmi Ramachandran Wafaa A. Ayad Yu Liu Angelica Lopez-Rodriguez Andrew L. Harris Jorge E. Contreras 《The Journal of general physiology》2015,146(3):245-254
Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol–MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC. 相似文献
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Selective chemical modification of arginyl residues 总被引:2,自引:0,他引:2
T P King 《Biochemistry》1966,5(11):3454-3459
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The modem state of chemical modification of hyaluronic acid, chondroitin sulfates, and heparin is considered, and the possible application of modified glycosaminoglycans as potential drugs is discussed. 相似文献
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I. Yu. Ponedel’kina E. S. Lukina V. N. Odinokov 《Russian Journal of Bioorganic Chemistry》2008,34(1):1-23
The modern state of chemical modification of hyaluronic acid, chondroitin sulfates, and heparin is considered, and the possible application of modified glycosaminoglycans as potential drugs is discussed. 相似文献
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As part of an investigation of the organization of cell surface macromolecular assemblies, we have treated intact central nervous system cells with chemical probes which react convalently with proteins and aminophospholipids. Selective alterations of the enzymatic activities of ecto-ATPases, ecto-5'-nucleotidases and cholinesterases were obtained under appropriate reaction conditions. The cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene, was a potent inactivator of ecto-ATPase of C6 glioblastoma, IMR-32 neuroblastoma and of a primary rat astroblast cell line (RB). Ecto-5'-nucleotidase and acetylcholinesterase were less sensitive to difluorodinitrobenzene. 1-Fluoro-2,4-dinitrobenzene at concentrations which inactivated ecto-ATPase had little effect on ecto-5'-nucleotidase. Conversely, 2,4,6-trinitrobenzenesulfonic acid was a potent inactivator of ecto-5'-nucleotidase but had no effect on ecto-ATPase. The difluorodinitrobenzene inactivation of ecto-ATPase and of ecto-5'-nucleotidase as well as the fluorodinitrobenzene inactivation of ecto-ATPase could be prevented by the presence of the appropriate substrates in the reaction medium. In the presence of protecting nucleotide substrates, a decrease in reactivity with proteins and lipids was observed when the isotopic probe fluorodinitro[3H]-benzene was used. 相似文献
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G-actin has been nitrated with tetranitromethane in conditions that lead to the modification of one tyrosine residue. The reactive residue was found by earlier workers to be Tyr-69. The nitrated actin is conformationally similar to native G-actin, as judged by sedimentation velocity and circular dichroism analysis. A small proportion only is in the form of covalently linked dimers and trimers. The nitrated G-actin will polymerise to form filaments, indistinguishable in the electron microscope from those of native F-actin, but the polymerisation process is slower. Reduction of the nitrophenol group to the corresponding aminophenol leaves the properties of the protein in respect of polymerisation unchanged. When a dansyl group is introduced at the same point, however, the ability of the actin to polymerise is lost. The nitrated actin and its reduced counterpart will also bind heavy meromyosin, and the characteristic arrowhead formation of the bound molecules along the filaments can be seen in the electron microscope. Neither of the modified F-actins, however, significantly activates or inhibits the myosin ATPase activity. The fluorescence of nitrated actin is strongly quenched through the presence of the nitrophenol chromophore. In soluble complexes with heavy meromyosin the fluorescence is indistinguishable from the sum of the separate contributions of the two protein components. There is thus no measurable excitation transfer between any tryptophan residues on the myosin heads, such as that inferred to be present in the ATPase site, and the nitrotyrosine in position 69 of the actin sequence. Implications of this observation are considered in relation to the different interaction sites in myosin and in actin. The activation of heavy meromyosin ATPase by copolymers containing actin and nitroactin in different proportions has been measured, and is not proportional to the fraction of native actin. The results are consistent with the view that the function of actomyosin depends on the interaction of the myosin heads with more than one actin subunit. 相似文献
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E Agradi L Libertini S Smith 《Biochemical and biophysical research communications》1976,68(3):894-900
Fatty acid synthetase from lactating rat mammary gland after limited proteolysis with chymotrypsin or trypsin synthesizes longer chain fatty acids than those produced by the native enzyme. Of the seven partial reactions of the multienzyme complex, only the thioesterase activity was decreased. The results suggest that modification of the fatty acid synthetase product specificity by chymotrypsin and trypsin results from a specific action of these proteases on the thioesterase component. Trypsin, but not chymotrypsin, cleaved a catalytically active thioesterase from the complex; it thus appears that limited trypsinization will be a useful tool for the isolation of the thioesterase component of the multienzyme. 相似文献
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Acrolein-dependent chemical modification is implicated in the etiology of postoperative cognitive dysfunction (POCD). We examined this process further using human serum albumin (HSA), which is a target of acrolein modification and contains anesthetic binding sites. We tested whether trifluoroethanol (TFE), which mimics inhaled anesthetics, affects the susceptibility of HSA to modification by acrolein. We observed that acrolein promoted the formation of fluorescent adducts. TFE (10%) increased the amount of acrolein-HSA adducts. TFE (40%) caused a 5-fold increase in adduct formation. Acrolein also increased tryptophan anisotropy of HSA, which was further increased by TFE (10%). Acrolein-induced protein cross-linking was also increased in the presence of TFE (40%). These observations suggest that TFE promotes acrolein-induced modification of HSA, supporting a putative mechanism for POCD. 相似文献
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Base modification during solid-phase phosphoramidite synthesis of oligodeoxynucleotides has been investigated. We have discovered chemical modification that converts dG and dG-containing oligomers to a fluorescent form. This modification has been linked to N,N-dimethylaminopyridine (DMAP), an acylation catalyst, which can displace phosphate triester adducts at the 6-position of guanine. Further, we have found that this fluorescent intermediate can be converted in ammonium hydroxide solution to 2,6 diaminopurine deoxyribonucleoside (2,6 DAP), a potentially mutagenic nucleoside analog. We have shown that N-methylimidazole (NMI) in place of DMAP eliminates the fluorescent species and reduces 2,6 DAP contamination. 相似文献
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Four 25-nt long oligonucleotides containing dA and dT (D1, D2, D3, and D4) which are capable of forming parallel-stranded (ps) or antiparallel-stranded (aps) duplexes have been synthesized [Rippe, K., Ramsing, N. B., & Jovin, T. M. (1989) Biochemistry 28, 9536-9541]. In the present study, the OsO4-pyridine complex (Os,py), diethyl pyrocarbonate (DEPC), KMnO4, and the 1,10-phenanthroline-cuprous complex [(OP)2Cu+] were used to investigate the conformation-dependent reactivity of ps, aps, and single-stranded (ss) oligonucleotides. The products were analyzed by polyacrylamide gel electrophoresis with single-nucleotide resolution. The results confirm the duplex nature of the ps combinations of oligonucleotides and reveal structural differences in comparison with the aps molecules. Under conditions in which ss-DNA is substantially sensitive to Os,py, both the ps and aps duplexes are very unreactive. A similar result was observed with KMnO4 and DEPC, although with the latter reagent the modification pattern of the labeled strands D1* and D4* was slightly different for the parallel than for the antiparallel duplex. The (OP)2Cu+ complex efficiently cleaves the aps but not the ps duplex and shows a preference for TAT steps. We also tested the effect of monovalent and divalent cation concentrations on the chemical reactivity of the ps, aps, and ss species. Elevated NaCl concentration leads to a dramatic increase in the Os,py and KMnO4 modification of ss molecules and the ps, but not the aps, duplex. We attribute the apparent reaction with ps-DNA to a destabilization of this conformation under the conditions of reaction. In contrast, all reactions with DEPC are somewhat depressed at high salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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(3-14C)-serine was fed through cut ends of 10-day-old shoots of normal (Ganga-2) and opaque (opaque-2) maize ( L.) and allowed to be metabolized in light for 24 hours. Subsequent methanolic extraction, radiochromatographic separation and radioautographic analysis showed that 10.4 and 10.3 percent of the absorbed 14C-serine activity was incorporated into L-tryptophan by the shoot tissues of the normal and opaque maize respectively.By coupling extraction of the enzyme with ammonium sulfate precipitation (75% saturation) it was possible to show a serine-dependent indole utilization by the enzyme preparations from coleoptiles (4-day-old), kernels (18 and 28 days after anthesis) and first nodal callus tissues (one-month-old) of the two corn varieties. Furthermore, the indole disappearance from reaction mixture occurred with a concomitant transfer of (3-14C)-serine appearance into tryptophan. 相似文献