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排序方式: 共有1765条查询结果,搜索用时 15 毫秒
1.
《Journal of molecular biology》2021,433(15):167097
DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding. 相似文献
2.
Monte Carlo simulations and a modified Poisson–Boltzmann (MPB) theory are used to investigate the temperature dependence of the capacitance (around the potential of zero charge) of an electric double layer in the presence of surface polarization due to a dielectric boundary. Within the context of the restricted primitive model planar double layer, whose solvent dielectric constant is ε2, the cases when the electrode is an insulator (ε1 = 1), when the electrode and the electrolyte have the same permittivity (ε1 = ε2, no polarization), and when the electrode is a conductor (ε1 → ∞) are studied for the case where the electrolyte concentration is 0.1 M. The simulations reveal a capacitance anomaly, that is, a positive temperature dependence of the capacitance at low temperatures for the former two situations. The MPB theory also shows this effect for these two situations and is in qualitative or better agreement with the simulation data. In these two cases, both the simulations and theory show a dramatic increase of the diffuse layer potential in the temperature regime where capacitance anomaly occurs. However, in the latter situation, where the electrode is metallic, the capacitance always has a negative temperature derivative for the MPB theory and probably also for the simulation data. 相似文献
3.
Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks. 相似文献
4.
A M O'Brien C O'Fágáin P F Nielsen K G Welinder 《Biotechnology and bioengineering》2001,76(4):277-284
The bifunctional compound, ethylene-glycol bis(N-hydroxysuccinimidylsuccinate) (EGNHS), stabilizes horseradish peroxidase C (HRP) by reaction with the enzyme's lysine residues. In this study we compare native and modified HRP by proteolytic fragmentation, peptide sequencing, and mass spectroscopy, and identify the sites of modification. Most significantly, EGNHS is shown to form a crosslink between Lys232 and Lys241 of HRP and modifies Lys174 without formation of a crosslink. These findings are in agreement with the lysine side-chain reactivities predicted from the surface accessibility of the amino groups, and the maximal span of 16 A of the EGNHS crosslinker. 相似文献
5.
A. K. Vidybida 《European biophysics journal : EBJ》1989,16(6):357-361
A theoretical model is proposed to describe the influence of a periodic electric field (PEF) upon a biopolymer. The biopolymer is treated as a classical mechanical system consisting of subsystems (molecular groups) which interact with each other through potential forces. The PEF is treated as a periodic driving force applied to a molecular group. The energy dissipation is considered using the model of fluid (viscous) friction. Arguments for the non-linear character of the friction-velocity dependence caused by the non-Newtonian rheology of a viscous medium are formulated.A forced molecular-group motion is investigated for the situation of a small driving-force period, with oscillations overdamped and a driving force consisting of more than one harmonic. As a result, it is established that the motion always gets to a terminal stage when only a small-scale vibration about some point, X
*, takes place. The terminal motion is preceded by a transient characterized by the presence of a directional velocity component and so by a drift along a potential profile. the drift goes on until a barrier is met which has a sufficiently large steepness (the barrier height is not important). As a result, the point X
* may happen to be remote from the conformation potential local minimum (conformational state). The physical reasons for the drift are described.If we consider the small-scale vibration about X
* in the framework of the hierarchy of scales for intramolecular mobility, it can be regarded as an equilibrium mobility, whereas the drift can be regarded as a functionally important motion, and X
* as a new conformational state which is realizable only in the presence of the PEF. It may be concluded, as the result of a consistent treatment and neglecting the small-scale vibration, that the conformational potential is modified by adding a linear term (in the one-dimensional case). In this connection, the set of conformational states can both deform (deviation of the positions of minima and their relative depth) and rearrange qualitatively (some minima can vanish and/or new minima can appear). In particular, the transition from one conformation to another one may happen due to rearrangement. 相似文献
6.
F N Zeytin F Reyl-Desmars T Rathbun 《Biochemical and biophysical research communications》1985,127(3):992-998
GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist. 相似文献
7.
N E Owen 《Biochemical and biophysical research communications》1984,125(2):500-508
The regulation of Na/K/Cl cotransport was investigated in vascular smooth muscle cells. That a Na/K/Cl cotransport system exists was established by the finding that the ouabain insensitive K influx was sensitive to the "loop" diuretic bumetanide. Furthermore, bumetanide sensitive K influx was dependent upon the presence of both Na and Cl in the extracellular milieu. Bumetanide sensitive K influx was inhibited by agents which elevate cellular cyclic AMP levels, and to a lesser extent by agents which elevate cellular cyclic GMP levels. When serum, EGF or TPA was added, bumetanide sensitive K influx was enhanced. These results suggest that vascular smooth muscle cells have a ouabain insensitive, bumetanide sensitive Na/K/Cl cotransport system which is stimulated by serum, EGF or TPA and inhibited by cAMP or cGMP. 相似文献
8.
Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells 总被引:3,自引:0,他引:3
In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells. 相似文献
9.
A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 106 cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 105 cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 106 cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture. 相似文献
10.
Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors. 相似文献