排序方式: 共有99条查询结果,搜索用时 359 毫秒
21.
The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities. 相似文献
22.
VM Hernández-Rocamora B Reija C García P Natale C Alfonso AP Minton S Zorrilla G Rivas M Vicente 《The Journal of biological chemistry》2012,287(36):30097-30104
The full-length ZipA protein from Escherichia coli, one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein, has been incorporated in single copy into nanodiscs formed by a membrane scaffold protein encircling an E. coli phospholipid mixture. This is an acellular system that reproduces the assembly of part of the cell division components. ZipA contained in nanodiscs (Nd-ZipA) retains the ability to interact with FtsZ oligomers and with FtsZ polymers. Interactions with FtsZ occur at similar strengths as those involved in the binding of the soluble form of ZipA, lacking the transmembrane region, suggesting that the transmembrane region of ZipA has little influence on the formation of the ZipA·FtsZ complex. Peptides containing partial sequences of the C terminus of FtsZ compete with FtsZ polymers for binding to Nd-ZipA. The affinity of Nd-ZipA for the FtsZ polymer formed with GTP or GMPCPP (a slowly hydrolyzable analog of GTP) is moderate (micromolar range) and of similar magnitude as for FtsZ-GDP oligomers. Polymerization does not stabilize the binding of FtsZ to ZipA. This supports the role of ZipA as a passive anchoring device for the proto-ring with little implication, if any, in the regulation of its assembly. Furthermore, it indicates that the tethering of FtsZ to the membrane shows sufficient plasticity to allow for its release from noncentral regions of the cytoplasmic membrane and its subsequent relocation to midcell when demanded by the assembly of a division ring. 相似文献
23.
We previously developed a calcium phosphate (CaP) calcifying solution that allows to deposit a uniform layer of nanocrystalline apatite on metallic implants in a few hours. In this work we modified the composition of the CaP solution by addition of Sr2+, Mg2+, and Mn2+, in order to improve the biological performance of the implants. The results of the investigation performed on the coatings, as well as on the powders precipitated in the absence of the substrates, indicate that both Sr2+ and Mg2+ reduce the extent of precipitation, although they are quantitatively incorporated into the nanocrystalline apatitic phase. The inhibitory effect on deposition is much more evident for Mn2+, which completely hinders the precipitation of apatite and yields just a small amount of amorphous phosphate relatively rich in manganese content. Human osteoblast-like MG-63 cells cultured on the different materials show that the Mg2+ and Sr2+ apatitic coatings promote proliferation and expression of collagen type I, with respect to bare Ti and to the thin layer of amorphous phosphate obtained in the presence of Mn2+. However, the relatively high content of Mn2+ in the phosphate has a remarkable beneficial effect on osteocalcin production, which is even greater than that observed for Sr2+. 相似文献
24.
Ádám Kupán Gábor Speier Michel Giorgi Ferenc Pollreisz 《Journal of inorganic biochemistry》2009,103(3):389-395
The attempted alkylation of 1,3-bis(2′-pyridylimino)isoindoline (indH) by the use of n-BuLi and subsequent alkyl halides led to quaternization of the pyridine nitrogens and the zwitterionic monodentate N-ligand (Me2ind)I was formed. By the use of the ligand the copper(I) complex [CuI(Me2ind)I2] was prepared and its structure determined. It was found to be good catalyst for the oxidation of 3,5-di-tert-butylcatechol (DTBCH2) to 3,5-di-tert-butyl-1,2-benzoquinone (DTBQ) and H2O2 by dioxygen. Detailed kinetic studies revealed first-order dependence on the catalyst and dioxygen concentration and saturation type behavior with respect to the substrate. 相似文献
25.
Li-Cheng Song Bin Gai Hu-Ting Wang Qing-Mei Hu 《Journal of inorganic biochemistry》2009,103(5):805-812
As an extension of our study on the H-cluster model compounds, a series of diiron propanediselenolate (PDS)-type models have been successfully synthesized. Reaction of diselenol HSe(CH2)3SeH with Fe3(CO)12 in THF (tetrahydrofuran) at reflux gave the parent model compound [μ-Se(CH2)3Se-μ]Fe2(CO)6 (1) in 48% yield. Further reaction of 1 with PPh3 or PPh2H in the presence of Me3NO in MeCN at room temperature afforded the phosphine-monosubstituted model compounds [μ-Se(CH2)3Se-μ]Fe2(CO)5(L) (2, L = PPh3; 3, L = PPh2H) in 76% and 68% yields, respectively. Similarly, the N-heterocyclic carbene IMes-monosubstituted model compound [μ-Se(CH2)3Se-μ]Fe2(CO)5(IMes) (4) could be prepared in 46% yield by reaction of imidazolium salt IMes · HCl with n-BuLi followed by treatment of the resulting IMes ligand with 1 in THF at room temperature. Compounds 1-4 were fully characterized by elemental analysis and various spectroscopic methods. While the structures of 1-4 were further confirmed by X-ray crystallography, the comparative study of 1 and its analog [μ-S(CH2)3S-μ]Fe2(CO)6 demonstrates that 1 is a better catalyst for TsOH proton reduction to hydrogen under electrochemical conditions. 相似文献
26.
Yufei Zhang Hong Wu Lin Li Jian Li Zhongyi Jiang Yanjun Jiang Ying Chen 《Journal of Molecular Catalysis .B, Enzymatic》2009,57(1-4):130-135
In this study, Baicalein was produced through an enzymatic conversion catalyzed by β-glucuronidase (GUS) encapsulated in biomimetic alginate/protamine/silica (APSi) capsules. Experimental results indicated that the thermal and pH tolerance as well as the storage and recycling stability of GUS were significantly improved after encapsulation. Under the optimum conversion conditions (37 °C, pH 7), a high productivity of Baicalein (73%) was obtained. No loss in enzyme activity was observed after 11-day storage and 90% of the initial activity remained after 26-day storage. No appreciable loss in activity was found during 10 repeated reaction cycles. The facile encapsulation process, the high conversion efficiency and the enhanced stability set an encouraging example for converting natural compounds into high value-added functional products. 相似文献
27.
Fabrice Thomas 《Inorganica chimica acta》2010,363(12):3122-11468
The three complexes [CoIIIL1Cl] (1), [CoIIIL2]+·ClO4− (2+·ClO4−), and [CuIIH2L2]2+·2ClO4− (H232+·2ClO4−) [where H2L1 = N,N′-dimethyl-N,N′-bis(2-hydroxy-3,5-di-tert-butylbenzyl)ethylenediamine, H2L2 = N,N′-bis(2-pyridylmethyl)-N,N′-bis(2-hydroxy-3,5-di-tert-butylbenzyl)ethylenediamine] have been prepared. The bis-phenolate and bis-phenol complexes, 2+ and H232+ respectively, have been characterized by X-ray diffraction, showing a metal ion within an elongated octahedral geometry. 1-2 exhibit in their cyclic voltammetry curves two anodic reversible waves attributed to the successive oxidation of the phenolates into phenoxyl radicals. The cobalt radical species (1)+, (2)2+, and (2)3+ have been characterized by combined UV-Vis and EPR spectroscopies. In the presence of one equivalent of base, one phenolic arm of H232+ is deprotonated and coordinates the metal. The resulting complex (H3+) exhibits a single reversible redox wave at ca. 0.3 V. The electrochemically generated oxidized species is EPR silent and exhibits the typical features of a radical compound, with absorption bands at 411 and 650 nm. The fully deprotonated complex 3 is obtained by addition of two equivalents of nBu4N+OH− to H232+. It exhibits a new redox wave at a lower potential (−0.16 V), in addition to the wave at ca. 0.3 V. We assigned the former to the one-electron oxidation of the uncoordinated phenolate into an unstable phenoxyl radical. 相似文献
28.
Nanostructural features of demosponge biosilica 总被引:6,自引:0,他引:6
Weaver JC Pietrasanta LI Hedin N Chmelka BF Hansma PK Morse DE 《Journal of structural biology》2003,144(3):271-281
Recent interest in the optical and mechanical properties of silica structures made by living sponges, and the possibility of harnessing these mechanisms for the synthesis of advanced materials and devices, motivate our investigation of the nanoscale structure of these remarkable biomaterials. Scanning electron and atomic force microscopic (SEM and AFM) analyses of the annular substructure of demosponge biosilica spicules reveals that the deposited material is nanoparticulate, with a mean particle diameter of 74+/-13 nm. The nanoparticles are deposited in alternating layers with characteristic etchant reactivities. Further analyses of longitudinally fractured spicules indicate that each deposited layer is approximately monoparticulate in thickness and exhibits extensive long range ordering, revealing an unanticipated level of nanoscale structural complexity. NMR data obtained from differentially heated spicule samples suggest that the etch sensitivity exhibited by these annular domains may be related to variation in the degree of silica condensation, rather than variability in the inclusion of organics. In addition, AFM phase imaging in conjunction with results obtained from HF and alkaline etching experiments suggest that at various stages in spicule biosynthesis, regions of unusually low silica condensation are deposited, indicating a possible interruption in normal spicule formation. While this discovery of nanoparticulate silica aggregation in demosponge skeletal elements is likely to reflect the intrinsic kinetic tendency of silica to form such particles during polycondensation, the heirarchical organization of these nanoparticles is biologically unique. 相似文献
29.
A hybrid bilayer membrane is a planar model membrane that is formed at an alkanethiol monolayer-coated gold surface by the spontaneous reorganization of phospholipid vesicles. Membrane vesicles from monkey kidney COS-1 cells also reorganize at an alkanethiol/lipid monolayer-coated surface resulting in the formation of a cell membrane hybrid bilayer. Atomic force microscopy and spectroscopic ellipsometry indicate that the cell membrane layer is equivalent to the thickness of one leaflet of the membrane and is continuous over large areas. Cell membrane hybrid bilayers were formed from membrane vesicles from COS-1 cells that were transiently transfected with a synthetic human CCR5 chemokine receptor gene. Preparations that contained "inside out" and "right side out" membrane vesicles were used. Binding of monoclonal antibodies to either the amino- or carboxyl-terminus of CCR5 was observed by surface plasmon resonance and confirmed the presence and the random orientation of these integral membrane receptors. Specific and concentration-dependent binding of the beta-chemokine RANTES to the cell membrane hybrid confirmed that CCR5 retained ligand-binding activity. The ability to form cell membrane hybrid bilayers that contain functional G-protein-coupled or other multispanning receptors without requiring protein isolation, purification, and reconstitution offers a promising method for the rapid screening of potential ligands. 相似文献
30.
《Nucleosides, nucleotides & nucleic acids》2013,32(5-8):519-534
Abstract Ribonucleoside 5′-diphosphate reductases (RDPRs) have been studied for several decades. Increasingly sophisticated mechanisms have been proposed for the reduction of natural substrate ribonucleotides to their 2′-deoxy counterparts and for mechanism-based inactivation of RDPRs with 2′-substituted-ribonucleotides. We now discuss biomimetic reactions of model substrate and inhibitor analogues, which clarify three aspects of previously proposed mechanisms postulated to occur at the active site of RDPRs. 相似文献