排序方式: 共有17条查询结果,搜索用时 15 毫秒
11.
Ronan Gealageas Natalia P. Malikova Sandrine Picaud Aren J. Borgdorff Ludmila P. Burakova Philippe Brûlet Eugene S. Vysotski Robert H. Dodd 《Analytical and bioanalytical chemistry》2014,406(11):2695-2707
The main analytical use of Ca2+-regulated photoproteins from luminous coelenterates is for real-time non-invasive visualization of intracellular calcium concentration ([Ca2+]i) dynamics in cells and whole organisms. A limitation of this approach for in vivo deep tissue imaging is the fact that blue light emitted by the photoprotein is highly absorbed by tissue. Seven novel coelenterazine analogues were synthesized and their effects on the bioluminescent properties of recombinant obelin from Obelia longissima and aequorin from Aequorea victoria were evaluated. Only analogues having electron-donating groups (m-OCH3 and m-OH) on the C6 phenol moiety or an extended resonance system at the C8 position (1-naphthyl and α-styryl analogues) showed a significant red shift of light emission. Of these, only the α-styryl analogue displayed a sufficiently high light intensity to allow eventual tissue penetration. The possible suitability of this compound for in vivo assays was corroborated by studies with aequorin which allowed the monitoring of [Ca2+]i dynamics in cultured CHO cells and in hippocampal brain slices. Thus, the α-styryl coelenterazine analogue might be potentially useful for non-invasive, in vivo bioluminescence imaging in deep tissues of small animals. 相似文献
12.
Frank LA Borisova VV Markova SV Malikova NP Stepanyuk GA Vysotski ES 《Analytical and bioanalytical chemistry》2008,391(8):2891-2896
Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution.
The mutant W92F-H22E emits violet light (λmax = 390 nm) and the mutant Y139F emits greenish light (λ
max = 498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration,
the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones—follicle-stimulating hormone
and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca2+ solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous
bioluminescence assay was close to that of a separate radioimmunoassay.
Figure Two kinds of Ca2+-regulated photoprotein obelin with altered color of bioluminescence were obtained and applied in dual-color simultaneous
immunoassay of two gonadotropic hormones. 相似文献
13.
Fernanda Ely José ES Nunes Evelyn K Schroeder Jeverson Frazzon Mário S Palma Diógenes S Santos Luiz A Basso 《BMC biochemistry》2008,9(1):13
Background
The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.Results
In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (Mt CS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant Mt CS. The bifunctionality of Mt CS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and Mt CS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.Conclusion
This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of Mt CS and should thus pave the way for the rational design of antitubercular agents.14.
The algebraic structure of chiral anomalies ismade globally valid on non-trivial bundles by the introduction of a fixed background connection. Some of the techniques used in the study of the anomaly are improved or generalized, including a systematic way of generating towers of ??descent equations??. 相似文献
15.
16.
Site‐directed mutagenesis is a powerful tool to investigate the structure–function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site‐directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+‐regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed. 相似文献
17.
S. T. Davitadze B. A. Strukov D. V. Vysotskiĭ V. V. Lemanov S. G. Shul’man Y. Uesu S. Asanuma 《Physics of the Solid State》2008,50(12):2303-2306
The temperature dependence of the heat capacity of thin epitaxial films BaTiO3/MgO is studied by the dynamic 3ω method in the thickness range 50–500 nm. It is revealed that the heat capacity exhibits diffuse anomalies due to phase transitions. The temperature of the ferroelectric phase transition T C increases with decreasing film thickness. The reasons for the strong diffuseness of the transition and the nonlinear dependence of the transition temperature on the film thickness are discussed. 相似文献