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Quantitative distinction between bound and free NADH in biological systems

机译:生物系统中结合和游离NADH的定量区别

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摘要

Without exceptions, in all living cells NADH is a key metabolite linking a large number of metabolic pathways. Flux rates through such pathways are an essential component in the understanding of the functioning of living cells. Knowledge about the way these fluxes depend on the concentrations of the metabolites involved (including NADH/NAD~+) allows calculation of these fluxes. Therefore, a method to determine the concentration of free NADH is necessary. A distinction between the free and protein-bound NADH can be made on the basis of fluorescence emission spectra and fluorescence lifetimes. A method for such measurements using a microscopic set-up for time-gated fluorescence spectroscopy has been introduced by Schneckenburger and co-workers (Paul RJ, Schneckenburger H. Naturwissenschaften 83, pp. 32-35, 1996). We further improve this method by first characterizing NADH binding to model proteins by isothermal titration calorimetry and fluorescence. This allows a precise calculation of bound and free NADH and their respective spectra. An analysis of experimental data is advanced by applying two-component deconvolution and subsequent fitting. Using this method we can detect a significant proportion of free NADH in isolated potato tuber mitochondria respiring malate. Taken together these improvements allow a more accurate characterization of the NADH turnover in biological systems.
机译:无一例外,在所有活细胞中,NADH是连接大量代谢途径的关键代谢产物。通过这种途径的通量率是理解活细胞功能的重要组成部分。有关这些通量取决于相关代谢物浓度(包括NADH / NAD〜+)的方式的知识可以计算这些通量。因此,需要一种确定游离NADH浓度的方法。可以根据荧光发射光谱和荧光寿命来区分游离的和结合蛋白的NADH。 Schneckenburger和他的同事们已经介绍了一种使用用于时间门控荧光光谱的显微装置进行此类测量的方法(Paul RJ,Schneckenburger H. Naturwissenschaften 83,第32-35页,1996年)。我们通过首先通过等温滴定量热法和荧光表征NADH与模型蛋白的结合来进一步改进该方法。这样可以精确计算结合的和游离的NADH及其各自的光谱。通过应用二分量反卷积和随后的拟合来推进对实验数据的分析。使用这种方法,我们可以在分离出的马铃薯块茎线粒体呼吸苹果酸中检测到很大比例的游离NADH。综合起来,这些改进可以更准确地表征生物系统中的NADH转换。

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