TAILIEUCHUNG - Báo cáo khoa học: Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry

Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype. Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose. Of particular interest are meth-ods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution. In this paper, we present data validating the use of mass spectrometry (MS) for the quantification of complex metabolic flux networks. | Eur. J. Biochem. 270 3525-3542 2003 FEBS 2003 doi Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry Maria I. Klapa Juan-Carlos Aont and Gregory Stephanopoulos Department of Chemical Engineering Massachusetts Institute of Technology Cambridge MA USA Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype. Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose. Of particular interest are methods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution. In this paper we present data validating the use of mass spectrometry MS for the quantification of complex metabolic flux networks. In the context of the lysine biosynthesis flux network of Corynebacterium glutamicum ATCC 21799 under glucose limitation in continuous culture operating at h-1 after the introduction of 50 1-13C glucose we deploy a bioreaction network analysis methodology for flux determination from mass isotopomer measurements of biomass hydrolysates while thoroughly addressing the issues of measurement accuracy flux observability and data reconciliation. The analysis enabled the resolution of the involved anaplerotic activity of the microorganism using only one labeled substrate the determination of the range of most of the exchange fluxes and the validation of the flux estimates through satisfaction of redundancies. Specifically we determined that phosphoenolpyruvate carboxykinase and synthase do not carry flux at these experimental conditions and identified a high futile cycle between oxaloacetate and pyruvate indicating a highly active in vivo oxaloacetate decarboxylase. Both results validated previous in vitro activity measurements. The flux estimates obtained passed the v statistical test. This is a very important result considering that prior flux analyses of extensive metabolic .

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