结构与功能

Flux variability analysis enables comprehensive exploration of alternate optimal routes in a metabolic network. This method is especially useful with models such as bna572 for the developing oilseed rape embryo which is highly compartmentalized. Here, we describe a protocol for ca ...

The analysis of plant metabolic networks essentially contributes to the understanding of the efficiency of plant systems in terms of their biotechnological usage. Metabolic fluxes are determined by biochemical parameters such as metabolite concentrations as well as enzyme pro ...

This chapter describes a practical procedure to dissect metabolic systems, simplify them, and use or derive enzyme rate equations in order to build a mathematical model of a metabolic system and run simulations. We first deal with a simple example, modeling a single enzyme that follows Michael ...

The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The implementation of microplate technology enables the determination of up to 50 enzymes in relatively large num ...

In this chapter we illustrate the methodology for high-throughput metabolic flux analysis. Central to this is developing an end to end data pipeline, crucial for integrating the wet lab experiments and analytics, combining hardware and software automation, and standardizing data re ...

An important aspect of kinetic modeling is the ability to provide predictive information on network control and dynamic responses to genetic or environmental perturbations based on innate enzyme kinetics. In a top-down approach to model assembly, unknown kinetic parameters are cal ...

Metabolic flux analysis (MFA) is a powerful approach for quantifying plant central carbon metabolism based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstati ...

Metabolic flux analysis (MFA) is a powerful tool for exploring and quantifying carbon traffic in metabolic networks. Accurate flux quantification requires (1) high-quality isotopomer measurements, usually of biomass components including proteinogenic/free amino acids or ...

This chapter describes a procedure to analyze 13C-labeled phosphorylated compounds by liquid chromatography tandem mass spectrometry. Phosphorylated compounds, intermediaries of the glycolysis and pentose phosphate pathway, are separated by anion exchange chromato ...

We describe an approach to extract 13C-labeled sugars (glucose, fructose, maltose, sucrose, myo-inositol as well as glucose from starch) from plant tissues and to analyze their isotopomer distribution by gas chromatography–mass spectrometry (GC-MS). Sugars are derivatized with N, ...

Plant metabolic pathways and the molecular and atomic fluxes through them can be deduced using stable isotopically labeled substrates. To this end one prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of ...

Comprehensive analysis of isotopic labeling patterns of metabolites in proteinogenic amino acids and starch for plant systems lay in the powerful tool of 2-Dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy. From 13C-labeling experiments, 2D NMR provides informati ...

The analysis of stable isotope incorporation following feeding of 13C-labeled precursors to plant tissues provides the constraints necessary for metabolic flux analysis. This protocol describes the use of one-dimensional 1H and 13C nuclear magnetic resonance spectroscopy f ...

While steady-state 13C metabolic flux analysis is a powerful method for deducing multiple fluxes in the central metabolic network of heterotrophic and mixotrophic plant tissues, it is also time-consuming and technically challenging. Key steps in the design and interpretation of ste ...

Steady state 13C-MFA is classically used to measure fluxes in complex metabolic networks. However, the modeling of steady state labeling allows the quantification of internal fluxes only and requires the estimation, by other methods, of the external fluxes, corresponding to substrate ...

The use of in vivo NMR within the framework of Metabolic Flux Analysis in plants is presented. In vivo NMR allows to visualize the active metabolic network, to determine metabolic and isotopic steady state and to measure metabolic fluxes which are not necessarily accessible by isotopic steady s ...

This chapter focuses on the way to build a metabolic network and how to analyze its structure. The first part of this chapter describes the methods of the network model reconstruction from biochemical data found in specialized databases and/or literature. The second part deals with metabolic ...

This volume compiles a series of chapters that cover the major aspects of plant metabolic flux analysis, such as but not limited to labeling of plant material, acquisition of labeling data, mathematical modeling of metabolic network at the cell, tissue, and plant level. A short revue, including m ...

The integration of mathematical modeling with analytical experimentation in an iterative fashion is a powerful approach to advance our understanding of the architecture and regulation of metabolic networks. Ultimately, such knowledge is highly valuable to support efforts aim ...

The importance of kinetic modeling for understanding the control and regulation of complex metabolic networks is increasingly being recognized. Kinetic models encapsulate the available kinetic information of all the enzymes in a pathway, and then calculate the complex behavior t ...