Abstract
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 numbers of samples and in various biological materials. Most of these enzymes are involved in central metabolism and several pathways are entirely covered. Direct or indirect assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, protocols for UDP-glucose pyrophosphorylase (E.C. 2.7.7.9) operating in real time and for pyrophosphate:fructose-6-phosphate 1-phosphotransferase (E.C. 2.7.1.90) are presented.
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Conflict of Interest The authors declare that they have no conflict of interest.
References
Buchner E (1897) Alkoholische Gärung ohne Hefezellen. Berichte der deutschen chemischen Gesellschaft 30:1110–1113
Kruckeber AL, Neuhaus HE, Feil R et al (1989) Decreased-activity mutants of phosphoglucose isomerase in the cytosol and chloroplast of Clarkia xantiana—Impact on mass-action ratios and fluxes to sucrose and starch, and estimation of flux control coefficients and elasticity coefficients. Biochem J 261:457–467
Stitt M, Schulze D (1994) Does Rubisco control the rate of photosynthesis and plant-growth—an exercise in molecular ecophysiology. Plant Cell Environ 17:465–487
Poolman MG, Olcer H, Lloyd JC et al (2001) Computer modelling and experimental evidence for two steady states in the photosynthetic Calvin cycle. Eur J Biochem 268:2810–2816
Kacser HJ, Burns A (1973) The control of flux. Symp Soc Exp Biol 27:65–104
Jenner HL (2003) Transgenesis and yield: what are our targets? Trends Biotechnol 21:190–192
Schomburg I, Chang A, Ebeling C et al (2004) BRENDA, the enzyme database: updates and major new developments. Nucleic Acids Res 32:D431–D433
Rohwer JM, Botha FC (2001) Analysis of sucrose accumulation in the sugar cane culm on the basis of in vitro kinetic data. Biochem J 358:437–445
Rowher JM (2012) Kinetic modelling of plant metabolic pathways. J Exp Bot 63:2275–2292
Gardossi L, Poulsen PB, Ballesteros A et al (2010) Guidelines for reporting of biocatalytic reactions. Trends Biotechnol 28:171–180
Kaiser WM, Huber SC (2001) Post-translational regulation of nitrate reductase: mechanism, physiological relevance and environmental triggers. J Exp Bot 52:1981–1989
Winter H, Huber SC (2000) Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit Rev Plant Sci 19:31–67
Bergmeyer HU (1987) Methods of enzymatic analysis. VCH, Weinheim, Germany
Gibon Y, Blaesing OE, Hannemann J et al (2004) A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: Comparison of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16:3304–3325
Junker BH, Lonien J, Heady LE et al (2007) Parallel determination of enzyme activities and in vivo fluxes in Brassica napus embryos grown on organic or inorganic nitrogen source. Phytochem 68:2232–2242
Gibon Y, Pyl E-T, Sulpice R et al (2009) Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods. Plant Cell Environ 32:859–874
Tschoep H, Gibon Y, Carillo P et al (2009) Adjustment of growth and central metabolism to a mild but sustained nitrogen-limitation in Arabidopsis. Plant Cell Environ 32:300–318
Gillespie KM, Rogers A, Ainsworth EA et al (2011) Growth at elevated ozone or elevated carbon dioxide concentration alters antioxidant capacity and response to acute oxidative stress in soybean (Glycine max). J Exp Bot 62:2667–2678
Biais B, Bernillon S, Deborde C et al (2012) Precautions for harvest, sampling, storage, and transport of crop plant metabolomics samples. Methods Mol Biol 860:51–63, Clifton NJ ed
Rogers A, Gibon Y (2009) Enzyme kinetics: theory and practice. In: Svhwender J (ed) Plant metabolic networks. Springer, New York, pp 71–103. ISBN 978-0-38-778744-2
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Financial support from INRA, Région Aquitaine, Eranet KBBE SAFQIM, Eranet Erasysbio+ FRIM, FP7 KBBE DROPS and SFR BIE is gratefully acknowledged.
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Ménard, G., Biais, B., Prodhomme, D., Ballias, P., Gibon, Y. (2014). Analysis of Enzyme Activities. In: Dieuaide-Noubhani, M., Alonso, A. (eds) Plant Metabolic Flux Analysis. Methods in Molecular Biology, vol 1090. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-688-7_15
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DOI: https://doi.org/10.1007/978-1-62703-688-7_15
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