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Measuring enzyme activities under standardized in vivo-like conditions for systems biology

Authors

  • Karen van Eunen,

    1.  Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands
    2.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
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  • Jildau Bouwman,

    1.  Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands
    2.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
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  • Pascale Daran-Lapujade,

    1.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    2.  Department of Biotechnology, Delft University of Technology, The Netherlands
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  • Jarne Postmus,

    1.  Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
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  • André B. Canelas,

    1.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    2.  Department of Biotechnology, Delft University of Technology, The Netherlands
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  • Femke I. C. Mensonides,

    1.  Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands
    2.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
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  • Rick Orij,

    1.  Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
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  • Isil Tuzun,

    1.  Department of Molecular Micriobial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
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  • Joost van den Brink,

    1.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    2.  Department of Biotechnology, Delft University of Technology, The Netherlands
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  • Gertien J. Smits,

    1.  Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
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  • Walter M. van Gulik,

    1.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    2.  Department of Biotechnology, Delft University of Technology, The Netherlands
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  • Stanley Brul,

    1.  Department of Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
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  • Joseph J. Heijnen,

    1.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    2.  Department of Biotechnology, Delft University of Technology, The Netherlands
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  • Johannes H. de Winde,

    1.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    2.  Department of Biotechnology, Delft University of Technology, The Netherlands
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  • M. Joost Teixeira de Mattos,

    1.  Department of Molecular Micriobial Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands
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  • Carsten Kettner,

    1.  Beilstein-Institut zur Förderung der Chemischen Wissenschaften, Frankfurt/Main, Germany
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  • Jens Nielsen,

    1.  Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
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  • Hans V. Westerhoff,

    1.  Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands
    2.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    3.  Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary BioCentre, The University of Manchester, UK
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  • Barbara M. Bakker

    1.  Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands
    2.  Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands
    3.  Department of Paediatrics, Centre for Liver, Digestive and Metabolic Diseases, University Medical Centre Groningen, University of Groningen, The Netherlands
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  • Note
    As a team and independently, the authors are actively engaged in ongoing efforts of the international scientific community to define standards for yeast and other organisms and to get them widely adopted. Hence, the authors would specifically welcome responses from readers who would like to be involved in such efforts and/or have specific comments on the proposed standards or the scientific strategy to define them.

B. M. Bakker, Department of Paediatrics, Centre for Liver, Digestive and Metabolic Diseases, University Medical Centre Groningen, University of Groningen, Hanzeplein 1, NL-9713 GZ Groningen, The Netherlands
Fax: +31 50 361 1746
Tel: +31 50 361 1542
E-mail: B.M.Bakker@med.umcg.nl

Abstract

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.

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