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.
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Measuring enzyme activities under standardized in vivo-like conditions for systems biology
Article first published online: 7 JAN 2010
DOI: 10.1111/j.1742-4658.2009.07524.x
© 2010 The Authors Journal compilation © 2010 FEBS
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How to Cite
van Eunen, K., Bouwman, J., Daran-Lapujade, P., Postmus, J., Canelas, A. B., Mensonides, F. I. C., Orij, R., Tuzun, I., van den Brink, J., Smits, G. J., van Gulik, W. M., Brul, S., Heijnen, J. J., de Winde, J. H., Teixeira de Mattos, M. J., Kettner, C., Nielsen, J., Westerhoff, H. V. and Bakker, B. M. (2010), Measuring enzyme activities under standardized in vivo-like conditions for systems biology. FEBS Journal, 277: 749–760. doi: 10.1111/j.1742-4658.2009.07524.x
Publication History
- Issue published online: 18 JAN 2010
- Article first published online: 7 JAN 2010
- (Received 7 October 2009, revised 20 November 2009, accepted 27 November 2009)
Keywords:
- glycolysis;
- in vivo enzyme kinetics;
- modelling;
- Saccharomyces cerevisiae;
- standardization
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.