Measuring enzyme activities under standardized in vivo-like conditions for Systems Biology

K. van Eunen, J. Bouwman, P.A.L. Daran-Lapujade, J. Postmus, A. Canelas, F.I.C. Mensonides, R. Orij, I. Tuzun, J. van der Brink, G.J. Smits, W.M. van Gulik, S. Brul, J.J. Heijnen, J.H. de Winde, M.J. Teixeira de Mattos, C. Kettner, J. Nielsen, H.V. Westerhoff, B.M. Bakker

Research output: Contribution to JournalArticleAcademicpeer-review

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
Original languageEnglish
Pages (from-to)749-760
JournalThe FEBS Journal
Volume277
DOIs
Publication statusPublished - 2010

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