Measuring enzyme activities under standardized in-vivo like conditions for systems biology
Artikel i vetenskaplig tidskrift, 2010
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 V(max) 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
modelling
in vivo enzyme kinetics
glycolysis
Saccharomyces cerevisiae
standardization
Författare
K. Van Eunen
Rijksuniversiteit Groningen
Vrije Universiteit Amsterdam
J. Bouwman
Rijksuniversiteit Groningen
Vrije Universiteit Amsterdam
P. Daran-Lapujade
Kluyver Centre for Genomics of Industrial Fermentation
Vrije Universiteit Amsterdam
J. Postmus
TU Delft
A.B. Canelas
Vrije Universiteit Amsterdam
Kluyver Centre for Genomics of Industrial Fermentation
F.I. Mensonides
Vrije Universiteit Amsterdam
Rijksuniversiteit Groningen
R. Orij
TU Delft
I Tuzun
Swammerdam Institute for Life Sciences
J. van den Brink
Vrije Universiteit Amsterdam
Kluyver Centre for Genomics of Industrial Fermentation
G.J. Smits
TU Delft
W.M. Van Gulik
Vrije Universiteit Amsterdam
Kluyver Centre for Genomics of Industrial Fermentation
S. Brul
TU Delft
J.J. Heijnen
Kluyver Centre for Genomics of Industrial Fermentation
Vrije Universiteit Amsterdam
J.H. de Winde
Vrije Universiteit Amsterdam
Kluyver Centre for Genomics of Industrial Fermentation
M.J. Teixeira de Mattos
Swammerdam Institute for Life Sciences
C. Kettner
Swammerdam Institute for Life Sciences
Jens B Nielsen
Chalmers, Kemi- och bioteknik, Livsvetenskaper
Hans W Westerhoff
University of Manchester
Vrije Universiteit Amsterdam
Rijksuniversiteit Groningen
Chalmers, Kemi- och bioteknik
B.M. Bakker
Rijksuniversiteit Groningen
Vrije Universiteit Amsterdam
FEBS Journal
1742-464X (ISSN) 17424658 (eISSN)
Vol. 277 3 749-760Styrkeområden
Livsvetenskaper och teknik (2010-2018)
Ämneskategorier
Bioinformatik och systembiologi
Annan industriell bioteknik
DOI
10.1111/j.1742-4658.2009.07524.x