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Measuring enzyme activities under standardized in vivo ‐like conditions for systems biology
Author(s) -
van Eunen Karen,
Bouwman Jildau,
DaranLapujade Pascale,
Postmus Jarne,
Canelas André B.,
Mensonides Femke I. C.,
Orij Rick,
Tuzun Isil,
van den Brink Joost,
Smits Gertien J.,
van Gulik Walter M.,
Brul Stanley,
Heijnen Joseph J.,
de Winde Johannes H.,
Teixeira de Mattos M. Joost,
Kettner Carsten,
Nielsen Jens,
Westerhoff Hans V.,
Bakker Barbara M.
Publication year - 2010
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07524.x
Subject(s) - in vivo , chemostat , biochemistry , saccharomyces cerevisiae , enzyme , yeast , chemistry , potassium , magnesium , sodium , biophysics , biology , microbiology and biotechnology , bacteria , genetics , organic chemistry
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 m m potassium, 50 m m phosphate, 245 m m glutamate, 20 m m sodium, 2 m m free magnesium and 0.5 m m 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.