title: Measuring enzyme activities under standardized in vivo-like conditions for systems biology.
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 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.
authors.affiliation: Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands.
authors.forename: Karen
authors.initials: K
authors.lastname: van Eunen
authors.forename: Jildau
authors.initials: J
authors.lastname: Bouwman
authors.forename: Pascale
authors.initials: P
authors.lastname: Daran-Lapujade
authors.forename: Jarne
authors.initials: J
authors.lastname: Postmus
authors.forename: André B
authors.initials: AB
authors.lastname: Canelas
authors.forename: Femke I C
authors.initials: FI
authors.lastname: Mensonides
authors.forename: Rick
authors.initials: R
authors.lastname: Orij
authors.forename: Isil
authors.initials: I
authors.lastname: Tuzun
authors.forename: Joost
authors.initials: J
authors.lastname: van den Brink
authors.forename: Gertien J
authors.initials: GJ
authors.lastname: Smits
authors.forename: Walter M
authors.initials: WM
authors.lastname: van Gulik
authors.forename: Stanley
authors.initials: S
authors.lastname: Brul
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Heijnen
authors.forename: Johannes H
authors.initials: JH
authors.lastname: de Winde
authors.forename: M Joost Teixeira
authors.initials: MJ
authors.lastname: de Mattos
authors.forename: Carsten
authors.initials: C
authors.lastname: Kettner
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.forename: Hans V
authors.initials: HV
authors.lastname: Westerhoff
authors.forename: Barbara M
authors.initials: BM
authors.lastname: Bakker
mesh_terms: D003470:Culture Media / Q000592:standards*; D003600:Cytosol / Q000201:enzymology; D005285:Fermentation / Q000235:genetics; D006019:Glycolysis / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D049490:Systems Biology / Q000592:standards*
chemical_list: D003470:Culture Media