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Measuring enzyme activities under standardized in-vivo like conditions for systems biology

K. Van Eunen ; J. Bouwman ; P. Daran-Lapujade ; J. Postmus ; A.B. Canelas ; F.I. Mensonides ; R. Orij ; I Tuzun ; J. van den Brink ; G.J. Smits ; W.M. Van Gulik ; S. Brul ; J.J. Heijnen ; J.H. de Winde ; M.J. Teixeira de Mattos ; C. Kettner ; Jens B. Nielsen (Institutionen för kemi- och bioteknik, Systembiologi) ; H.V. Westerhoff ; B.M. Bakker
FEBS Journal (1742-464X). Vol. 277 (2010), 3, p. 749-760.
[Artikel, refereegranskad vetenskaplig]

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

Nyckelord: glycolysis; in vivo enzyme kinetics; modelling; Saccharomyces cerevisiae; standardization



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Denna post skapades 2010-07-05. Senast ändrad 2015-07-01.
CPL Pubid: 123658

 

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Institutioner (Chalmers)

Institutionen för kemi- och bioteknik, Systembiologi (2008-2014)

Ämnesområden

Livsvetenskaper
Bioinformatik och systembiologi
Genteknik inkl. funktionsgenomik
Biokemisk och bioteknisk processteknik

Chalmers infrastruktur