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

glycolysis

Saccharomyces cerevisiae

in vivo enzyme kinetics

standardization

modelling

Författare

K. Van Eunen

University of Groningen

Vrije Universiteit Amsterdam

J. Bouwman

University of Groningen

Vrije Universiteit Amsterdam

P. Daran-Lapujade

Kluyver Centre for Genomics of Industrial Fermentation

Vrije Universiteit Amsterdam

J. Postmus

Delft University of Technology

A.B. Canelas

Vrije Universiteit Amsterdam

Kluyver Centre for Genomics of Industrial Fermentation

F.I. Mensonides

University of Groningen

Vrije Universiteit Amsterdam

R. Orij

Delft University of Technology

I Tuzun

Swammerdam Institute for Life Sciences

J. van den Brink

Vrije Universiteit Amsterdam

Kluyver Centre for Genomics of Industrial Fermentation

G.J. Smits

Delft University of Technology

W.M. Van Gulik

Kluyver Centre for Genomics of Industrial Fermentation

Vrije Universiteit Amsterdam

S. Brul

Delft University of Technology

J.J. Heijnen

Vrije Universiteit Amsterdam

Kluyver Centre for Genomics of Industrial Fermentation

J.H. de Winde

Kluyver Centre for Genomics of Industrial Fermentation

Vrije Universiteit Amsterdam

M.J. Teixeira de Mattos

Swammerdam Institute for Life Sciences

C. Kettner

Swammerdam Institute for Life Sciences

Jens B Nielsen

Kemi- och bioteknik, Livsvetenskaper, Systembiologi

H.V. Westerhoff

University of Manchester

Chalmers University of Technology

University of Groningen

Vrije Universiteit Amsterdam

B.M. Bakker

University of Groningen

Vrije Universiteit Amsterdam

FEBS Journal

1742-464X (ISSN) 1432-1033 (eISSN)

Vol. 277 749-760

Styrkeområden

Livsvetenskaper och teknik

Ämneskategorier

Bioinformatik och systembiologi

Annan industriell bioteknik

DOI

10.1111/j.1742-4658.2009.07524.x