1
Measuring enzyme activities under standardized in vivo-like conditions for systems biology.
FEBS J. 2010 Feb; 277(3):749-60
All selected articles haveThis article has been added to your saved articles
You can follow/unfollow articles via MyF1000 or via the article or browse pages.
27 Jul 2010
Technical Advance
DOI: 10.3410/f.3782960.3512060
Recognizing that the intracellular milieu has a drastically different make up from buffers typically employed to study the kinetics of enzymes in solution, van Eunen et al. make measurements to estimate the concentrations of major ionic components of the cytosol of a specific strain of yeast under specific...Continue reading
Recognizing that the intracellular milieu has a drastically different make up from buffers typically employed to study the kinetics of enzymes in solution, van Eunen et al. make measurements to estimate the concentrations of major ionic components of the cytosol of a specific strain of yeast under specific aerobic growth conditions. Furthermore, assaying the maximal fluxes catalyzed by the glycolytic enzymes, they report significant deviation from results obtained under in vitro conditions designed for individual enzyme assays, demonstrating the critical need to achieve standardized conditions.
By proposing a "defined in vivo-like medium" that is "as close as possible to the in vivo situation for the yeast", van Eunen et al. make a major step towards systematizing experimental systems to provide consistent and useful data for system simulation and analysis. As a point of criticism, it should be noted that, in conducting kinetic assays under defined conditions to mimic the intracellular environment, the authors violate their proposed standard of reporting "a precise description of the assay conditions". For example, we do not know which salts of phosphate and glutamate are added to achieve the reported concentrations of the anions. This makes it impossible to compute how much of various salts (NaCl, KCl) were added to the buffers. Thus it is not possible to estimate the ionic strength, which is not reported. Nor am I able to determine the temperature at which the experiments were performed. These oversights actually reinforce the novelty and difficulty of the overall goal of this study and remind us of the drastic cultural change that is called for. Thus, regardless of these somewhat picky criticisms, the approach of Nedul et al. provides a blueprint to seriously improve working practices in systems biology.
By proposing a "defined in vivo-like medium" that is "as close as possible to the in vivo situation for the yeast", van Eunen et al. make a major step towards systematizing experimental systems to provide consistent and useful data for system simulation and analysis. As a point of criticism, it should be noted that, in conducting kinetic assays under defined conditions to mimic the intracellular environment, the authors violate their proposed standard of reporting "a precise description of the assay conditions". For example, we do not know which salts of phosphate and glutamate are added to achieve the reported concentrations of the anions. This makes it impossible to compute how much of various salts (NaCl, KCl) were added to the buffers. Thus it is not possible to estimate the ionic strength, which is not reported. Nor am I able to determine the temperature at which the experiments were performed. These oversights actually reinforce the novelty and difficulty of the overall goal of this study and remind us of the drastic cultural change that is called for. Thus, regardless of these somewhat picky criticisms, the approach of Nedul et al. provides a blueprint to seriously improve working practices in systems biology.
Acknowledgments
I would like to thank Karen van Eunen for their assistance in the preparation of this evaluation.
Disclosures
None declared
Add Comment
Beard D: F1000Prime Recommendation of [van Eunen K et al., FEBS J 2010, 277(3):749-60]. In F1000Prime, 27 Jul 2010; DOI: 10.3410/f.3782960.3512060. F1000Prime.com/3782960#eval3512060
F1000Prime Recommendations, Dissents and Comments for [van Eunen K et al., FEBS J 2010, 277(3):749-60]. In F1000Prime, 25 May 2013; F1000Prime.com/3782960
Only registered/signed-in users can make comments.
Sign in or Subscribe to create your user account and join the discussion.
Sign in or Subscribe to create your user account and join the discussion.
Comment by:
Daniel Beard,
Medical College of Wisconsin
Evaluator | 27 Jul 2010, 12:00 AM
Evaluator | 27 Jul 2010, 12:00 AM
Since the publication of the original review, the authors of the paper contacted the reviewer to graciously point out that indeed, the experimental temperature (30 deg Celsius) was reported in the original paper and to provide the following additional details on the experimental buffers used in the experiments. The assay medium was stored in small batches at 4 deg Celsius as three separate components: (a) Buffer at pH 6.8 containing 0.9M potassium, 0.735M glutamate, and 0.11M phosphate -- this buffer was made by mixing KH2PO4, K2HPO4 and L-glutamic acid potassium salt, such that a pH of 6.8 was obtained. This was a 3-times concentrated stock solution and was made by dissolving 7.48g/l KH2PO4 (Baker product 0240), 9.58g/l K2HPO4 (Baker product 0241) and 149.4g/l L-glutamic acid potassium salt (Sigma product G1501) in demineralized water. The pH of the buffer was always checked after preparation. (b) Buffer at pH 6.8 containing 1.5M sodium and 1M phosphate -- this buffer was made by mixing NaH2PO4 and Na2HPO4 such that, again, a pH of 6.8 was obtained. This buffer was a 75-times stock solution and was made by dissolving 69g/l NaH2PO4 (Baker product 0306) and 71g/l Na2HPO4.H2O (Baker product 0303) in demineralized water. The pH of the buffer was always checked after preparation. (c) 0.01M calcium sulphate -- this was a 20-times stock solution and was made by dissolving 1.72g/l CaSO4 (Baker product 0081) in demineralized water. For each assay, a fresh mix of these three components was prepared and the final concentrations of the components in the assay medium were 245mM glutamate, 20mM sodium, 0.5mM calcium, at a pH of 6.8.
Disclosures:
None declared.
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.
DOI: 10.1111/j.1742-4658.2009.07524.x
PMID: 20067525
