Department of Molecular Cell Physiology, Vrije Universiteit Amsterdam, The Netherlands.
FEBS J. 2010 Feb;277(3):749-60. doi: 10.1111/j.1742-4658.2009.07524.x. Epub 2010 Jan 7.
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.
现实的定量模型需要来自多个实验室的数据。因此，实验系统和测定条件的标准化是至关重要的。此外，标准应代表体内条件。然而，通常情况下，酶动力学参数是在测定每种酶最大活性的条件下测量的。实际上，这意味着不同酶的动力学参数是在不同的缓冲液、不同的 pH 值、不同的离子强度等条件下测量的。在荷兰垂直基因组学联盟、欧洲酵母系统生物学网络和酶学数据报告标准委员会的共同努力下，我们已经开发出一种用于测定酵母中酶动力学参数的单一测定介质。该培养基尽可能接近酿酒酵母的体内情况，同时在实验上也是可行的。体内条件是根据在有氧葡萄糖限制恒化培养物中生长的酿酒酵母菌株 CEN.PK113-7D 进行估算的，其胞外 pH 值为 5.0，特定生长速率为 0.1 h(-1)。测定了胞质 pH 值以及钙、钠、钾、磷、硫和镁的浓度。基于这些数据和文献数据，我们提出了一种含有 300 mM 钾、50 mM 磷酸盐、245 mM 谷氨酸、20 mM 钠、2 mM 游离镁和 0.5 mM 钙的类似于体内的定义培养基，pH 值为 6.8。在新培养基中测量了酿酒酵母糖酵解和发酵酶的 V(max)值。对于一些酶，结果与在特定酶最佳条件下进行的测定明显不同。
