Dynamic simulation of an in vitro multi-enzyme system.
http://www.ncbi.nlm.nih.gov/pubmed/17239859
FEBS Letters
2007
Nobuyoshi Ishii, Yoshihiro Suga, Akiko Hagiya, Hisami Watanabe, Hirotada Mori, Masataka Yoshino, Masaru Tomita
Institute for Advanced Biosciences, Keio University, Japan
dynamic simulation, enzyme kinetics, metabolism, glycolysis, in vitro
https://kimosys.org/rails/active_storage/blobs/eyJfcmFpbHMiOnsibWVzc2FnZSI6IkJBaHBBbThFIiwiZXhwIjpudWxsLCJwdXIiOiJibG9iX2lkIn19--b8e3128d37c561482ee23924cd4a52c3647a6ff8/Ishii_2007.pdf
not specified
Escherichia coli
K-12 AG1
time-series data of metabolites
mM
not specified
37.0 °C
not specified
glucose
batch
aerobic
— h⁻¹
0.2 L
not specified g/L
LB medium supplemented with 50 µg/ml chloramphenicol to OD600=0.5
Sampling Method: Samples were obtained 2 h after addition of 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG); Quenching: cells were collected by centrifugation (6000 r.p.m., 5 min at 4 ºC) and resuspended in 10 ml of cold buffer I [50 mM sodium phosphate (pH 7.0), 200 mM NaCl, Protease inhibitor (Complete Mini EDTA-Free, Roche Diagnostics, Basel, Switzerland)].; Extraction list: sonication; Analysis list: enzymatic;
Measurement of metabolite concentrations - A sample was added to 100 mM MOPS buffer (pH 7.2) containing 0.1 mM NADH, 20 mM MgCl2, 5 mM ATP, and 2 mM DTT (I), and maintained at 37 ºC. Then, 1.8 U/ml ALD, 31 U/ml G3PDH, and 90 U/ml TIM (II), 3 U/ml PfkA (III), 18 U/ml Pgi (IV), and 10 U/ml Glk (V) were added sequentially, and A340 was recorded at each step. Concentrations of fructose-1,6-diphosphate (FDP), F6P, G6P, and glucose were calculated from the difference in A340 between I and II, II and III, III and IV, and IV and V, respectively. The FDP concentration of each sample was calibrated by the measured value of a known concentration standard solution of FDP treated using the same procedure employed for sample preparation.
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