A sensitive radiometric assay to measure D-xylulose kinase activity.
A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.
Institut Le Bel, Université Louis Pasteur/CNRS UMR 7123, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France. dtritsch@chimie.u-strasbg.fr
Denis
D
Tritsch
Andréa
A
Hemmerlin
Michel
M
Rohmer
Thomas J
TJ
Bach
D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011874:Radiometry / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase