Capillary electrophoresis-based assay of phosphofructokinase-1 |
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Authors: | Andrew Malina Sherrisse K Bryant Simon H Chang Grover L Waldrop S Douglass Gilman |
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Institution: | 1. Division of Biochemistry and Molecular Biology, Louisiana State University, Baton Rouge, LA 70803, USA;2. Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA |
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Abstract: | An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg–ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg–ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg–ATP and Mg–ADP. The separation was enhanced by the addition of Mg2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors. |
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Keywords: | Phosphofructokinase-1 Inhibition Capillary electrophoresis Enzyme assay |
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