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 Mg 2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC 50 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. © 2013 Elsevier Inc. All rights reserved.
Publication Source (Journal or Book title)
Malina, A., Bryant, S., Chang, S., Waldrop, G., & Gilman, S. (2014). Capillary electrophoresis-based assay of phosphofructokinase-1. Analytical Biochemistry, 447 (1), 1-5. https://doi.org/10.1016/j.ab.2013.10.028