I am trying to assess the activity of PFK enzyme in mESC lines. Recently in the metabolomics analysis, I discovered that although the cells show an increased uptake of glucose 6-P and fructose 6-P, there is a major drop in the levels of downstream metabolites starting from fructose-1,6-bisP all the way up to pyruvate. (except glyceraldehyde-3-P, 1,3-bisPglycerate and 2-phosphoglycerate)
There is also an increase in the levels of glucosamine-6-P, mannose, mannose-6-P and of 6-P gluconate and ribose 5-P. These levels could be increased as G 6-P and F 6-P could be shunted into the pentoseP pathways.
Therefore, I am planning to check if there is a blockage between the F 6-P and fructose 1,6-P by detecting the activity of the PFKinase enzyme.
So far the kits I've found to do this, are based on the calorimetric assay where PFK activity is determined by a coupled enzyme assay, in which fructose-6-phosphate and ATP is converted to fructose1,6-diphosphate and ADP by PFK. The ADP is converted by the enzyme mix to AMP and NADH. The resulting NADH reduces a colorless probe resulting in a colorimetric (450 nm) product proportional to the PFK activity present. One unit of PFK is the amount of enzyme that will generate 1.0 mmole of NADH per minute at pH 7.4 at 37 °C.
I was wondering if there is an in cell method, more precise for cell extracts.. I am not sure how sensitive or specific this calorimetric assay would be.. Any comments/ suggestions would be much appreciated.
Many thanks.
Best,
Pooja.
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