Assay of D-Fructose 2,6-Bisphosphate in Biological Extracts

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Abstract
Tissues or cells are extracted in mildly alkaline solutions and reduced by sodium borohydride. The fructose 6-phosphate released from the intact D-fructose 2,6-bisphosphate by gentle acid hydrolysis is determined fluorometrically in a coupled enzyme system with phosphoglucoisomerase (EC 5.3.1.9), NAD+:glucose 6-phosphate dehydrogenase (EC 1.1.1.49), diaphorase (EC 1.6.99.1) and resazurine. To demonstrate the application of this method, the concentration of D-fructose 2,6-bisphosphate in liver, yeast and legume-seedling extracts at different physiological states was measured and compared to the existing pool level of other intermediates of glycolysis.