A competitive binding assay for fructose 2,6-bisphosphate

A new direct assay method for fructose 2,6-bisphosphate has been developed based on competitive binding of labeled and unlabeled fructose 2,6-P₂ to phosphofructokinase. Phosphofructokinase (0.5-1.3 pmol protomer) is incubated with saturating concentrations (5.0-5.5 pmol) of fructose 2,6-[2-³²P]P₂ and samples containing varying concentrations of fructose 2,6-P₂. The resulting stable binary complex is retained on nitrocellulose filters with a binding efficiency of up to 70%. Standard curves obtained with this assay show strict linearity with varying fructose 2,6-P₂ in the range of 0.5 to 45 pmol, which exceeds the sensitivity of most of the previously described assay methods. Fructose 2,6-P₂, ATP, and high concentrations of phosphate interfere with this assay. However, the extent of this inhibition is negligible since their tissue contents are one-half to one-tenth that examined. This new assay is simple, direct, rapid, and does not require pretreatment of tissue extracts.