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.
D000818:Animals; D001667:Binding, Competitive; D003101:Collodion; D004789:Enzyme Activation; D005374:Filtration / Q000295:instrumentation; D005635:Fructosediphosphates / Q000032:analysis* / Q000378:metabolism; D006598:Hexosediphosphates / Q000032:analysis*; D066298:In Vitro Techniques; D008297:Male; D010732:Phosphofructokinase-1 / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D014018:Tissue Distribution
D005635:Fructosediphosphates; D006598:Hexosediphosphates; C027652:fructose 2,6-diphosphate; D003101:Collodion; D010732:Phosphofructokinase-1