Studies on the ATP-sensitivity of yeast phosphofructokinase by means of affinity partitioning using polymer bound Cibacron blue F3G-A.
Yeast phosphofructokinase was partitioned in an aqueous two-phase system composed of polyethylene glycol and dextran in which a small amount of the first polymer was replaced by Cibacron Blue F3G-A substituted polyethylene glycol. It was found that the partition coefficient, K, of the enzyme determined immediately after adding the enzyme to the system and within a series of time intervals (min) shifts from a lower to a higher value. This effect was amplified when the enzyme was preincubated with fructose 6-phosphate in millimolar concentration range. Other effectors, like ATP, ADP, AMP, fructose 1,6-bisphosphate and protons were without significant influence on the partition. A similar behaviour in lowering the partition coefficient was found with an enzyme form desensitized to ATP-inhibition. The existence of enzyme forms of different affinity to Cibacron Blue depending on the fructose 6-phosphate could be manifested by counter-current distribution. Because of the competition in the binding of Cibacron Blue and ATP to the nucleotide binding sites the conclusion was drawn that the partition of phosphofructokinase reflects the alteration of the affinity of the enzyme to ATP.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D001667:Binding, Competitive; D003377:Countercurrent Distribution; D007700:Kinetics; D010732:Phosphofructokinase-1 / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014227:Triazines / Q000378:metabolism*
D000345:Affinity Labels; D014227:Triazines; C012842:Cibacron Blue F 3GA; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1