A New Method of Assessing Rates of the Futile Cycle during Glycolytic and Gluconeogenic Metabolism

A method for assessing rates of the futile cycle is presented, and it is illustrated in vitro. Glycolytic- and gluconeogenic-type futile cycles are simulated for the reactions catalyzed by phosphofructokinase (EC 2.7.1.11) and fructose-1,6-bisphosphatase (EC 3.1.3.11) in assays systems in which the cycle rates in either direction can be varied and determined. While either system is sustaining a net flux of carbons in a given direction, the flux of radioactively labeled carbons in the opposite direction is determined. Different cycle rates are obtained by varying phosphofructokinase activity while keeping fructose-1,6-bisphosphatase activity constant in the "gluconeogenic" simulation and varying fructose-1,6-bisphosphatase while keeping phosphofructokinase activity constant in the "glycolytic" simulation. A direct, linear relationship was found between the cycle rate and the radioactive labeling of fructose 1,6-bisphosphate from [U-14C]glucose 6-phosphate during net gluconeogenic carbon flux. Also, a direct, linear relationship was found between cycle rate and radioactive labeling of fructose-6-P from [U-14C]fructose-1,6-bisP during net glycolytic carbon flux. The applicability, advantages, and problems of the method are discussed.