Enzymatic preparation of high-specific-activity β-d-[6,6′-3H]fructose-2,6-bisphosphate: Application to a sensitive assay for fructose-2,6-bisphosphatase

β-d-Fructose-2,6-bisphosphate (Fru-2,6-P2) is an important regulator of eukaryotic glucose homeostasis, functioning as a potent activator of 6-phosphofructo-1-kinase and inhibitor of fructose-1,6-bisphosphatase. Pharmaceutical manipulation of intracellular Fru-2,6-P2 levels, therefore, is of interest for the treatment of certain diseases, including diabetes and cancer. [2-32P]Fru-2,6-P2 has been the reagent of choice for studying the metabolism of this effector molecule; however, its short half-life necessitates frequent preparation. Here we describe a convenient, economical, one-pot enzymatic preparation of high-specific-activity tritium-labeled Fru-2,6-P2. The preparation involves conversion of readily available, carrier-free d-[6,6′-3H]glucose to [6,6′-3H]Fru-2,6-P2 using hexokinase, glucose-6-phosphate isomerase, and 6-phosphofructo-2-kinase. The key reagent in this preparation, bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from human liver, was produced recombinantly in Escherichia coli and purified in a single step using an appendant C-terminal hexa-His affinity tag. Following purification by anion exchange chromatography using triethylammonium bicarbonate as eluant, radiochemically pure [6,6′-3H]Fru-2,6-P2 having a specific activity of 50 Ci/mmol was obtained in yields averaging 35%. [6,6′-3H]Fru-2,6-P2 serves as a stable, high-specific-activity substrate in a facile assay capable of detecting fructose-2,6-bisphosphatase in the range of 10−14 to 10−15 mol, and it should prove to be useful in many studies of the metabolism of this important biofactor.