A radiotracer enzyme assay for phosphofructokinase using ( , , -32P) adenosine triphosphate

A radiotracer enzyme assay for phosphofructokinase using adenosine 5′-triphosphateα,β,γ-³²P] is described in this paper. Here the rates of appearance of both 1-³²P]d-fructose 1,6-diphosphate and α,β-³²P] adenosine 5′-diphosphate were followed to establish enzyme activity. The unique advantages of multiple rate determinations in a single reaction sequence which accrue from the use of a readily available multiply labeled cosubstrate are discussed. By an extension of this approach other labeled(¹) nucleotides of the type, N(¹P)_n, and enzymes in the Enzyme Commision categories, EC 2.7(phosphotransferases) and EC 6.1–6.4(ligases) are equally amenable to radionuclide assay.