Detection of glycinamide ribonucleotide by HPLC with pulsed amperometry: application to the assay for glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14).

An assay method is described for measuring the catalytic activity of glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase (EC 2.4.2.14), the first enzyme in the de novo purine biosynthetic pathway. Phosphoribosylamine, the unstable product of the enzymatic reaction, is trapped by glycinamide ribonucleotide synthetase (EC 6.3.4.13), the second enzyme in the pathway, to form the stable product glycinamide ribonucleotide. Glycinamide ribonucleotide is resolved by ion-exchange HPLC at alkaline pH and quantified by the use of a pulsed amperometric detector. The assay is specific, reproducible, rapid (15 min chromatography, including column recycling and stabilization), convenient (no radioactive materials used), linear at a wide range of glycinamide ribonucleotide concentrations, and sensitive at subnanomole levels even with crude extracts. Both the glutamine- and ammonia-dependent activity of glutamine: 5-phosphoribosyl-1-pyrophosphate amidotransferase can be accurately quantified.