Analysis of purine ribonucleotides and deoxyribonucleotides in cell extracts by high-performance liquid...

A high-performance liquid chromatographic (HPLC) assay for the fourteen major cellular purine ribonucleotides and 2'-deoxyribonucleotides is presented. Following an initial separation by anion-exchange HPLC, the nucleotides are hydrolyzed to their respective nucleosides by alkaline phosphatase and quantified by reversed-phase HPLC and UV absorbance detection. The assay is reproducible, specific, and has a detection limit of 10 pmol/sample. The recovery of nucleosides derived from nucleotides is 85%. Purine nucleotide pool sizes have been measured in cultured mouse T lymphoma (S-49) cells before and after treatment with 2.0 microM mycophenolic acid, an inhibitor of the enzyme IMP dehydrogenase, for 3 h. Control nucleotide levels obtained by this method are consistent with those reported for S-49 cells using other methods, and the observed decrease in guanine nucleotides and increase in IMP after treatment with mycophenolic acid agree with previous reports.