Assay of cyclic nucleotide phosphodiesterase activity in rat brain by high-performance liquid chromatography using (3,4-dimethoxyphenyl)glyoxal as a fluorescence derivatizing reagent

Abstract

A selective and sensitive HPLC measurement of 3′,5′-cyclic nucleotide phosphodiesterase (PDE; EC 3.1.4.17) activity in rat cerebral cortex is described using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorogenic reagent for guanine and its nucleosides and nucleotides. cGMP, a substrate of PDE, and GMP, which was produced by the enzyme reaction, are selectively converted with DMPG to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The Michaelis constant for cGMP was obtained with homogenates of cerebral cortex as 9.8 μM, and the limits of detection at a signal-to-noise ratio of 3 of GMP were 50 fmol on the column. More than 0.5 pmol min −1 mg −1 protein of PDE activity can be measured.