A radiochemical enzymatic endpoint assay for GTP and GDP

We present here a radiochemical enzymatic endpoint assay for the guanine nucleotides GTP and GDP that is suitable for use with cell extracts. The major coupling enzyme used is phosphoenolpyruvate carboxykinase purified from chicken liver. The ancillary coupling enzyme, aspartate aminotransferase, was used to generate a low steady-state concentration of oxalacetate. GTP was determined by the overall conversion of [U-14C]aspartate into [14C]phosphoenolpyruvate. This reaction was also scaled-up as a preparative method for [U-14C]phosphoenolpyruvate. This was used with the same coupling enzymes in reverse to measure GDP by the formation of [14C]aspartate. The assay method was applied to isolated rat hepatocytes. The total GTP and GDP concentrations found were within the range reported by others for rat liver. The advantages of this assay are its sensitivity, specificity, and applicability to large numbers of samples.