Radioassay of bifunctional 5-aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase by thin-layer chromatography.

A radioassay has been developed for the bifunctional enzyme, AICAR transformylase-IMP cyclohydrolase, which catalyzes reactions 9 and 10 of the de novo pathway for biosynthesis of purine nucleotides (AICAR-->FAICAR-->IMP). 3H-labeled AICAR or FAICAR is converted enzymically to product(s) which are separated by one-dimensional thin-layer chromatography prior to quantification by scintillation counting. Using this sensitive radioassay, a dissociation constant of IMP cyclohydrolase for FAICAR of 0.87 microM has been determined and AICAR, FAICAR, and IMP can be quantified in assay mixtures for AICAR transformylase-IMP cyclohydrolase. The ratio of specific enzymic activities for AICAR transformylase:IMP cyclohydrolase is 1:44.