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

Anal Biochem. 1994 Sep;221(2):401-4. doi: 10.1006/abio.1994.1433.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / analysis*
  • Aminoimidazole Carboxamide / analogs & derivatives
  • Aminoimidazole Carboxamide / chemical synthesis
  • Aminoimidazole Carboxamide / metabolism
  • Chromatography, Thin Layer / methods
  • Hydroxymethyl and Formyl Transferases*
  • Indicators and Reagents
  • Inosine Monophosphate / metabolism
  • Kinetics
  • Nucleotide Deaminases / analysis*
  • Phosphoribosylaminoimidazolecarboxamide Formyltransferase
  • Phosphorylation
  • Phosphotransferases
  • Radioisotope Dilution Technique
  • Ribonucleosides
  • Ribonucleotides / chemical synthesis
  • Ribonucleotides / metabolism
  • Sensitivity and Specificity
  • Serratia marcescens / enzymology
  • Time Factors
  • Tritium

Substances

  • Indicators and Reagents
  • Ribonucleosides
  • Ribonucleotides
  • Tritium
  • 5-formamidoimidazole-4-carboxamide ribotide
  • Inosine Monophosphate
  • Aminoimidazole Carboxamide
  • acadesine
  • Hydroxymethyl and Formyl Transferases
  • Phosphoribosylaminoimidazolecarboxamide Formyltransferase
  • Acyltransferases
  • Phosphotransferases
  • nucleoside phosphotransferase
  • Nucleotide Deaminases
  • IMP cyclohydrolase
  • AICA ribonucleotide