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.
Szabados
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Department of Biochemistry, University of Sydney, New South Wales, Australia.
Christopherson
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RI
D000217:Acyltransferases / Q000032:analysis*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D002855:Chromatography, Thin Layer / Q000379:methods; D019877:Hydroxymethyl and Formyl Transferases; D007202:Indicators and Reagents; D007291:Inosine Monophosphate / Q000378:metabolism; D007700:Kinetics; D009709:Nucleotide Deaminases / Q000032:analysis*; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D010766:Phosphorylation; D010770:Phosphotransferases; D011865:Radioisotope Dilution Technique; D012263:Ribonucleosides; D012265:Ribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012680:Sensitivity and Specificity; D012706:Serratia marcescens / Q000201:enzymology; D013997:Time Factors; D014316:Tritium
D007202:Indicators and Reagents; D012263:Ribonucleosides; D012265:Ribonucleotides; D014316:Tritium; C062030:5-formamidoimidazole-4-carboxamide ribotide; D007291:Inosine Monophosphate; D000620:Aminoimidazole Carboxamide; C011651:acadesine; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D010770:Phosphotransferases; C021892:nucleoside phosphotransferase; D009709:Nucleotide Deaminases; C021436:IMP cyclohydrolase; C031143:AICA ribonucleotide