Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-14C]phosphoribosylpyrophosphate.
Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-alpha-D-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction. 5-beta-phosphoribosyl-1-amine, using [1-14C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent Km values of the human lymphoblast enzyme are 0.46 mM for glutamine and 0.71 mM for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-L-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.
D000582:Amidophosphoribosyltransferase / Q000032:analysis*; D002250:Carbon Radioisotopes; D006801:Humans; D007553:Isotope Labeling; D008214:Lymphocytes / Q000201:enzymology; D010428:Pentosephosphates; D010430:Pentosyltransferases / Q000032:analysis*; D010754:Phosphoribosyl Pyrophosphate; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D010428:Pentosephosphates; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; D000582:Amidophosphoribosyltransferase