Demonstration of ammonia utilization for purine biosynthesis by the intact cell and characterization of the enzymatic activity catalyzing this reaction

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Previous studies have suggested that ammonia may be used in the first reaction of purine biosynthesis in man and that this might play a role in the stimulation of purine biosynthesis de novo, which results from an increase in nitrogen ingestion in human subjects. Due to the potential importance of ammonia utilization for purine biosynthesis, we have performed studies to determine whether ammonia is used in the intact cell, and if so, which enzymatic activity catalyzes this reaction. Chinese hamster fibroblasts were used for the studies with intact cells due to the availability of a mutant deficient in PP-ribose-P amidotransferase and PP-ribose-P aminotransferase activities. In the wild-type cell, ammonia stimulates the rate of synthesis of phosphoribosylamine (PRA) even when PP-ribose-P amidotransferase activity (glutamine-utilizing) is inhibited, eliminating the possibility that ammonia stimulates PRA synthesis through an increase in glutamine availability. In the mutant cell, with normal activity of ribose-5-phosphate aminotransferase, ammonia does not stimulate the rate of PRA synthesis. These observations indicate that the eukaryotic cell uses ammonia directly in the first reaction of purine biosynthesis and that PP-ribose-P aminotransferase is the activity that catalyzes this reaction. In human placental cells, PP-ribose-P aminotransferase and PP-ribose-P amidotransferase are found only in the cytosol, and both activities coelute from diethylaminoethyl (DEAE-) cellulose and gel filtration columns, and cosediment in sucrose gradients. In addition, both activities demonstrate identical interconversion properties after incubation with the ligands, PP-ribose-P and purine ribonucleotides. We conclude from these results that only one enzyme, amidophosphoribosyl-transferase (E.C.2.4.2.14), catalyzes the synthesis of PRA in the eukaryotic cell, and this enzyme utilizes either glutamine or ammonia as a nitrogen source.