Proc. Natl. Acad. Sci. USA Vol. 73, No. 7, pp. 2458-2461, July 1976 Genetics A purine auxotroph deficient in phosphoribosylpyrophosphate amidotransferase and phosphoribosylpyrophosphate aminotransferase activities with normal activity of ribose-5- phosphate aminotransferase (Chinese hamster fibroblasts/isolated defect in phosphoribosylamine synthesis) EDWARD W. HOLMES, GEORGE L. KING, ALBERT LEYVA, AND SARA C. SINGER Departments of Medicine and Biochemistry, Division of Rheumatic and Genetic Diseases, Duke University Medical Center, Durham, North Carolina Communicated by James B. Wyngaarden, April 28,1976 ABSTRACT Three enzyme reactions have been reported rated from P-Rib-P-P amidotransferase on gel filtration to catalyze the synthesis of phosphoribosylamine in eukaryotic chromatography (4). This activity may represent a distinct cells. These activities are glutamine phosphoribosylpyrophos- protein or a subunit of P-Rib-P-P amidotransferase. A third phate (P-Rib-P-F) amidotransferase [amidophosphoribosyl- transferase; 5-phosphoribosylamine: pyrophosphate phospho- enzyme, ammonia ribose-5-phosphate aminotransferase ribostransferase (glutamate-amidating) EC 2.4±2.141 ammonia (Rib-5-P aminotransferase), has also been reported to catalyze P-Rib-P-P aminotransferase, and ammonia ribose5-Iphosphate the synthesis of P-RibN (reaction 3) (2-5). However, the de- aminotransferase. A purine auxotroph derived from a cell line termination of P-RibN in this reaction has required an assay of Chinese hamster fibroblasts was shown to be deficient in coupled with the second enzyme in the purine biosynthetic catalytic activities of glutamine P-Rib-P-P amidotransferase pathway. Since other studies have suggested that P-RibN can and ammonia P-Rib-P-P aminotransferase. Extracts from this cell line had normal ammonia ribose--phosphate aminotrans- be synthesized nonenzymatically from NH3 and Rib-5-P (6-9), ferase activity. The defect in purine biosynthesis in the mutant the physiological significance of the Rib-5-P aminotransferase cell line was localized to the synthesis of phosphoribosylamine. reaction in eukaryotic cells has been questioned. The recent These results indicate that glutamine P-Rib-P-P amidotrans- isolation by Chu et al. (10) of a eukaryotic cell line deficient in ferase or ammonia P-Rib-P-P aminotransferase or both are P-Rib-P-P amidotransferase activity (11) provided the unique important for phosphoribosylamine synthesis, but that ammonia opportunity to evaluate the potential role of each of these three ribose-phosphate aminotransferase activity probably does not The present report has play a significant role in this eukaryotic cell line. The simulta- reactions in purine biosynthesis de novo. neous disappearance of both P-Rib-P-Pdependent activities characterized each of these three reactions in mutant and suggests these two enzyme activities are closely related wild-type cells. In addition, the remaining steps in the pathway structurally or genetically. of purine biosynthesis de novo, as well as some reactions in the purine reutilization pathway, have been studied. The synthesis of phosphoribosylamine (P-RibN) is the first committed reaction unique to purine biosynthesis de novo (1). MATERIALS AND METHODS Traditionally the catalysis of this reaction has been attributed Cell Lines. Chinese hamster fibroblast cell lines, wild-type to the enzyme glutamine phosphoribosylpyrophosphate ami- (743) and mutant (P-1-2), were gifts from Dr. E. H. Y. Chu, dotransferase (P-Rib-P-P amidotransferase, reaction 1) [ami- Department of Genetics, University of Michigan. The proce- dophosphoribosyltransferase; EC 2.4.2.14; 5-phosphoribo- dure for mutagenesis and selection of this purine auxotroph has sylamine:pyrophosphate phosphoribosyltransferase (gluta- been described by Chu et al. (10). mate-amidating)]. However, recent studies have suggest- Cells were routinely grown in monolayer in Falcon plastic petri dishes or glass roller bottles using Eagle's minimum es- Glutamine + P-Rib-P-P + H20 sential medium (F-15, Gibco) supplemented with 10% fetal calf P-Rib-P-P amidotransferas serum (Irvine) and 10-4 M hypoxanthine. Experiments per- Pa P-RibN + glutamate + PPj [1] formed in purine-free medium used fetal calf serum that had NH3 + P-Rib-P-P + H20 been dialyzed twice against 40 volumes of 0.15 M NaCl for 12 hr. P-Rib-P-P amintransferawe, PRibN + PPj [2] Enzyme Assays. Cells were harvested with trypsin and + ATP + washed twice with phosphate-buffered saline immediately prior Rib-S5-P NH3 to use. The cell pellet was resuspended in the buffer indicated Rib-5-P aninotransferase P-RibN + ADP + [3] in the text and freeze-thawed twice in a dry ice-acetone bath. P, The lysates were centrifuged at 10,000 X g for 20 min, and the ed that two other enzymatic activities also catalyze the synthesis supernatant fluid was dialyzed for 2 hr at 40 against 1000 vol- of P-RibN in eukaryotic cells (2-5). The first of these (reaction umes of the indicated buffer. 2) has been called ammonia P-Rib-P-P aminotransferase (P- P-Rib-P-P amidotransferase was assayed in a 100-il reaction Rib-P-P aminotransferase) (3-5). This enzyme utilizes am- mixture that contained the following: 5 mM P-Rib-P-P, 4 mM monia rather than glutamine as substrate and has been sepa- [14C]glutamine, 5 mM MgCl2, 0.75 mM dithiothreitol, and 50 ,l of cell extract (0.49-0.94 ,ug of protein) in 37.5 mm potassium Abbreviations: P-RibN, phosphoribosylamine; P-Rib-P-P amido- phosphate buffer, pH 7.4. This assay, which has been previously transferase, amidophosphoribosyltransferase (EC 2.4.2.14); P-Rib-P-P to determine P-Rib-P-P aminotransferase, ammonia phosphoribosylpyrophosphate amino- described, used a P-Rib-P-P blank transferase; Rib-S5-P aminotransferase, ammonia ribose-5-phosphate amidotransferase activity (12). The P-Rib-P-P-independent aminotransferase; P-Rib-GlyN, phosphoribosylglycinamide. conversion of [14C]glutamine to [14C]glutamate was attributed 2458 Downloaded by guest on September 30, 2021 Genetics: Holmes et al. Proc. Natl. Acad. Sci. USA 73 (1976) 2459 to glutaminase (12). P-Rib-P-P aminotransferase was 80 assayed A B in a 100-gl reaction mixture that contained the following: 5 mM 60 P-Rib-P-P, 100 mM NH4C1 (1.26 mM NH3), 5mM MgCl2, 1.4 mM dithiothreitol, 40mM [35S]cysteine, and 50 gd of cell extract , 40 401- (0.49-0.94 ,ug of protein) in 25 mM potassium phosphate buffer, 0. pH 8.4. An NH4C1 blank was used to determine the P-Rib-P-P aminotransferase activity. This assay for P-RibN used a newly Z 20 described reaction between [35C]cysteine and P-RibN*. Pro- duction of P-RibN that was dependent on NH3, Rib-5-P, and ATP was arbitrarily attributed to Rib-5-P aminotransferase 0 24 48 0 24 48 activity, since it is not known whether the synthesis of P-RibN hours after subculture hours after subculture under these conditions is an enzymatic or nonenzymatic process. FIG. 1. Growth requirements of mutant and wild-type cells. Cells Since the newly described direct assay for P-RibN could not were grown in a purine-free medium without supplementation (0) be used in the presence of Rib-5-P*, the assay for Rib-5-P or with 10-4 M hypoxanthine (A). (A) Mutant cells; (B) wild-type aminotransferase was performed in a 100-gl reaction mixture cells. that contained the following: 27 mM Rib-5-P, 22 mM NH40H (1.1 mM NH3), 2 mM ATP, 2mM [14C]glycine, 10 mM MgCI2, tected in extracts from the mutant cells. When the cells were 1 mM dithiothreitol, and 40 gl of cell extract (0.5-1.2 gg of cultured in a purine-free medium for 24 hr, there was a 2-fold protein) in 50 mM Tris-HCI buffer, pH 8.0. The Rib-5-P and increase in the activity of P-Rib-P-P amidotransferase in the NH40H were preincubated at 370 for 60 min in 50 mM Tris- wild-type extract, but there was no effect on the activity of HCI buffer, pH 8.0. The blank for this assay omitted the Rib- P-Rib-P-P amidotransferase or P-Rib-P-P aminotransferase in mutant 5-P and NH40H, and the [14C]glycine was separated from the extract. In mixing experiments of extracts from phosphoribosyl['4C]glycinamide (P-Rib-GlyN) on a Dowex mutant and wild-type cells there was no evidence for the column (9). Preliminary studies indicated that P-Rib-GlyN presence of an inhibitor of P-Rib-P-P amidotransferase or synthetase (EC 6.3.4.13) activity from the cell lysate was not P-Rib-P-P aminotransferase (Table 2). limiting, and consequently an exogenous source of this enzyme In contrast to these findings, extracts from both the mutant was not added to the reaction mixture. and wild-type cells, dialyzed against Tris-HCI, demonstrated Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) (13), an equal ability to synthesize P-RibN and P-Rib-GlyN from adenine phosphoribosyltransferase (EC 2.4.2.7) (14), inosinic Rib-5-P, NH3, ATP, and glycine (Table 1). The synthesis of acid dehydrogenase (IMP dehydrogenase) (EC 1.2.1.14) (15), P-Rib-GlyN in this reaction was linear with respect to time of adenylosuccinate synthetase (EC 6.3.4.4) (16), adenosine incubation and concentration of extract protein (Fig. 2). deamninase (EC 3.5.4.4), xanthine oxidase (18) (EC 1.2.3.2), and If the cell extracts were not dialyzed against Tris-HCI buffer were P-Rib-P-P synthetase (EC 2.7.6.1) (19) were determined as before these studies performed, the rate of synthesis of was previously described. All of the above assays were linear with P-Rib-GlyN unchanged in the mutant extract, but it was respect to time of incubation and protein concentration.