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-5phosphate 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 to catalyze the synthesis of phosphoribosylamine in eukaryotic cells. These activities are glutamine phosphoribosylpyrophosphate (P-Rib-P-F) amidotransferase [amidophosphoribosyltransferase; 5-phosphoribosylamine: pyrophosphate phosphoribostransferase (glutamate-amidating) EC 2.4±2.141 ammonia P-Rib-P-P aminotransferase, and ammonia ribose5-Iphosphate aminotransferase. A purine auxotroph derived from a cell line of Chinese hamster fibroblasts was shown to be deficient in catalytic activities of glutamine P-Rib-P-P amidotransferase and ammonia P-Rib-P-P aminotransferase. Extracts from this cell line had normal ammonia ribose--phosphate aminotransferase activity. The defect in purine biosynthesis in the mutant cell line was localized to the synthesis of phosphoribosylamine. These results indicate that glutamine P-Rib-P-P amidotransferase or ammonia P-Rib-P-P aminotransferase or both are important for phosphoribosylamine synthesis, but that ammonia ribose-phosphate aminotransferase activity probably does not play a significant role in this eukaryotic cell line. The simultaneous disappearance of both P-Rib-P-Pdependent activities suggests these two enzyme activities are closely related structurally or genetically.

The synthesis of phosphoribosylamine (P-RibN) is the first committed reaction unique to purine biosynthesis de novo (1). Traditionally the catalysis of this reaction has been attributed to the enzyme glutamine phosphoribosylpyrophosphate amidotransferase (P-Rib-P-P amidotransferase, reaction 1) [amidophosphoribosyltransferase; EC 2.4.2.14; 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (glutamate-amidating)]. However, recent studies have suggestGlutamine + P-Rib-P-P + H20 P-Rib-P-P amidotransferas

a P-RibN + glutamate + PPj [1] P NH3 + P-Rib-P-P + H20 P-Rib-P-P amintransferawe,

PRibN + PPj

[2]

Rib-S5-P + ATP + NH3 Rib-5-P aninotransferase

P-RibN + A DP + P, [3] also catalyze the synthesis activities other two that ed enzymatic of P-RibN in eukaryotic cells (2-5). The first of these (reaction 2) has been called ammonia P-Rib-P-P aminotransferase (PRib-P-P aminotransferase) (3-5). This enzyme utilizes ammonia rather than glutamine as substrate and has been sepaAbbreviations: P-RibN, phosphoribosylamine; P-Rib-P-P amidotransferase, amidophosphoribosyltransferase (EC 2.4.2.14); P-Rib-P-P aminotransferase, ammonia phosphoribosylpyrophosphate aminotransferase; Rib-S5-P aminotransferase, ammonia ribose-5-phosphate aminotransferase; P-Rib-GlyN, phosphoribosylglycinamide.

rated from P-Rib-P-P amidotransferase on gel filtration chromatography (4). This activity may represent a distinct protein or a subunit of P-Rib-P-P amidotransferase. A third enzyme, ammonia ribose-5-phosphate aminotransferase (Rib-5-P aminotransferase), has also been reported to catalyze the synthesis of P-RibN (reaction 3) (2-5). However, the determination of P-RibN in this reaction has required an assay coupled with the second enzyme in the purine biosynthetic pathway. Since other studies have suggested that P-RibN can be synthesized nonenzymatically from NH3 and Rib-5-P (6-9), the physiological significance of the Rib-5-P aminotransferase reaction in eukaryotic cells has been questioned. The recent isolation by Chu et al. (10) of a eukaryotic cell line deficient in P-Rib-P-P amidotransferase activity (11) provided the unique opportunity to evaluate the potential role of each of these three reactions in purine biosynthesis de novo. The present report has characterized each of these three reactions in mutant and wild-type cells. In addition, the remaining steps in the pathway of purine biosynthesis de novo, as well as some reactions in the purine reutilization pathway, have been studied. MATERIALS AND METHODS Cell Lines. Chinese hamster fibroblast cell lines, wild-type (743) and mutant (P-1-2), were gifts from Dr. E. H. Y. Chu, Department of Genetics, University of Michigan. The procedure for mutagenesis and selection of this purine auxotroph has been described by Chu et al. (10). Cells were routinely grown in monolayer in Falcon plastic petri dishes or glass roller bottles using Eagle's minimum essential medium (F-15, Gibco) supplemented with 10% fetal calf serum (Irvine) and 10-4 M hypoxanthine. Experiments performed in purine-free medium used fetal calf serum that had been dialyzed twice against 40 volumes of 0.15 M NaCl for 12 hr. Enzyme Assays. Cells were harvested with trypsin and washed twice with phosphate-buffered saline immediately prior to use. The cell pellet was resuspended in the buffer indicated in the text and freeze-thawed twice in a dry ice-acetone bath. The lysates were centrifuged at 10,000 X g for 20 min, and the supernatant fluid was dialyzed for 2 hr at 40 against 1000 volumes of the indicated buffer. P-Rib-P-P amidotransferase was assayed in a 100-il reaction mixture that contained the following: 5 mM P-Rib-P-P, 4 mM [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 phosphate buffer, pH 7.4. This assay, which has been previously described, used a P-Rib-P-P blank to determine P-Rib-P-P amidotransferase activity (12). The P-Rib-P-P-independent conversion of [14C]glutamine to [14C]glutamate was attributed 2458

Genetics: Holmes et al.

Proc. Natl. Acad. Sci. USA 73 (1976)

to glutaminase (12). P-Rib-P-P aminotransferase was assayed

80

RESULTS Growth requirements Fig. 1 demonstrates that after 24 hr of subculture in a purinefree medium the mutant cells were unable to replicate, while the wild-type cells continued to grow well. The slight increase in DNA synthesis by mutant cells when initially transferred to purine-free medium was probably a reflection of the purine pool that had accumulated during culture of the cells in a medium supplemented with 10-4 M hypoxanthine. As shown, both cell lines grew equally well when the medium was supplemented with 10-4 M hypoxanthine. Although not presented in Fig. 1, 10-4 M adenine also supported growth of the mutant cells. Synthesis of P-RibN Table 1 lists the three activities in mutant and wild-type extracts reported to synthesize P-RibN. Neither P-Rib-P-P amidotransferase nor P-Rib-P-P aminotransferase activity was de*

G. L. King and E. W. Holmes, manuscript submitted.

B

A

in a 100-gl reaction mixture that contained the following: 5 mM

P-Rib-P-P, 100 mM NH4C1 (1.26 mM NH3), 5 mM MgCl2, 1.4 mM dithiothreitol, 40 mM [35S]cysteine, and 50 gd of cell extract (0.49-0.94 ,ug of protein) in 25 mM potassium phosphate buffer, 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 described reaction between [35C]cysteine and P-RibN*. Production of P-RibN that was dependent on NH3, Rib-5-P, and ATP was arbitrarily attributed to Rib-5-P aminotransferase activity, since it is not known whether the synthesis of P-RibN under these conditions is an enzymatic or nonenzymatic process. Since the newly described direct assay for P-RibN could not be used in the presence of Rib-5-P*, the assay for Rib-5-P aminotransferase was performed in a 100-gl reaction mixture that contained the following: 27 mM Rib-5-P, 22 mM NH40H (1.1 mM NH3), 2 mM ATP, 2 mM [14C]glycine, 10 mM MgCI2, 1 mM dithiothreitol, and 40 gl of cell extract (0.5-1.2 gg of protein) in 50 mM Tris-HCI buffer, pH 8.0. The Rib-5-P and NH40H were preincubated at 370 for 60 min in 50 mM TrisHCI buffer, pH 8.0. The blank for this assay omitted the Rib5-P and NH40H, and the [14C]glycine was separated from the phosphoribosyl['4C]glycinamide (P-Rib-GlyN) on a Dowex column (9). Preliminary studies indicated that P-Rib-GlyN synthetase (EC 6.3.4.13) activity from the cell lysate was not limiting, and consequently an exogenous source of this enzyme was not added to the reaction mixture. Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) (13), adenine phosphoribosyltransferase (EC 2.4.2.7) (14), inosinic acid dehydrogenase (IMP dehydrogenase) (EC 1.2.1.14) (15), adenylosuccinate synthetase (EC 6.3.4.4) (16), adenosine deamninase (EC 3.5.4.4), xanthine oxidase (18) (EC 1.2.3.2), and P-Rib-P-P synthetase (EC 2.7.6.1) (19) were determined as previously described. All of the above assays were linear with respect to time of incubation and protein concentration. 5-Aminoimidazole-4-carboxamide ribonucleotide was determined by the method of Ravel et al. (20). Protein was determined by the method of Lowry et al. (21), with bovine serum albumin as standard. DNA was determined by the method of Leyva and Kelley (22). All radioisotopes were purchased from New England Nuclear Corp., except for [14C]glutamine and [asS]cystine, which were obtained from Amersham-Searle. All chemicals were of the highest grade commercially available.

2459

60

, 40 0.

Z

401-

20

0

24

hours after subculture

48

0

24

48

hours after subculture

FIG. 1. Growth requirements of mutant and wild-type cells. Cells purine-free medium without supplementation (0) hypoxanthine (A). (A) Mutant cells; (B) wild-type

were grown in a or with 10-4 M

cells.

tected in extracts from the mutant cells. When the cells were cultured in a purine-free medium for 24 hr, there was a 2-fold increase in the activity of P-Rib-P-P amidotransferase in the wild-type extract, but there was no effect on the activity of P-Rib-P-P amidotransferase or P-Rib-P-P aminotransferase in mutant extract. In mixing experiments of extracts from mutant and wild-type cells there was no evidence for the presence of an inhibitor of P-Rib-P-P amidotransferase or P-Rib-P-P aminotransferase (Table 2). In contrast to these findings, extracts from both the mutant and wild-type cells, dialyzed against Tris-HCI, demonstrated an equal ability to synthesize P-RibN and P-Rib-GlyN from Rib-5-P, NH3, ATP, and glycine (Table 1). The synthesis of P-Rib-GlyN in this reaction was linear with respect to time of incubation and concentration of extract protein (Fig. 2). If the cell extracts were not dialyzed against Tris-HCI buffer before these studies were performed, the rate of synthesis of P-Rib-GlyN was unchanged in the mutant extract, but it was 5-fold greater in the wild-type extract. Thus, the activity of P-Rib-GlyN synthetase was not limiting in this coupled reaction. One explanation for this reduction in P-Rib-GlyN synthesis may be the loss of P-Rib-P-P synthetase activity after dialysis in Tris.HCl buffer (23). Without P-Rib-P-P synthetase the wild-type cells cannot synthesize P-RibN by the P-Rib-P-P amidotransferase or P-Rib-P-P aminotransferase reaction, and consequently the wild-type extract resembles the mutant extract in its ability to synthesize P-RibN. These results suggest that even in cell lysates P-RibN is more readily synthesized by the combined reactions of P-Rib-P-P synthetase and P-Rib-P-P amidotransferase or P-Rib-P-P aminotransferase than by the single reaction of Rib-5-P aminotransferase. Intracellular Rib-5P aminotransferase activity Table 3 lists the results of cell growth studies performed in purine-free medium that was supplemented with potential Table 1. Activity of P-RibN-synthesizing enzymes in mutant and wild-type extracts

Enzyme P-Rib-P-P amidotransferaset P-Rib-P-P aminotransferaset Rib-5-P aminotransferaset

Mutant* Wild-type* (nmol/hr-mg) (nmol/hr-mg) 88.5 256 4.32