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. 5-fold greater in the wild-type extract. Thus, the activity of 5-Aminoimidazole-4-carboxamide ribonucleotide was de- P-Rib-GlyN synthetase was not limiting in this coupled reac- termined by the method of Ravel et al. (20). tion. One explanation for this reduction in P-Rib-GlyN synthesis Protein was determined by the method of Lowry et al. (21), may be the loss of P-Rib-P-P synthetase activity after dialysis with bovine serum albumin as standard. DNA was determined in Tris.HCl buffer (23). Without P-Rib-P-P synthetase the by the method of Leyva and Kelley (22). wild-type cells cannot synthesize P-RibN by the P-Rib-P-P All radioisotopes were purchased from New England Nuclear amidotransferase or P-Rib-P-P aminotransferase reaction, and Corp., except for [14C]glutamine and [asS]cystine, which were consequently the wild-type extract resembles the mutant extract obtained from Amersham-Searle. All chemicals were of the in its ability to synthesize P-RibN. These results suggest that highest grade commercially available. 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 RESULTS amidotransferase or P-Rib-P-P aminotransferase than by the Growth requirements single reaction of Rib-5-P aminotransferase. Fig. 1 demonstrates that after 24 hr of subculture in a purine- Intracellular Rib-5P aminotransferase activity free medium the mutant cells were unable to replicate, while Table 3 lists the results of cell growth studies performed in the wild-type cells continued to grow well. The slight increase purine-free medium that was supplemented with potential in DNA synthesis by mutant cells when initially transferred to Table 1. Activity of enzymes purine-free medium was probably a reflection of the purine P-RibN-synthesizing in mutant and wild-type extracts pool that had accumulated during culture of the cells in a me- dium supplemented with 10-4 M hypoxanthine. As shown, both Wild-type* Mutant* cell lines grew equally well when the medium was supple- Enzyme (nmol/hr-mg) (nmol/hr-mg) mented with 10-4 M hypoxanthine. Although not presented in Fig. 1, 10-4 M adenine also supported growth of the mutant P-Rib-P-P amidotransferaset 88.5 <1 cells. P-Rib-P-P aminotransferaset 256 < 5 Synthesis of P-RibN Rib-5-P aminotransferaset 4.32 5.82 Table 1 lists the three activities in mutant and wild-type extracts * Cells were grown in regular medium supplemented with 1O-4 M reported to synthesize P-RibN. Neither P-Rib-P-P amido- hypoxanthine. Cell extracts were transferase nor P-Rib-P-P t dialyzed against 50 mM potassium phosphate aminotransferase activity was de- buffer, pH 7.4, containing 1 mM dithiothreitol. $ Extracts were dialyzed against 50 mM Tris-HCl buffer, pH 7.4, * G. L. King and E. W. Holmes, manuscript submitted. containing 1 mM dithiothreitol. Downloaded by guest on September 30, 2021 2460 Genetics: Holmes et al. Proc. Nati. Acad. Sci. USA 73 (1976)

Table 2. Mixing of mutant and wild-type extracts* Table 3. Growth of mutant and wild-type cells in supplemented medium Mutant Wild-type P-Rib-P-P P-Rib-P-P extractt extractt amidotransferase aminotransferase % Change in (M1l) (Ml) (nmol/hr) (nmol/hr) DNA/petri dish 25 4.41 3.01 Supplement Wild-type Mutant 25 - <0.50 <0.50 25 25 4.86 2.81 None +400 -16 1 mM Ribose +340 -10 1 mM * Cells were grown in regular medium supplemented with 10-4 M NH4,Cl +330 -5 hypoxanthine. Extracts were dialyzed against 50 mM potassium 1 mM Ribose + 1 mM NH,4Cl +340 -14 phosphate buffer, pH 7.4, containing 1 mM dithiothreitol for P-Rib-P-P amidotransferase and 50 mM potassium phosphate * Cells were grown in purine-free medium with the indicated sup- buffer, pH 7.4, containing 5 mM MgCl2 and 60 mM 2-mercapto- plement for 48 hr. The percent change in DNA is expressed as the ethanol for P-Rib-P-P aminotransferase. Mg of DNA per petri dish at 48 hr relative to that present at time 0. t The protein concentrations of the mutant cell extracts were 14.4 mg/ml and 12.5 mg/ml for the P-Rib-P-P amidotransferase and P-Rib-P-P aminotransferase experiments, respectively. The pro- drogenase, adenylosuccinate synthetase, hypoxanthine phos- tein concentrations of the wild-type extracts were 18.8 mg/ml phoribosyltransferase, adenine phosphoribosyltransferase, and 9.8 mg/ml, respectively. adenosine deaminase, P-Rib-P-P synthetase, xanthine oxidase, and glutaminase were comparable in the extracts from mutant substrates for Rib-5-P aminotransferase. Wild-type cells grew and wild-type cells. under all of the experimental conditions, but the mutant cells did not replicate even when the medium was supplemeented DISCUSSION with 1 mM ribose, 1 mM NH4Gl, or the combination of both P-RibN is the first intermediate unique to purine biosynthesis of these agents. Higher concentrations of NH4Cl inhibited de novo. Three activities havebeen reported to catalyze P-RibN growth of the wild-type cells and did not support growth of the synthesis in eukaryotic cells: P-Rib-P-P amidotransferase, P- mutant cells. Rib-P-P aminotransferase, and Rib-5-P aminotransferase. The isolation of a eukaryotic cell line by Chu et al. (10) deficient in P-RibN utilization P-Rib-P-P amidotransferase activity (1) provided the ex- The results presented in Table 4 indicate that the mutant cells perimental model for evaluating the role of each of these re- were capable of catalyzing the remainder of the reactions in actions in purine biosynthesis de novo. the pathway of purine biosynthesis de novo, if they were sup- Rib-5-P aminotransferase activity was comparable in the plied with P-RibN. P-RibN was synthesized by incubating the extracts from mutant and wild-type cells. Since the mutant cells extract from mutant or wild-type cells with Rib-5-P and NH3. were demonstrated to be deficient in P-Rib-P-P amidotrans- As shown in part A of Table 4, extract from mutant, as well as ferase and P-Rib-P-P aminotransferase activities, the Rib-5-P wild-type cells, was capable of synthesizing 5-aminoimida- zole-4-carboxamide ribonucleotide, the eighth intermediate Table 4. Synthesis of intermediates of purine metabolism in the pathway of purine biosynthesis de novo. Part B of this by mutant and wild-type cells table demonstrates that mutant cells grew well when the pu- rine-free medium was supplemented with aminoimidazole- (A) Synthesis of 5-aminoimidazole-4-carboxamide carboxamide. Aminoimidazolecarboxamide is metabolized to ribonucleotide the ribonucleotide by adenine phosphoribosyltransferase (14), and the ribonucleotide is then converted in two enzymatic re- (ng formed1/90 min-mg ofprotein) actions to the parent purine ribonucleotide, inosinic acid. Mutant extract 46.7 Wild-type extract 81.3 Enzyme activities Utilization of The activities of a number of other enzymes important to purine (B) 5-aminoimidazole-4-carboxamide biosynthesis are listed in Table 5. The activities of IMP dehy- (% change in DNA/petri dish) Mutant cells +510 V Wild-type cells t E 1.2 N.D. 0 z 0.8 In part A, the undialyzed extracts from freshly harvested cells - were incubated with 27 mM Rib-5-P, 1.1 mM NH3, 2 mM ATP, ._ 10 mM MgCl2, 2 mM glycine, 2 mM formate, 10 mM glutamine, _. 4 0.4 1 mM aspartate, 10 mM KCl, and 25 mM bicarbonate in 50 mM 0 Tris-HCl buffer, pH 7.4, containing 1 mM dithiothreitol for 90 E min at 370. A Rib-5-P/NH3 blank was used to calculate 5-amino- 30 0.4 0.8 1.2 imidazole-4-carboxamide ribonucleotide produced during the Minutes jg of protein 90-min incubation. In part B, mutant cells were grown for 48 hr in a purine-free FIG. 2. Rib-5-P aminotransferase activity. Extracts from the medium that was supplemented with 1 mM 5-aminoimidazole-4- mutant cell line were dialyzed against 50 mM Tris-HCl buffer, pH 7.4. carboxamide. The percent change in DNA is expressed as the Ag of Assay for P-Rib-GlyN synthesis from Rib-5-P, NH3, ATP, and DNA/petri dish at 48 hr relative to that present at time 0. [14CIglycine was performed as described in Materials and Methods. *The actual concentration of the ribonucleotide in the extract The left-hand panel depicts product formed versus time of incubation varied from 1.26 to 2.5 Mg/ml. at 370 with 1.2 Mug of extract protein; the right-hand panel depicts. t N.D. = not determined since wild-type cells grew in purine-free product formed versus protein concentration for a 60-min incubation. medium. Downloaded by guest on September 30, 2021 Genetics: Holmes et al. Proc. Natl. Acad. Sct. USA 73 (1976) 2461

Table 5. Enzyme activities in mutant and these two proteins were structurally related through a common wild-type extracts* subunit, such as demonstrated for several glutamine-utilizing enzymes (25, 26). An alteration in the structure, rate of syn- Wild-type Mutant thesis, or rate of degradation of this common subunit could Enzyme (nmol/hr-mg) (nmol/hr-mg) explain the concurrent loss of these two enzyme activities. It is Hypoxanthine also possible that P-Rib-P-P amidotransferase and P-Rib-P-P phosphoribosyltransferase 252 303 aminotransferase are distinct proteins whose synthesis or in- Adenine activation are closely coordinated at the genetic level. Further phosphoribosyltransferase 452 583 studies are needed to understand the relationship between these IMP dehydrogenase 1.55 1.68 two enzyme activities. Adenylosuccinate synthetase 6.48 9.09 Adenosine deaminase 596 575 E.W.H. is an Investigator, Howard Hughes Medical Institute. Sup- Xanthine oxidase 54.6 48.3 ported by a Basil O'Connor Starter Research Grant from the National P-Rib-P-P synthetase 154 167 Foundation-March of Dimes, 5-35. Glutaminase 31.7 30.1 1. Wyngaarden, J. B. (1972) Current Topics in Cellular Regulation * All extracts were dialyzed against 50 mM potassium phosphate 5,135-176. buffer, pH 7.4, containing 1 mM dithiothreitol, except those used 2. Reem, G. H. (1968) J. Biol. Chem. 243,5695-5701. for the xanthine oxidase assays; these were dialyzed against 50 3. Reem, G. H. (1972) J. CGn. Invest. 51, 1058-1062. mM Tris.HCl buffer, pH 7.4 with 1 mM dithiothreitol. 4. Reem, G. H. (1974) J. Biol. Chem. 249,1696-1703. 5. Reem, G. H., and Friend, C. (1975) Proc. Natl. Acad. Sci. USA aminotransferase reaction represents the only known mecha- 72, 1630-1634. nism for P-RibN synthesis in these cells. However, the mutant 6. Westby, C. A. & Gots, J. S. (1969) J. Biol. Chem. 244,2095-2102. cells were confirmed to be strict purine auxotrophs even when 7. Henderson, J. F. (1963) Biochim. Biophys. Acta 76, 173-180. grown in medium with concentrations 8. Nierlich, D. P. & Magasanik, B. (1965) J. Biol. Chem. 240, supplemented maximal 366-374. of ribose and NH3, potential substrates for Rib-5-P amino- 9. Malloy, G. R., Sitz, T. 0. & Schmidt, R. R. (1973) J. Biol. Chem. transferase. The failure of the mutant cells to grow under these 248, 1970-1975. conditions cannot be explained by additional enzymatic defects 10. Chu, E. H. Y., Sun, N. C. & Chang, C. C. (1972) Proc. Natl. Acad. in the pathway of purine biosynthesis de novo, since these cells Sci. USA 69,3459-3463. can utilize P-RibN for the synthesis of aminoimidazolecar- 11. Feldman, R. I. & Taylor, M. W. (1973) Biochem. Genet. 13, boxamide ribonucleotide and this ribonucleotide can be con- 227-234. verted to purine ribonucleotides. Thus, the Rib-5-P amino- 12. Holmes, E. W., McDonald, J. A., McCord, J. M., Wyngaarden, transferase activity observed in cell lysates does not play a sig- J. B. & Kelley, W. N. (1973) J. Blol. Chem. 248,144-150. nificant role in the intracellular synthesis of P-RibN in this 13. Kelley, W. N. & Meade, J. C. (1971) J. Biol. Chem. 246,2953- eukaryotic cell line under the in vitro conditions studied. 2958. 14. Thomas, C. B., Arnold, W. J. & Kelley, W. N. (1973) J. Biol. The present studies clearly establish a role for P-Rib-P-P Chem. 248,2529-2535. amidotransferase or P-Rib-P-P aminotransferase or both in the 15. Holmes, E. W., Pehlke, D. M. & Kelley, W. N. (1974) Blochim. synthesis of P-RibN, since the mutant cells, deficient only in Blophys. Acta 364,209-217. the activity of these two enzymes, are strict purine auxotrophs. 16. Van Der Weyden, M. B. & Kelley, W. N. (1974) J. Biol. Chem. The relative contribution of each of these enzyme activities to 249,7282-7289. P-RibN synthesis is not known, but probably depends upon the 17. Van Der Weyden, M. B., Buckley, R. H. & Kelley, W. N. (1974) intracellular concentration of glutamine relative to that of NH3 Biochem. Blophys. Res. Commun. 57,590-595. as well as the affinity of the protein(s) for each of these sub- 18. Holmes, E. W., Mason, D. H., Goldstein, L. I., Blount, R. E. & strates. Kelley, W. N. (1974) Clin. Chem. 20, 1076-1079. In 19. Leyva, A. (1974) Doctoral Dissertation, Duke University Medical the absence of other demonstrable abnormalities of purine Center. biosynthesis, the deficiency of both P-Rib-P-P amidotransferase 20. Ravel, J. M., Eakin, R. E. & Shive, W. (1948) J. Biol. Chem. 172, and P-Rib-P-P aminotransferase activities in the mutant cells 67-70. suggests a close relationship between these two enzyme ac- 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. tivities. It is possible that these two reactions are catalyzed by (1951) J. Biol. Chem. 193,265-275. a single protein, since the highly purified chicken liver P- 22. Leyva, A. & Kelley, W. N. (1974) Anal. Biochem. 62, 173- Rib-P-P amidotransferase has been reported to utilize NH3 as 179. well as glutamine as substrate (24). Given this circumstance, 23. Fox, L. H. & Kelley, W. N. (1971) J. Bid. Chem. 246,5739-5748. the concurrent loss of these two enzyme activities might be 24. Hartman, S. C. (1963) J. Biol. Chem. 238,3024-3035. attributed to a mutation affecting a single protein. However, 25. Hartman, S. C. (1973) in The Enzymes of Glutamine Metabo- Reem lism, eds. Prusiner, S. & Stadtman, E. R. (Academic Press, New has reported that human P-Rib-P-P amidotransferase York), pp. 319-30. can be separated from P-Rib-P-P aminotransferase on gel fil- 26. Trotta, P. P., Pinkus, L. M., Wellner, V. P., Estis, L., Haschem- tration chromatography (4). To account for the concurrent loss eyer, R. H. & Meister, A. (1973) in The Enzymes of Glutamine of P-Rib-P-P amidotransferase and P-Rib-P-P aminotrans- Metabolism, eds. Prusiner, S. & Stadtman, E. R. (Academic Press, ferase activities in this circumstance it could be postulated that New York), pp. 431-482. Downloaded by guest on September 30, 2021