Rate-Limiting Steps in the Interconversion of Purine Ribonucleotides in Ehrlich Ascites Tumor Cells in Vitro'

(CANCER RESEARCH 31, 985â€”991, July 1971]

Rate-limiting Steps in the Interconversion of Purine Ribonucleotides in Ehrlich Ascites Tumor Cells in Vitro'

G. W. Crabtree2 and J. Frank Henderson3

University ofAlberta Cancer Research Unit (McEachern Laboratory) and Department of Biochemistry, Edmonton, Alberta, Canada

SUMMARY would conclude from their results that the synthesis of adenine nucleotides from inosinate would take place much The conversion of inosinate to guanylate in Ehrlich ascites more readily than the synthesis of guanine ribonucleotides. tumor cells incubated in vitro in Krebs-Ringer phosphate Furthermore, the rate-limiting reactions for these two medium is limited first by the concentration of glutamine and processes would be adenylosuccinate lyase and inosinate then by the concentration of inosinate. The conversion of dehydrogenase, respectively. Santos et a!. (10) also suggested inosinate to adenylate is limited by the concentration of that inosinate dehydrogenase activity might limit the aspartate. Adenylate conversion to guanylate is limited first by conversion of adenylate to guanylate in rat brain extracts. the concentration of glutamine and then probably by Rates of interconversion of adenine and guanine adenylate deaminase activity. Guanylate conversion to ribonucleotides are slow in rabbit erythrocytes in vitro (6), adenylate is limited by guanylate reductase activity. and activities of adenylate deaminase and guanylate reductase may be limiting. However, it is uncertain whether substrate and cofactor INTRODUCTION concentrations for these enzymes are saturating in Ehrlich ascites tumor cells in vitro. The concentration of glutamine, Purine ribonucleotides are interconverted by a system of 6 for example, has already been shown to be limiting for protein enzymes arranged in 2 cycles which have a common synthesis (8) and for purine biosynthesis de novo (4), and intermediate in inosinate:4 Hershko et a!. (6) have proposed that the availability of this adenylate guanylate amino acid may also limit guanylate synthesis in rabbit erythrocytes in vitro. Finally, Fontenelle and Henderson (3) have suggested that intracellular concentrations of aspartate /\N7h \ may be limiting for adenylate synthesis from inosinate. Numerous studies (reviews in Refs. 1 and 12) have also \ + shown that most of the enzymes of purine ribonucleotide @ Adenylosuccinate inosinate xanthylate aspartate NAD interconversion are activated or inhibited by one or another + purine nucleotide. Although these reactions and their regulations have been studied individually in some detail in cell These reactions may at least potentially be regulated by the extracts and with partially purified enzymes, relatively little amounts of the enzymes involved, by the concentrations of work has been done to elucidate the controls of these the nucleotide intermediates and of coenzyme and amino acid reactions as they operate as an integrated system in intact substrates, and by allosteric activation and inhibition. cells. In this study, the rate-limiting steps in the pathways of The relative activities of several enzymes of purine purine ribonucleotide interconversion in Ehrlich ascites tumor ribonucleotide interconversion have been measured by McFall cells have been identified under several conditions of and Magasanik (7) in extracts of L-cells and of Ehrlich ascites incubation in vitro. tumor cells. If total enzyme activities were rate limiting, one This study has been greatly facilitated by the development of procedures for the rapid analysis of radioactivity in purine I This work was supported by the National Cancer Institute of ribonucleotides, ribonucleosides, and bases in large numbers of Canada. small samples; these methods are given in detail. 2 Research Fellow of the National Cancer Institute of Canada. Present address: Division of Biological and Medical Sciences, Brown University, Providence, Ri. 3To whom inquiries should be addressed. MATERIALS AND METHODS 4The enzymes of purine ribonucleotide interconversion are: inosinate dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14]; guanylate synthetase (xanthosine-5'-phosphate ligase (AMP), EC 4 C (52.6 mCi/mmole), 4 C @ 6.3.4.1] ; guanylate reductase (reduced NADP:GMP oxidoreductase (49.5 mCi/mmole), and C (31 .7 mCi/mmole) (deaminating), EC 1.6.6.8] ; adenylosuccinate synthetase were obtained from Schwarz BioResearch, Inc., Orangeburg, (IMP:L-aspartate ligase (GDP), EC 6.3.4.4] ; adenylosuccinate lyase N. Y.; purine bases and ribonucleosides were from Sigma (adenylosuccinate AMP lyase, EC 4.3.2.2] ; adenylate deaminase (AMP aminohydrolase, EC 3.5.4.6]. Chemical Company, St. Louis, Mo.; purine ribonucleotides Received December 11, 1970; accepted March 5, 1971. were from P-L Biochemicals, Milwaukee, Wis.; L-glutamine

JULY 1971 985

was from Calbiochem, Los Angeles, Calif. ; and L-aspartic acid from the top were scraped off and discarded. The plates were was from Mann Research Laboratories, New York, N. Y. rotated 90Â° and developed in the 2nd direction with Hadaci di n (N-formylhydroxyaminoacetic acid) and 1-butanol: methanol:water:ammonia (60:20:20: 1); after 6-diazo-5-oxo-L-norleucine were gifts of the Cancer drying, the 2nd dimension was redeveloped with the same Chemotherapy National Service Center, National Cancer solvent. Purine-containing areas were visualized with UV light. Institute, Bethesda, Md. With this method, the following compounds were well Six days after i.p implantation in ICR Swiss mice of separated (the distance of each from the origin is given in cm approximately 106 Ehrlich ascites tumor cells, cells were with the 1st dimension followed by the 2nd dimension): removed and washed 3 times with buffered saline (140 mM adenine (10.8, 11.7), adenosine (13.2, 10.0), hypoxanthine NaCl:lO mM Tris buffer, pH 7.4:4 mM sodium phosphate (10.6, 7.8), guanine (6.9, 5.8), inosine (12.3, 6.7), guanosine buffer, pH 7.4) containing 5.5 mM glucose. A 2% cell (10.9, 5.7), xanthine (6.9, 4.7), xanthosine (1 1.3, 4.5), and suspension was incubated in modified Krebs-Ringer phosphate uric acid (5.6, 3.2). Nucleotides remained as a streak along the medium ( I 10 mM NaCl:4.9 mM KC1:1.2 mM MgSO4 :25 mM line of 1st development. sodium phosphate buffer, pH 7.4) containing 5.5 mM glucose UV-absorbing areas of the chromatography sheets were cut and other additions as required. All incubations were carried out and placed in counting vials, phosphor solution was added out in a water bath at 37Â°with shaking at 80 oscillations/min (4 g PPO and 0.1 g POPOP per liter of toluene), and and air as the gas phase. In all experiments, cells were radioactivity measurements were made at 72% counting incubated with glucose alone for 20 mm before radioactive efficiency. precursors were added. Preliminary experiments had shown, Results presented below are measurements of the amounts that prior incubation with glucose in a high-phosphate medium of radioactivity in each metabolite, expressed as nmoles/g of gave maximal rates of purine ribonucleotide synthesis from cells, rather than the total amount of each metabolite. Average purine bases. values from duplicate samples are reported. The results are After various periods of incubation, 0.5-ml samples of representative of those obtained in at least 2 experiments. incubation media containing cells were transferred to tubes containing 25 p1 of cold 4.2 M perchioric acid; acid extracts were subsequently neutralized with 25 @.zlof4.42 N KOH. RESULTS After centrifugation , samples were chromatographed. Baker-Flex polyethyleneimine cellulose thin layers on Mylar Factors that are rate limiting for the conversion of inosinate sheets (Fisher Scientific, Edmonton, Alta., Canada) were to adenylate and guanylate were studied first. The data used to separate purine ribonucleotides by I-dimensional presented in Chart 1 show that Ehrlich ascites tumor cells in @@ chromatography in a modification of the method of vitro converted C more extensively to Randerath and Randerath (9). Sheets were first developed for adenine nucleotides (including NAD) than to guanine 5 hr with 4 M sodium formate buffer, pH 3.4, dried, and then developed overnight with methanol:water (1 : 1). After drying, 10 or 20 @zlofcell extract plus about 30 nmole of each purine LI w ribonucleotide carrier were applied as a I-cm streak 2 cm from 90 I-. the bottom of the sheet. A wick of Whatman No. 3MM paper 0 LU was stapled to the top of the sheet, and it was developed -j overnight with methanol:water (1 : 1) to wash salts, purine L) :@ 60 bases, and ribonucleosides onto the paper wick; the wick was z then discarded. For separation of the ribonucleotides, the sheets were developed with increasing concentrations of 0 sodium formate buffers, pH 3.4, as follows: 0.5 M formate U. 30 buffer to a line 2.5 cm above the origin, then 2.0 M formate 0 buffer to a line 7.0 cm above the origin, and finally 4.0 M z LU J. formate buffer to the top of the plate. The sheets were dried, L) @ and nucleotide-containing areas were visualized under UV LU I a- light. With this method, the following nucleotides were well _0 30 60 90 separated (the distance of each from the origin is given in cm): MINUTES GTP (1 .3), ATP (3.5), GDP (4.5), ADP (9.0), GMP (10.0), Chart 1. Relative incorporation of anin4 C into adenine XMP (1 1.0), IMP (12.3), AMP (14.0), and NAD (15.5). Eight and guanine nucleotides. Ehrlich ascites tumor cells, 2% by volume, samples were usually analyzed per sheet. were incubated in 25-mi Erlenmeyer flasks at 37Â°with shaking with an Eastman Kodak unsubstituted cellulose thin layers on Mylar atmosphere of air in 5.0 ml of Krebs-Ringer medium containing 25 mM sodium phosphate buffer, pH 7.4, and 5.5 mM glucose. After 20 mm, sheets (Fisher Scientific) were used to separate purine bases hypoxanthine-' 4C was added to final concentrations of 5 MM(o, i@)or and ribonucleosides by 2-dimensional chromatography. Each 100 jsM (., Â£).At various times, portions were removed for analysis of sheet was developed in the first direction for about 50 mm radioactivity in adenine nucleotides (., o) and guanine nucleotides (a, with acetonitrite:0.l M ammonium acetate, pH 7.0:ammonia a').Eachpoint representsthemean of separateanalyse@of duplicate (60:30: 10). After the plates were dried, areas below the origin flasks in I experiment; the results are representative of those obtained (which was 2.5 cm in each dimension from 1 corner) and 3 cm in 4 experiments.

986 CANCER RESEARCH VOL. 31

nucleotides (plus xanthylate). With a low concentration of a- precursor (5 pM), the ratio of incorporation into adenine LI nucleotides relative to that into guanmne nucleotides was LU

approximately 3 after 30 mm incubation, whereas this ratio I- 20 0 was approximately 6 at the same time when the initial LU extracellular concentration of hypoxanthine was 100 jiM. LI 15 Further studies were conducted to identify the rate-limiting z steps and factors on the pathway of guanylate synthesis from inosinate. Inosinate or xanthylate might be expected to 0 10 accumulate if inosinate dehydrogenase or xanthylate aminase, Iâ€” U. respectively, were rate limiting. However, Chart 2 shows that 0 the concentrations of radioactive inosinate and xanthylate Iâ€” 5 z remained very low throughout the incubation period. LU LI Because nucleotides might be dephosphorylated if they LU 0- began to accumulate, the formation of nucleosides and bases a- 0 30 60 90 by cells incubated with hypoxanthine-'4C was measured. MINUTES After 90 mm of incubation, 1190 nmoles/g cells of radioactive Chart 2. Formation of radioactive inosinate and xanthylate from xanthosine plus xanthine accumulated under these conditions; hypoxanthine-' 4C. Tumor cells were incubated as described in Chart 1 this amounted to almost 30% of the total acid-soluble with 100 @LMhypoxanthine-' 4C. Portions were removed at various radioactivity present in the sample. It will be shown below times for analysis of inosinate (.) and xanthylate (0). that the main pathway of xanthosine and xanthine synthesis from hypoxathine in these cells is via xanthylate rather than by xanthine oxidase action on hypoxanthmne. These observations both imply that significant amounts of a- xanthylate were very readily dephosphorylated. Xanthylate LI aminase, therefore, appears to limit the conversion of inosinate 0 to guanylate under these conditions.5 Iâ€” It thus became apparent that the flow of radioactive U. compounds along the mnosinate-guanylate pathway could not 0 I- be accurately estimated by measurements of radioactive z LU nucleotides only. Chart 3 shows the relative distribution of LI LU radioactivity in â€œadeninecompoundsâ€• (adenine nucleotides, a- NAD, and adenosine) and in â€œguaninecompoundsâ€•(guanmne nucleotides, xanthylate, xanthosine , guanosine, xanthine , and guanine) after incubation of cells with pothi4 C. At a precursor concentration of S jiM the incorporation of MINUTES @ radioactivity into guanmne compounds was greater than that Chart 3. Relative conversion of hypoxanthine-' C to â€œadÃ«nine into adenine compounds, although the rate of incorporation compoundsâ€•and â€œguaninecompoundsâ€•(see text). Tumor cells were into guanine compounds decreased more rapidly than did the incubated as described in Chart 1 with 5 MM(o, @)or 100 @iM(., a) other process. After 90 mm of incubation, the ratio of hypoxanthine-' 4C. Portions @reremoved at various times for analysis radioactivity in adenine compounds relative to that in guanine of adenine compounds (., o) and guanine compounds (a, @). compounds was about 1:3. When the initial extracellular @ concentration of C was 100 jiM, the rates of acid to incubation media on the synthesis of guanine its incorporation into adenine compounds and guanine nucleotides and of xanthosine plus xanthine were therefore compounds were similar for the first 30 mm of incubation. At studied. The accumulation of radioactive xanthosine plus later times, the rate of incorporation into adenine compounds xanthine after 90 mm of incubation decreased from 1210 decreased relative to that into guanine compounds. nmoles/g in the absence of glutammne to 74 nmoles/g in the The rate-limiting character of xanthylate aminase presence of 2 mM glutamine. (These data also show that demonstrated above might have been due to the total activity almost no xanthine is being formed via xanthine oxidase of this enzyme or to the concentration of another substrate of activity on hypoxanthine under these conditions.) As would @ this reaction, glutamine. The effects of addition of this amino be expected, the incorporation of C into guanine nucleotides was markedly increased in the presence of glutamine (Chart 4). S Measurements of radioactive inosine formation in cells incubated @ with hypoxanthine-' C or C do not necessarily measure the Further information regarding the role of glutamine in possible rate-limiting character of IMP dehydrogenase because inosine regulating xanthylate aminase activity came from studies with may be synthesized by pathways alternative to dephosphorylation of diazooxonorleucine, an antimetabolite of glutamine, and with inosinate. Thus, inosine may be made directly from hypoxanthine by purine nucleoside phosphorylase and from adenine via adenylate and methionine sulfoximine, an inhibitor of glutamine synthetase. adenosine. At the present time, the relative rates of the alternative Chart S shows that the concentration of diazooxonorleucine pathways involved have not been evaluated. used almost completely inhibited the synthesis of guanine

JULY 1971 987

The decrease in the amount of radioactivity found in 225 xanthosine plus xanthine when glutamine was added was not equaled by the increase in the radioactivity found in guanine nucleotides under the same conditions. This discrepancy was due in part to a doubling in the amount of radioactive 150 0 â€˜I) guanosine plus guanine formed. However, this amounted to LU -I only I to 2% of the total radioactivity involved. Another cause 0 of the discrepancy was a marked decrease in the amount of C 4 C converted to ribonucleotides (Chart 6). 75 This apparent decrease in hypoxanthine phosphoribosyltransferase activity may be caused by diversion of phosphoribosylpyrophosphate to the pathway of purine biosynthesis de novo, which is still operating to some extent 0 ) 30 60 90 even at 100 jiM hypoxanthine (4). MINUTES Chart 7 shows another alteration in purine metabolism upon Chart 4. Effect of glutamine on the synthesis of radioactive guanine the addition of glutamine. A considerable amount of @ nucleotides from hypoxanthine-' C. Tumor cells were incubated as radioactive inosinate, which in the absence of glutamine would described in Chart 1 with 100 pM hypoxanthine-' 4C with (0) and have been converted to xanthosine and xanthine, was in the without (.) 2 mM glutamine. presence of this amino acid converted to adenine nucleotides rather than to guanine nucleotides. Although the mechanism 1@

, I 100 @ 4200

0 75 â€˜I, LU @1 @ 0 !@2800 50

C 00oo@ @ 25 1400

0 30 60 90 I I I 0 MINUTES 0 30 60 90 Chart 5. Effect of diazooxonorleucine on the synthesis of radioactive MINUTES guanine nucleotides from hypoxanthine-' 4C. Tumor cells were Chart 6. Effect of glutamine on the utilization of nm4 C. @ incubated as described in Chart 1 with 100 @M C with Tumor cells were incubated as described in Chart 1 with 100 @M (0) and without (.) 35 @M diazooxonorleucine. hypoxanthine-' 4C with (o) and without (.) 2 mM glutamine.

Table 1 I I I Effect ofmethionine sulfoximine on the synthesis of radioactive xanthine plus xant ho sine from hypoxanthine-' 4C 1500 Tumor cells were incubated as described in Chart 1 with 100 @M hypoxanthine with and without 5 mM methionine sulfoximine. 0

â€˜I, LU plus -I 1000 time xanthosine 0 (nmoles/g)NoneAdditionsIncubation (mm)Xanthine C

500 60 978 Methioninesulfoximine30 30 960 60750 1242 @ 0 I I C 30 60 90 @ nucleotides from C. The data in Table 1 show MINUTES that the formation of radioactive xanthine plus xanthosine was Chart 7. Effect of glutamine on the synthesis of radioactive adenine also increased when glutammne synthesis was inhibited by nucleotides from hi4 C. Tumor cells were incubated as methionine sulfoximine. (Glutamine completely overcame the described in Chart 1 with 100 @zMhypoxanthine-' 4C with (o) and effect of this amino acid analog.) without (.) 2 mM glutamine.

988 CANCER RESEARCH VOL.31

of this stimulation of adenine nucleotide synthesis will be high initial extracellular concentrations of aspartate were used, discussed below, this observation implies that in the presence but the intracellular concentration attained has not been of glutamine total inosinate dehydrogenase activity was still determined. Chart 9 shows that the addition of aspartate not rate limiting for the conversion of inosinate to guanylate. increased the conversion of an4 C to adenine Instead, the amount of inosinate available to this enzyme in nucleotides almost 2-fold, whereas guanine nucleotide the face of increased adenylosuccinate synthetase activity synthesis was scarcely affected. The synthesis of xanthine plus appeared to be the more important regulating factor. xanthosine decreased from 1190 to 876 nmoles/g after 90 mm If adenylosuccinate synthetase did have a competitive of incubation, suggesting again that inosinate was diverted advantage over inosinate dehydrogenase with respect to away from the pathway of guanylate synthesis in the presence utilization of inosinate, then inhibition of the former enzyme of aspartate. with hadacidin ( 11) might alter this situation. Chart 8 shows Although radioactivity in adenylosuccinate was not that, although this analog of aspartate did inhibit the synthesis routinely measured in these experiments, preliminary of radioactive adenine nucleotides by more than 80%, there experiments have shown that it does not appear to accumulate was little or no stimulation of the synthesis of radioactive in the presence or absence of aspartate. Adenylosuccmnate guanine nucleotides. It would be expected, however, that most synthetase would appear therefore to be the rate-limiting step of the product of the inosinate dehydrogenase reaction would for adenylate synthesis from inosinate. @ accumulate as xanthosine plus xanthine under these When C was used as substrate of nucleotide conditions; after 90 mm of incubation, accumulation of these synthesis in Ehrlich ascites tumor cells, almost 95% of the products increased from 1254 to 1490 nmoles/g in the total nucleotide fraction was composed of adenine nucleotides presence of hadacidin. An increase in inosine formation, as themselves. Even when its conversion into bases and @ well as an apparent decreased utilization of C, nucleosides not containing adenine was measured (Table 2), 89 were also observed in the presence of hadacidin. These changes might be due, at least in part, to increased dephosphorylation I I of inosinate ; whether this increased dephosphorylation is due 2100 to accumulation of inosinate consequent upon saturation of inosinate dehydrogenase with this substrate, or simply to dephosphorylation of increased amounts of inosinate without 0 saturation of inosinate dehydrogenase, is not clear. ..%. 1400 The stimulation of radioactive adenine nucleotide synthesis LU from hypoxanthine-' 4C upon addition of glutammne to 0 incubation media (Chart 6) was probably due to the rapid C conversion of this amino acid to aspartate, a substrate of 700 adenylosuccinate synthetase; this process has previously been shown to occur in these cells (5). Aspartate itself was therefore added to incubation media and its effects on U mn4 C metabolism were measured. Becausethe 0 30 60 90 cells are not very permeable to dicarboxylic amino acids (2), MINUTES Chart 9. Effect of aspartate on the synthesis of radioactive adenine 1200 - and guanine nucleotides from hypoxanthine-' 4C. Tumor cells were incubated as described in Chart 1 with 100 @Mhypoxanthine-' 4C with

(0, i@@)and without (., a) 20 mM aspartate. Portions were removed at various times for analysis of adenine nucleotides (., o) and guanine nucleotides (a, is). 0800 -

LU -I Table2 @ 0 Conversion â€˜C into metabolites not containing adenineÂ° @ C 400 - Tumor cells were incubated as described in Chart 1. Adenine-'4C not concentration time containing adeninea radioactivity)2010 (mm)Metabolites (% total 0@-@ (NM)Incubation

MINUTES 14.25010 307.2 Chart 8. Effect of hadacidin on the synthesis of radioactive adenine 303.4 and guanine nucleotides from hypoxanthine-' 4C. Tumor cells were 6.710010 incubated as described in Chart 1 with 100 @Mhypoxanthine-14C with 302.5 5.5 (0, t@) and without (5, a) 100 @g/ml of hadacidin. Portions were removed at various times for analysis of adenine nucleotides (., o) and a Inosinate, xanthylate, guanine nucleotides, hypoxanthine, inosine, guanine nucleotides (a, â€˜s). xanthine, xanthosine, guanine, and guanosine.

JULY 1971 989

@ to 97% of the total radioactivity was in adenine and related the action of guamine deaminase on the precursor guanine-' C nucleosides and nucleotides. as well as by the catabolism of guanine nucleotides.) Table 3 shows that neither radioactive inosinate nor It is apparent that the conversion of guanylate to adenylate radioactive xanthylate accumulate in cells incubated with took place very slowly in these cells. Because neither imosinate adenine-'4C, but 221 nmoles/g of radioactive xanthosine plus nor hypoxanthine plus inosine accumulated under these xanthine accumulated after 90 mm of incubation with 100 conditions, the rate-limiting step in this process appears to be mM adenine-'4C. Other experiments have shown that this guanylate reductase. accumulation did not occur in the presence of added glutamine and that the amount of radioactive guanine DISCUSSION nucleotides was concomitantly increased. Some inosine and hypoxanthine were formed both in the presence and absence It is apparent from these studies that the flow of material of glutamine, but, because these could be formed via the along the various pathways of purine ribonucleotide dephosphorylation of adenylate as well as by that of inosinate, rate-limiting steps could not be evaluated. interconversion cannot be accurately gauged by measurement of radioactivity in the ribonucleotide intermediates and An experiment was done with adenine-'4C to determine whether any radioactive inosinate formed from adenylate was products only. The nucleosides and bases derived from these converted back to adenylate via adenylosuccinate synthet.ase. compounds may contain significant amounts of radioactivity, The conversion of radioactivity from adenine into inosine was which may not only influence conclusions regarding increased from 74 to 102 nmoles/g after a 90-mm incubation identification of rate-limiting steps but may also change of hadacidin. If we assume that hadacidin has no effect on the markedly depending on experimental conditions. dephosphorylation of adenylate and deamination of adeno The conversion of inosinate both to guanylate and to sine, it may tentatively be concluded that the increased adenylate in cells incubated in this salts:glucose medium was amount of radioactive inosine was derived from inosinate limited primarily by the intracellular concentrations of the which was not utilized by adenylosuccinate synthetase in the amino acid substrates of these reactions, glutamine and presence of this inhibitor. aspartate, respectively. No firm evidence was obtained to When Ehrlich cells were incubated in vitro with indicate that inosinate concentrations ever rose to the point guanine-' 4C, less than 10% of the precursor was converted to where total inosinate dehydrogenase activity became rate compounds that did not contain the guanine moiety per se limiting. Instead, inosinate appeared to be either (Table 4). (Xanthine, a possible catabolite of xanthylate, was dephosphorylated or converted to adenylosuccinate and not included by these figures because it may also be formed by adenylate. The results of supplementation with glutammne and the Table3 effects of diazooxonorleucine and methionine sulfoximime are Concentrations of radioactive inosinate and xanthylate in agreement with previous studies with Ehrlich ascites tumor synthesized from @4C cells which showed that glutamine concentrations were Tumor cells were incubated as described in Chart 1. limiting for other processes as well (2â€”5,8). These results also support previous suggestions (3) that aspartate concentrations Incubationconcentration14 c might limit adenylosuccinate synthetase activity. Xanthylate(NM) time Inosinate Although no evidence was obtained to indicate that total (nmoles/g)20 (mm) (nmoles/g) activities of inosinate dehydrogenase or of adenylosuccinate 2.03010 4.0 synthetase were rate limiting, some evidence does support the 3.050 5.0 idea that total activities of adenylate deaminase and guanylate 2.53010 7.5 reductase may be of greater regulatory significance. 4.0100 12.5 103010 20 Unfortunately, this point may remain unclear until means are 15Table 25 found to distinguish between the dephosphorylation of adenylate and that of inosinate. 4Conversion Studies to evaluate the possible regulation of the enzymes metabolitesnotofguanine-'4C into of purine ribomucleotide interconversion by variation in the a14 containing guanine concentrations of purine ribonucleoside di- and triphosphates are being begun for further study of regulatory factors in this notconcentration C Incubation Metabolites intact cell system. guaninea(@zM) time containing radioactivity)20 (mm) (% of total REFERENCES 105.630 6.850 1. Blakeley, R. L., and Vitols, E. The Control of Nucleotide 102.630 Biosynthesis. Ann. Rev. Biochem., 37: 201â€”224,1968. 2.7100 102.230 2. Coles, N. W., and Johnstone, R. M. Glutamine Metabolism in 2.4 Ehrlich Ascites-Carcinoma Cells. Biochem. J., 83: 284â€”291, 1962. 3. Fontenelle, L. J., and Henderson, J. F. Sources of Nitrogen as a Inosinate, adenine nucleotides, xanthylate, hypoxanthine, inosine, Rate-limiting Factors for Purine Biosynthesis de Novo in Ehrlich adenine, adenosine, and xanthosine. Ascites Tumor Cells. Biochim. Biophys. Acta, 1 77: 88â€”93,1969.

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4. Henderson, J. F. Feedback Inhibition of Purine Biosynthesis in Glutamine in Protein Synthesis by the Ehrlich Ascites Carcinoma. Ascites Tumor Cells. J. Biol. Chem., 237: 2631â€”2635, 1962. J. Biol. Chem., 222: 879â€”893,1956. 5. Henderson, J. F. Dual Effects of Ammonium Chloride on Purine 9. Randerath, K., and Randerath, E. Ion-Exchange Chromatography Biosynthesis de Novo in Ehrlich Ascites-Tumor Cells in Vitro. of Nucleotides on Poly(ethyleneimine)cellulose Thin Layers. J. Biochim. Biophys. Acta, 76: 173â€”180,1963. Chromatog., 16: 111â€”125,1964. 6. Hershko, A., Razin, A., Shoshani, T., and Mager, J. Turnover of 10. Santos, J. N., Hempstead, K. W., Kopp, L. E., and Miech, R. P. Purine Nucleotides in Rabbit Erythrocytes. II. Studies in Vitro. Nucleotide Metabolism in Rat Brain. J. Neurochem., 15: 367â€”376, Biochim. Biophys. Acts, 149: 59â€”73,1967. 1968. 7. McFall, E., and Magasanik, B. The Control of Purine Biosynthesis 11. Shigeura, H. T., and Gordon, C. N. The Mechanism of Action of in Cultured Mammalian Cells. J. Biol. Chem., 235: 2103â€”2108, Hadacidin. J. Biol. Chem., 237: 1937â€”1940, 1962. 1960. 12. Stadtman, E. R. Allosteric Regulation or Enzyme Activity. 8. Rabinovitz, M., Olsen, M. E., and Greenberg, D. M. Role of Advan. Enzymol., 28: 41â€”154,1966.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1971 American Association for Cancer Research. Rate-limiting Steps in the Interconversion of Purine Ribonucleotides in Ehrlich Ascites Tumor Cells in Vitro

G. W. Crabtree and J. Frank Henderson

Cancer Res 1971;31:985-991.

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