(CANCER RESEARCH 31, 985—991, July 1971]

SUMMARY

The conversion of inosinate to guanylate in Ehrlich ascitestumor cells incubated in vitro in Krebs-Ringer phosphatemedium is limited first by the concentration of glutamine andthen by the concentration of inosinate. The conversion ofinosinate to adenylate is limited by the concentration ofaspartate. Adenylate conversion to guanylate is limited first bythe concentration of glutamine and then probably byadenylate deaminase activity. Guanylate conversion toadenylate is limited by guanylate reductase activity.

INTRODUCTION

Purine ribonucleotides are interconverted by a system of 6enzymes arranged in 2 cycles which have a commonintermediate in inosinate:4

adenylate guanylate

/\N7h

Adenylosuccinate@ inosinate xanthylateaspartate NAD

+

These reactions may at least potentially be regulated by theamounts of the enzymes involved, by the concentrations ofthe nucleotide intermediates and of coenzyme and amino acidsubstrates, and by allosteric activation and inhibition.

The relative activities of several enzymes of purineribonucleotide interconversion have been measured by McFalland Magasanik (7) in extracts of L-cells and of Ehrlich ascitestumor cells. If total enzyme activities were rate limiting, one

I This work was supported by the National Cancer Institute of

Canada.2 Research Fellow of the National Cancer Institute of Canada.

Present address: Division of Biological and Medical Sciences, BrownUniversity, Providence, Ri.

3To whom inquiries should be addressed.4The enzymes of purine ribonucleotide interconversion are:

inosinate dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14];guanylate synthetase (xanthosine-5'-phosphate ligase (AMP), EC6.3.4.1] ; guanylate reductase (reduced NADP:GMP oxidoreductase(deaminating), EC 1.6.6.8] ; adenylosuccinate synthetase(IMP:L-aspartate ligase (GDP), EC 6.3.4.4] ; adenylosuccinate lyase(adenylosuccinate AMP lyase, EC 4.3.2.2] ; adenylate deaminase (AMPaminohydrolase, EC 3.5.4.6].

Received December 11, 1970; accepted March 5, 1971.

would conclude from their results that the synthesis ofadenine nucleotides from inosinate would take place muchmore readily than the synthesis of guanine ribonucleotides.Furthermore, the rate-limiting reactions for these twoprocesses would be adenylosuccinate lyase and inosinatedehydrogenase, respectively. Santos et a!. (10) also suggestedthat inosinate dehydrogenase activity might limit theconversion of adenylate to guanylate in rat brain extracts.Rates of interconversion of adenine and guanineribonucleotides are slow in rabbit erythrocytes in vitro (6),and activities of adenylate deaminase and guanylate reductasemay be limiting.

However, it is uncertain whether substrate and cofactorconcentrations for these enzymes are saturating in Ehrlichascites tumor cells in vitro. The concentration of glutamine,for example, has already been shown to be limiting for proteinsynthesis (8) and for purine biosynthesis de novo (4), andHershko et a!. (6) have proposed that the availability of thisamino acid may also limit guanylate synthesis in rabbiterythrocytes in vitro. Finally, Fontenelle and Henderson (3)have suggested that intracellular concentrations of aspartatemay be limiting for adenylate synthesis from inosinate.

Numerous studies (reviews in Refs. 1 and 12) have alsoshown that most of the enzymes of purine ribonucleotideinterconversion are activated or inhibited by one or anotherpurine nucleotide. Although these reactions and theirregulations have been studied individually in some detail in cellextracts and with partially purified enzymes, relatively littlework has been done to elucidate the controls of thesereactions as they operate as an integrated system in intactcells. In this study, the rate-limiting steps in the pathways ofpurine ribonucleotide interconversion in Ehrlich ascites tumorcells have been identified under several conditions ofincubation in vitro.

This study has been greatly facilitated by the developmentof procedures for the rapid analysis of radioactivity in purineribonucleotides, ribonucleosides, and bases in large numbers ofsmall samples; these methods are given in detail.

MATERIALS AND METHODS

4 C (52.6 mCi/mmole), 4 C

(49.5 mCi/mmole), and@ C (31 .7 mCi/mmole)were obtained from Schwarz BioResearch, Inc., Orangeburg,N. Y.; purine bases and ribonucleosides were from SigmaChemical Company, St. Louis, Mo.; purine ribonucleotideswere from P-L Biochemicals, Milwaukee, Wis. ; L-glutamine

\\ +

985JULY 1971

Rate-limiting Steps in the Interconversion of PurineRibonucleotides 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

on May 27, 2018. © 1971 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

G. W. Crabtree and J. Frank Henderson

was from Calbiochem, Los Angeles, Calif. ; and L-aspartic acidwas from Mann Research Laboratories, New York, N. Y.Hadaci di n (N-formylhydroxyaminoacetic acid) and6-diazo-5-oxo-L-norleucine were gifts of the CancerChemotherapy National Service Center, National CancerInstitute, Bethesda, Md.

Six days after i.p implantation in ICR Swiss mice ofapproximately 106 Ehrlich ascites tumor cells, cells wereremoved and washed 3 times with buffered saline (140 mMNaCl:lO mM Tris buffer, pH 7.4:4 mM sodium phosphatebuffer, pH 7.4) containing 5.5 mM glucose. A 2% cellsuspension was incubated in modified Krebs-Ringer phosphatemedium ( I 10 mM NaCl:4.9 mM KC1:1.2 mM MgSO4 :25 mMsodium phosphate buffer, pH 7.4) containing 5.5 mM glucoseand other additions as required. All incubations were carriedout in a water bath at 37°with shaking at 80 oscillations/minand air as the gas phase. In all experiments, cells wereincubated with glucose alone for 20 mm before radioactiveprecursors were added. Preliminary experiments had shown,that prior incubation with glucose in a high-phosphate mediumgave maximal rates of purine ribonucleotide synthesis frompurine bases.

After various periods of incubation, 0.5-ml samples ofincubation media containing cells were transferred to tubescontaining 25 p1 of cold 4.2 M perchioric acid; acid extractswere subsequently neutralized with 25 @.zlof 4.42 N KOH.After centrifugation , samples were chromatographed.

Baker-Flex polyethyleneimine cellulose thin layers on Mylarsheets (Fisher Scientific, Edmonton, Alta., Canada) wereused to separate purine ribonucleotides by I -dimensionalchromatography in a modification of the method ofRanderath and Randerath (9). Sheets were first developed for5 hr with 4 M sodium formate buffer, pH 3.4, dried, and thendeveloped overnight with methanol:water (1 : 1). After drying,10 or 20 @zlof cell extract plus about 30 nmole of each purineribonucleotide carrier were applied as a I -cm streak 2 cm fromthe bottom of the sheet. A wick of Whatman No. 3MM paperwas stapled to the top of the sheet, and it was developedovernight with methanol:water (1 : 1) to wash salts, purinebases, and ribonucleosides onto the paper wick; the wick wasthen discarded. For separation of the ribonucleotides, thesheets were developed with increasing concentrations ofsodium formate buffers, pH 3.4, as follows: 0.5 M formatebuffer to a line 2.5 cm above the origin, then 2.0 M formatebuffer to a line 7.0 cm above the origin, and finally 4.0 Mformate buffer to the top of the plate. The sheets were dried,and nucleotide-containing areas were visualized under UVlight. With this method, the following nucleotides were wellseparated (the distance of each from the origin is given in cm):GTP (1 .3), ATP (3.5), GDP (4.5), ADP (9.0), GMP (10.0),XMP (1 1.0), IMP (12.3), AMP (14.0), and NAD (15.5). Eightsamples were usually analyzed per sheet.

Eastman Kodak unsubstituted cellulose thin layers on Mylarsheets (Fisher Scientific) were used to separate purine basesand ribonucleosides by 2-dimensional chromatography. Eachsheet was developed in the first direction for about 50 mmwith acetonitrite:0.l M ammonium acetate, pH 7.0:ammonia(60:30: 10). After the plates were dried, areas below the origin(which was 2.5 cm in each dimension from 1 corner) and 3 cm

from the top were scraped off and discarded. The plates wererotated 90° and developed in the 2nd direction with1-butanol: methanol:water:ammonia (60:20:20: 1); afterdrying, the 2nd dimension was redeveloped with the samesolvent. Purine-containing areas were visualized with UV light.With this method, the following compounds were wellseparated (the distance of each from the origin is given in cmwith the 1st dimension followed by the 2nd dimension):adenine (10.8, 11.7), adenosine (13.2, 10.0), hypoxanthine(10.6, 7.8), guanine (6.9, 5.8), inosine (12.3, 6.7), guanosine(10.9, 5.7), xanthine (6.9, 4.7), xanthosine (1 1.3, 4.5), anduric acid (5.6, 3.2). Nucleotides remained as a streak along theline of 1st development.

UV-absorbing areas of the chromatography sheets were cutout and placed in counting vials, phosphor solution was added(4 g PPO and 0.1 g POPOP per liter of toluene), andradioactivity measurements were made at 72% countingefficiency.

Results presented below are measurements of the amountsof radioactivity in each metabolite, expressed as nmoles/g ofcells, rather than the total amount of each metabolite. Averagevalues from duplicate samples are reported. The results arerepresentative of those obtained in at least 2 experiments.

RESULTS

Factors that are rate limiting for the conversion of inosinateto adenylate and guanylate were studied first. The datapresented in Chart 1 show that Ehrlich ascites tumor cells invitro converted@@ C more extensively toadenine nucleotides (including NAD) than to guanine

_0 30 60 90

MINUTES

Chart 1. Relative incorporation of anin4 C into adenineand guanine nucleotides. Ehrlich ascites tumor cells, 2% by volume,were incubated in 25-mi Erlenmeyer flasks at 37°with shaking with anatmosphere of air in 5.0 ml of Krebs-Ringer medium containing 25 mMsodium phosphate buffer, pH 7.4, and 5.5 mM glucose. After 20 mm,hypoxanthine-' 4C was added to final concentrations of 5 MM(o, i@)or100 jsM (., £).At various times, portions were removed for analysis ofradioactivity in adenine nucleotides (., o) and guanine nucleotides (a,a').Each point representsthe mean of separateanalyse@of duplicate

flasks in I experiment; the results are representative of those obtainedin 4 experiments.

LIw

I-.

0LU-jL):@z

0

U.

0

zLU

L)LUa-

90

60

30

J.

@ I

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Purine Ribonucleotide Interconversions

nucleotides (plus xanthylate). With a low concentration ofprecursor (5 pM), the ratio of incorporation into adeninenucleotides relative to that into guanmne nucleotides wasapproximately 3 after 30 mm incubation, whereas this ratiowas approximately 6 at the same time when the initialextracellular concentration of hypoxanthine was 100 jiM.

Further studies were conducted to identify the rate-limitingsteps and factors on the pathway of guanylate synthesis frominosinate. Inosinate or xanthylate might be expected toaccumulate if inosinate dehydrogenase or xanthylate aminase,respectively, were rate limiting. However, Chart 2 shows thatthe concentrations of radioactive inosinate and xanthylateremained very low throughout the incubation period.

Because nucleotides might be dephosphorylated if theybegan to accumulate, the formation of nucleosides and basesby cells incubated with hypoxanthine-'4C was measured.After 90 mm of incubation, 1190 nmoles/g cells of radioactivexanthosine plus xanthine accumulated under these conditions;this amounted to almost 30% of the total acid-solubleradioactivity present in the sample. It will be shown belowthat the main pathway of xanthosine and xanthine synthesisfrom hypoxathine in these cells is via xanthylate rather thanby xanthine oxidase action on hypoxanthmne. Theseobservations both imply that significant amounts ofxanthylate were very readily dephosphorylated. Xanthylateaminase, therefore, appears to limit the conversion of inosinateto guanylate under these conditions.5

It thus became apparent that the flow of radioactivecompounds along the mnosinate-guanylate pathway could notbe accurately estimated by measurements of radioactivenucleotides only. Chart 3 shows the relative distribution ofradioactivity in “adeninecompounds― (adenine nucleotides,NAD, and adenosine) and in “guaninecompounds― (guanmnenucleotides, xanthylate, xanthosine , guanosine, xanthine , andguanine) after incubation of cells with pothi4 C. At aprecursor concentration of S jiM the incorporation ofradioactivity into guanmne compounds was greater than thatinto adenine compounds, although the rate of incorporationinto guanine compounds decreased more rapidly than did theother process. After 90 mm of incubation, the ratio ofradioactivity in adenine compounds relative to that in guaninecompounds was about 1:3. When the initial extracellularconcentration of@ C was 100 jiM, the rates ofits incorporation into adenine compounds and guaninecompounds were similar for the first 30 mm of incubation. Atlater times, the rate of incorporation into adenine compoundsdecreased relative to that into guanine compounds.

The rate-limiting character of xanthylate aminasedemonstrated above might have been due to the total activityof this enzyme or to the concentration of another substrate ofthis reaction, glutamine. The effects of addition of this amino

S Measurements of radioactive inosine formation in cells incubated

with hypoxanthine-'@ C or@ C do not necessarily measure thepossible rate-limiting character of IMP dehydrogenase because inosinemay be synthesized by pathways alternative to dephosphorylation ofinosinate. Thus, inosine may be made directly from hypoxanthine bypurine nucleoside phosphorylase and from adenine via adenylate andadenosine. At the present time, the relative rates of the alternativepathways involved have not been evaluated.

a-LILU

I-0LU

LI

z

0I—U.

0I—zLULILUa-

20

15

10

5

0-

0 30 60 90

MINUTES

Chart 2. Formation of radioactive inosinate and xanthylate fromhypoxanthine-' 4C. Tumor cells were incubated as described in Chart 1with 100 @LMhypoxanthine-' 4C. Portions were removed at varioustimes for analysis of inosinate (.) and xanthylate (0).

Chart 3. Relative conversion of hypoxanthine-'@ C to “adëninecompounds― and “guaninecompounds― (see text). Tumor cells wereincubated as described in Chart 1 with 5 MM(o, @)or 100 @iM(., a)hypoxanthine-' 4C. Portions @reremoved at various times for analysisof adenine compounds (., o) and guanine compounds (a, @).

acid to incubation media on the synthesis of guaninenucleotides and of xanthosine plus xanthine were thereforestudied. The accumulation of radioactive xanthosine plusxanthine after 90 mm of incubation decreased from 1210nmoles/g in the absence of glutammne to 74 nmoles/g in thepresence of 2 mM glutamine. (These data also show thatalmost no xanthine is being formed via xanthine oxidaseactivity on hypoxanthine under these conditions.) As wouldbe expected, the incorporation of@ C intoguanine nucleotides was markedly increased in the presence ofglutamine (Chart 4).

Further information regarding the role of glutamine inregulating xanthylate aminase activity came from studies withdiazooxonorleucine, an antimetabolite of glutamine, and withmethionine sulfoximine, an inhibitor of glutamine synthetase.Chart S shows that the concentration of diazooxonorleucineused almost completely inhibited the synthesis of guanine

a-LI

0I—U.

0I-zLULILUa-

MINUTES

JULY 1971 987

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I I I

I I I

AdditionsIncubationtime(mm)Xanthine

plusxanthosine

(nmoles/g)None

Methioninesulfoximine30603060750

978960

1242

G. W. Crabtree and J. Frank Henderson

‘I)LU-I

0

C

225

150

75

0

0

) 30 60 90

MINUTES

Chart 4. Effect of glutamine on the synthesis of radioactive guaninenucleotides from hypoxanthine-'@ C. Tumor cells were incubated asdescribed in Chart 1 with 100 pM hypoxanthine-' 4C with (0) andwithout (.) 2 mM glutamine.

0‘I,LU

@1

0

C

0 30 60 90

MINUTES

Chart 5. Effect of diazooxonorleucine on the synthesis of radioactiveguanine nucleotides from hypoxanthine-' 4C. Tumor cells wereincubated as described in Chart 1 with 100 @M@ C with(0) and without (.) 35 @M diazooxonorleucine.

Table 1Effect ofmethionine sulfoximine on the synthesis of

radioactive xanthine plus xant ho sinefrom hypoxanthine-' 4C

Tumor cells were incubated as described in Chart 1 with 100 @Mhypoxanthine with and without 5 mM methionine sulfoximine.

1@

100

75

50

25

00oo@

The decrease in the amount of radioactivity found inxanthosine plus xanthine when glutamine was added was notequaled by the increase in the radioactivity found in guaninenucleotides under the same conditions. This discrepancy wasdue in part to a doubling in the amount of radioactiveguanosine plus guanine formed. However, this amounted toonly I to 2% of the total radioactivity involved. Another causeof the discrepancy was a marked decrease in the amount of

4 C converted to ribonucleotides (Chart 6).

This apparent decrease in hypoxanthinephosphoribosyltransferase activity may be caused by diversionof phosphoribosylpyrophosphate to the pathway of purinebiosynthesis de novo, which is still operating to some extenteven at 100 jiM hypoxanthine (4).

Chart 7 shows another alteration in purine metabolism uponthe addition of glutamine. A considerable amount ofradioactive inosinate, which in the absence of glutamine wouldhave been converted to xanthosine and xanthine, was in thepresence of this amino acid converted to adenine nucleotidesrather than to guanine nucleotides. Although the mechanism

, I

4200@

!@2800@1400@

00 30 60 90

MINUTES

Chart 6. Effect of glutamine on the utilization of nm4 C.Tumor cells were incubated as described in Chart 1 with 100 @Mhypoxanthine-' 4C with (o) and without (.) 2 mM glutamine.

1500

1000

500

0@ I I

C 30 60 90

MINUTES

Chart 7. Effect of glutamine on the synthesis of radioactive adeninenucleotides from hi4 C. Tumor cells were incubated asdescribed in Chart 1 with 100 @zMhypoxanthine-' 4C with (o) andwithout (.) 2 mM glutamine.

0‘I,LU-I0

C

nucleotides from@ C. The data in Table 1 showthat the formation of radioactive xanthine plus xanthosine wasalso increased when glutammne synthesis was inhibited bymethionine sulfoximine. (Glutamine completely overcame theeffect of this amino acid analog.)

988 CANCER RESEARCH VOL.31

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I I

0 30 60 90U

Adenine-'4Cconcentration

(NM)Incubationtime(mm)Metabolites

notcontaining adeninea

(% totalradioactivity)2010

307.214.25010

303.46.710010

302.5 5.5

Purine Ribonucleotide Interconversions

of this stimulation of adenine nucleotide synthesis will bediscussed below, this observation implies that in the presenceof glutamine total inosinate dehydrogenase activity was stillnot rate limiting for the conversion of inosinate to guanylate.Instead, the amount of inosinate available to this enzyme inthe face of increased adenylosuccinate synthetase activityappeared to be the more important regulating factor.

If adenylosuccinate synthetase did have a competitiveadvantage over inosinate dehydrogenase with respect toutilization of inosinate, then inhibition of the former enzymewith hadacidin ( 11) might alter this situation. Chart 8 showsthat, although this analog of aspartate did inhibit the synthesisof radioactive adenine nucleotides by more than 80%, therewas little or no stimulation of the synthesis of radioactiveguanine nucleotides. It would be expected, however, that mostof the product of the inosinate dehydrogenase reaction wouldaccumulate as xanthosine plus xanthine under theseconditions; after 90 mm of incubation, accumulation of theseproducts increased from 1254 to 1490 nmoles/g in thepresence of hadacidin. An increase in inosine formation, aswell as an apparent decreased utilization of@ C,were also observed in the presence of hadacidin. These changesmight be due, at least in part, to increased dephosphorylationof inosinate ; whether this increased dephosphorylation is dueto accumulation of inosinate consequent upon saturation ofinosinate dehydrogenase with this substrate, or simply todephosphorylation of increased amounts of inosinate withoutsaturation of inosinate dehydrogenase, is not clear.

The stimulation of radioactive adenine nucleotide synthesisfrom hypoxanthine-' 4C upon addition of glutammne toincubation media (Chart 6) was probably due to the rapidconversion of this amino acid to aspartate, a substrate ofadenylosuccinate synthetase; this process has previously beenshown to occur in these cells (5). Aspartate itself was thereforeadded to incubation media and its effects on

mn4 C metabolism were measured. Becausethecells are not very permeable to dicarboxylic amino acids (2),

high initial extracellular concentrations of aspartate were used,but the intracellular concentration attained has not beendetermined. Chart 9 shows that the addition of aspartateincreased the conversion of an4 C to adeninenucleotides almost 2-fold, whereas guanine nucleotidesynthesis was scarcely affected. The synthesis of xanthine plusxanthosine decreased from 1190 to 876 nmoles/g after 90 mmof incubation, suggesting again that inosinate was divertedaway from the pathway of guanylate synthesis in the presenceof aspartate.

Although radioactivity in adenylosuccinate was notroutinely measured in these experiments, preliminaryexperiments have shown that it does not appear to accumulatein the presence or absence of aspartate. Adenylosuccmnatesynthetase would appear therefore to be the rate-limiting stepfor adenylate synthesis from inosinate.

When@ C was used as substrate of nucleotidesynthesis in Ehrlich ascites tumor cells, almost 95% of thetotal nucleotide fraction was composed of adenine nucleotidesthemselves. Even when its conversion into bases andnucleosides not containing adenine was measured (Table 2), 89

MINUTES

Chart 9. Effect of aspartate on the synthesis of radioactive adenineand guanine nucleotides from hypoxanthine-' 4C. Tumor cells wereincubated 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 guaninenucleotides (a, is).

Table2Conversion ‘@ C into metabolites

not containing adenine°Tumor cells were incubated as described in Chart 1.

2100

0..%. 1400

LU

0

C700

1200 -

0800 -

LU-I

0

C 400@ -

0@-@

MINUTES

Chart 8. Effect of hadacidin on the synthesis of radioactive adenineand guanine nucleotides from hypoxanthine-' 4C. Tumor cells wereincubated as described in Chart 1 with 100 @Mhypoxanthine-1 4C with(0, t@) and without (5, a) 100 @g/ml of hadacidin. Portions were

removed at various times for analysis of adenine nucleotides (., o) andguanine nucleotides (a, ‘s).

a Inosinate, xanthylate, guanine nucleotides, hypoxanthine, inosine,xanthine, xanthosine, guanine, and guanosine.

JULY 1971 989

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1.

14 cIncubationconcentration

time InosinateXanthylate(NM)(mm) (nmoles/g)(nmoles/g)20

10 4.02.0305.03.050

10 7.52.53012.54.0100

10 2010302515Table

4Conversionofguanine-'4C intometabolitesnot

containing guaninea14

C Incubation Metabolitesnotconcentration

time containingguaninea(@zM)(mm) (% of totalradioactivity)20

105.6306.850

102.6302.7100

102.2302.4

G. W.CrabtreeandJ. Frank Henderson

to 97% of the total radioactivity was in adenine and relatednucleosides and nucleotides.

Table 3 shows that neither radioactive inosinate norradioactive xanthylate accumulate in cells incubated withadenine-'4C, but 221 nmoles/g of radioactive xanthosine plusxanthine accumulated after 90 mm of incubation with 100mM adenine-'4C. Other experiments have shown that thisaccumulation did not occur in the presence of addedglutamine and that the amount of radioactive guaninenucleotides was concomitantly increased. Some inosine andhypoxanthine were formed both in the presence and absenceof glutamine, but, because these could be formed via thedephosphorylation of adenylate as well as by that of inosinate,rate-limiting steps could not be evaluated.

An experiment was done with adenine-'4C to determinewhether any radioactive inosinate formed from adenylate wasconverted back to adenylate via adenylosuccinate synthet.ase.The conversion of radioactivity from adenine into inosine wasincreased from 74 to 102 nmoles/g after a 90-mm incubationof hadacidin. If we assume that hadacidin has no effect on thedephosphorylation of adenylate and deamination of adenosine, it may tentatively be concluded that the increasedamount of radioactive inosine was derived from inosinatewhich was not utilized by adenylosuccinate synthetase in thepresence of this inhibitor.

When Ehrlich cells were incubated in vitro withguanine-' 4C, less than 10% of the precursor was converted tocompounds that did not contain the guanine moiety per se(Table 4). (Xanthine, a possible catabolite of xanthylate, wasnot included by these figures because it may also be formed by

Table3Concentrations of radioactive inosinate and xanthylate

synthesized from @4CTumor cells were incubated as described in Chart

the action of guamine deaminase on the precursor guanine-'@ Cas well as by the catabolism of guanine nucleotides.)

It is apparent that the conversion of guanylate to adenylatetook place very slowly in these cells. Because neither imosinatenor hypoxanthine plus inosine accumulated under theseconditions, the rate-limiting step in this process appears to beguanylate reductase.

DISCUSSION

It is apparent from these studies that the flow of materialalong the various pathways of purine ribonucleotideinterconversion cannot be accurately gauged by measurementof radioactivity in the ribonucleotide intermediates andproducts only. The nucleosides and bases derived from thesecompounds may contain significant amounts of radioactivity,which may not only influence conclusions regardingidentification of rate-limiting steps but may also changemarkedly depending on experimental conditions.

The conversion of inosinate both to guanylate and toadenylate in cells incubated in this salts:glucose medium waslimited primarily by the intracellular concentrations of theamino acid substrates of these reactions, glutamine andaspartate, respectively. No firm evidence was obtained toindicate that inosinate concentrations ever rose to the pointwhere total inosinate dehydrogenase activity became ratelimiting. Instead, inosinate appeared to be eitherdephosphorylated or converted to adenylosuccinate andadenylate.

The results of supplementation with glutammne and theeffects of diazooxonorleucine and methionine sulfoximime arein agreement with previous studies with Ehrlich ascites tumorcells which showed that glutamine concentrations werelimiting for other processes as well (2—5,8). These results alsosupport previous suggestions (3) that aspartate concentrationsmight limit adenylosuccinate synthetase activity.

Although no evidence was obtained to indicate that totalactivities of inosinate dehydrogenase or of adenylosuccinatesynthetase were rate limiting, some evidence does support theidea that total activities of adenylate deaminase and guanylatereductase may be of greater regulatory significance.Unfortunately, this point may remain unclear until means arefound to distinguish between the dephosphorylation ofadenylate and that of inosinate.

Studies to evaluate the possible regulation of the enzymesof purine ribomucleotide interconversion by variation in theconcentrations of purine ribonucleoside di- and triphosphatesare being begun for further study of regulatory factors in thisintact cell system.

REFERENCES

1. Blakeley, R. L., and Vitols, E. The Control of NucleotideBiosynthesis. Ann. Rev. Biochem., 37: 201—224,1968.

2. Coles, N. W., and Johnstone, R. M. Glutamine Metabolism inEhrlich Ascites-Carcinoma Cells. Biochem. J., 83: 284—291, 1962.

3. Fontenelle, L. J., and Henderson, J. F. Sources of Nitrogen asRate-limiting Factors for Purine Biosynthesis de Novo in EhrlichAscites Tumor Cells. Biochim. Biophys. Acta, 1 77: 88—93,1969.

a Inosinate, adenine nucleotides, xanthylate, hypoxanthine, inosine,adenine, adenosine, and xanthosine.

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Purine Ribonucleotide Interconversions

Glutamine in Protein Synthesis by the Ehrlich Ascites Carcinoma.J. Biol. Chem., 222: 879—893,1956.

9. Randerath, K., and Randerath, E. Ion-Exchange Chromatographyof Nucleotides on Poly(ethyleneimine)cellulose Thin Layers. J.Chromatog., 16: 111—125,1964.

10. Santos, J. N., Hempstead, K. W., Kopp, L. E., and Miech, R. P.Nucleotide Metabolism in Rat Brain. J. Neurochem., 15: 367—376,1968.

11. Shigeura, H. T., and Gordon, C. N. The Mechanism of Action ofHadacidin. J. Biol. Chem., 237: 1937—1940, 1962.

12. Stadtman, E. R. Allosteric Regulation or Enzyme Activity.Advan. Enzymol., 28: 41—154,1966.

4. Henderson, J. F. Feedback Inhibition of Purine Biosynthesis inAscites Tumor Cells. J. Biol. Chem., 237: 2631—2635, 1962.

5. Henderson, J. F. Dual Effects of Ammonium Chloride on PurineBiosynthesis de Novo in Ehrlich Ascites-Tumor Cells in Vitro.Biochim. Biophys. Acta, 76: 173—180, 1963.

6. Hershko, A., Razin, A., Shoshani, T., and Mager, J. Turnover ofPurine Nucleotides in Rabbit Erythrocytes. II. Studies in Vitro.Biochim. Biophys. Acts, 149: 59—73,1967.

7. McFall, E., and Magasanik, B. The Control of Purine Biosynthesisin Cultured Mammalian Cells. J. Biol. Chem., 235: 2103—2108,1960.

8. Rabinovitz, M., Olsen, M. E., and Greenberg, D. M. Role of

991JULY 1971

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1971;31:985-991. Cancer Res   G. W. Crabtree and J. Frank Henderson 

in VitroRibonucleotides in Ehrlich Ascites Tumor Cells Rate-limiting Steps in the Interconversion of Purine

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