Alternative Pathways of Glucose Metabolism II. Nucleotides from The

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Alternative Pathways of Glucose Metabolism II . Nucleotides from the Acid-soluble Fraction of Normal and Tumor Tissues and Studies on Nucleic Acid Synthesis in Tumors*t

HANNS SCHMITZ4 VAN R. POTTER, ROBERT B. HTJRLBERT,@ AND DWAIN M. WHITE

(McArdie Memovial Laboratory, the Medical School, University of Wisconain, Madison, WI..)

The first paper (18) in this series on the alterna MATERIALS AND METHODS tive pathways of glucose metabolism established The present study has involved the measure the fact that radioactivity from glucose-i-C'4 was ment of the specific activities of the free 5' mono-, readily incorporated into the pentose moiety of the di-, and triphosphates of adenosine, guanosine, ribonucleic and desoxyribonucleic acids of Flexner cytidine, and uridine from the acid-soluble extract Jobling tumors in rats, and described the over-all of tumor tissue in relation to the specific activities distribution of radioactivity in the acid-soluble of the corresponding nucleotides that were oh and acid-insoluble fractions of tumor and liver tis tamed by chemical or enzymatic hydrolysis of the sue at various time periods. The acid-soluble frac nucleic acids from the same tissue samples, at tion of tissue contains an appreciable amount of specified time intervals after the injection of glu free nucleotides which are possible intermediary cose-1-C'4. The experimental plan corresponds compounds in the synthesis of the nucleic acids exactly to that described in the preceding paper; (6, 7, 8, 9, 13, 16). It was noted (18) that as the C'4 many of the radioactive samples that were used for disappeared from the acid-soluble fraction, the the over-all survey (18) were also used for the de nucleic acids of the acid-insoluble residue showed tailed study reported here, and the plating and the increases in radioactivity, but it was evident that counting procedures were the same.' only by the isolation of the individual nucleotides Chromatography of acid-solublefractions.â€”The acid-soluble in pure form could any information on nucleic acid extracts were prepared as previously described (18), and the synthesis from the possible intermediates be ob PCA2 extract was used for chromatography without previous tained. This task was greatly facilitated by con fractionation with barium or other procedures. The chromatog raphy was usually carried out by means of the mechanically comitant studies in this laboratory in which operated two-column system of anion exchange with Dower-i Cohn's methods (4) for nucleotide separations in the formate form in both columns, and with a device for have been adapted to the study of unknown mix automatically changing the concentration of eluant (9). The tures (9), and in which the isolation of a number of two columns employed are referred to as the formic acid sys tem and the ammonium formate system or as the type I and hitherto unrecognized nucleotides has been ac type II systems, respectively. They are described fully else complished (16). where (9). In using the two columns, selected fractions from the formic acid system were pooled and rechromatographed S A preliminary report has appeared (17). t This workwassupportedinpartby a grant(No.C-646) â€˜Much of the plating and counting was done by Mrs. from the National Cancer Institute, National Institutes of Dorothy McManus and Mrs. Edith Wallestad under the Health, United States Public Health Service, and in part by general supervision of Dr. Charles Heidelberger of this de an institutional grant (No. 71) from the American Cancer partment. This skilled assistance is gratefully acknowledged. Society. Throughout this paper â€œc.p.m.â€•willbe used to mean counts @ Present address: Institut fÃ¼r expenimentelle Krebs per minute in the internal gas flowproportional counters used forschung, UniversitÃ¤tHeidelberg. in this study. Â§Present address: Kemiska Institutionen, Karolinska S Abbreviations are as follows: PCA, perchioric acid; RNA, Institutet, Stockholm. nibonucleic acid; DNA, desoxynibonucleic acid; MP, mono phosphate; DP, diphosphate; TP, triphosphate; A, adenosine; Received for publication September 9, 1953. G, guanosine; I, inosine; C, cytidine; U, uridine. 66

on the ammonium formate system to increase the purity of than one nucleotide, and this heterogeneity is individual nucleotides before determining their radioactivity. noted on the chart on the basis of the results oh In the present work, the longer columns (1X20 cm.) were em ployed in combination with a 500-ml. mixing flask. This pro tamed by rechromatographing these peaks on the cedure improves the resolution obtained with the formic acid type II columns insofar as the components have column and yields less heterogenous fractions for rechro been identified. The complexity of the fractions of matography on the type II column. a given tissue extract is of course much greater As a preface to the extensive fractionation of the radio than is indicated in Chart 1, since this chromato active samples, the nature of the nucleotide mixture in the acid-soluble fraction of Flexner-Jobling rat carcinoma was gram gives no information about the compounds compared to that of several nontumor tissues, by means of that do not absorb ultraviolet light. It is evident, the formicacid system. however, from this chart that the tumor contains Nudeotid.. o@ained from nucleic acids.â€”The mononucleo a large number of different nucleotides, many of tides of the RNA2 were obtained by treating the mixed sodium nucleates (7) with 0.1 M NaOH (1.0 ml/iO mg) for 20â€”24 which have not been reported in tumor or in non hours at 88 C. to hydrolyze the RNA. The DNA was then pre tumor tissues prior to the present studies (16). cipitated by making the solution 0.1N with respect to HC1, For this reason it was important to note whether and the supernatant containingthe RNA nucleotideswas neu the nucleotide .pattern shown in Chart 1 was tralized and placed on the type I columns (TX1O cm. with unique for tumor, or for growing or actively syn 250-mi. mixing flask). Since this mixture of nueleotides is much simpler than that of the acid-soluble fractions, re thesizing tissues, and, accordingly, two nongrow chromatography was unnecessary. The precipitated DNA was ing tissues in addition to liver have been included @ washed with 0.01 N HC1, reprocessed several times with 0.1 N for comparison with the tumor. Chart represents NaOH at 800C. for 20 minutes, reprecipitated with HC1 a chromatogram for liver, while the results for each time as before, and finally washed with alcohol and dried. The mononucleotidesfrom the DNA were obtained by en brain and muscle are shown in Charts 3 and 4, re. zymatic hydrolysis with a combination of desoxyribonuclease' spectively. All these tissues were from tumor (14) and a phosphodiesterase (10, ii) obtained from snake bearing rats. venom.4 The latter enzyme was not entirely free from phos These chromatograms will not be described in phomonoesterase, since the ion exchange chromatograms detail, since their features are evident by inspec yielded a relatively large amount of nucleosides. The incuba tion mixtures were extracted with cold PCA at a final con tion. However, it may be noted that the nucleotide centration of 0.4 N, the perchlorate was precipitated as the pattern for liver from tumor-bearing rats shown in @ potassium salt, and the supernatant fluid at pH 6.8 was placed Chart is quite similar to that for normal liver (9) on type I columns that were identical with those used for the and that in both cases there is a significant peak RNA hydrolysates. The sample effluents as well as the water wash (200 ml.) was collected in 8-ml. fractions. These frac (labeled ADP-X on Chart @)that does not appear tions contained the nucleosides as well as a portion of all four in the chromatograms or rechromatograms for tu of the bases. Suitable fractions containing the nucleosides mor, brain, or skeletal muscle. This peak contains were then subjected to paper chromatography according to one or more derivatives of an adenosine polyphos Carter (8) with the use of the disodium phosphate isoamyl phate that are as yet not fully identified (9). The alcohol solventsystem.' Specific activities were calculated from radioactivity measurements on individual nucleosides or chromatograms for brain and tumor are quite sim nucleotides depending on the quantity available. ilar and resemble each other more than they re Identification of nucleotides.â€”The identification of the semble liver or muscle. Both apparently lack the individual nucleotides that were chromatographically sepa ADP-Xpeakthat is foundin liver,andbothpos rated was made on the basis of their positions on the type I and type U columns, their ultraviolet absorption spectra in sess the UDP derivatives that are not seen in the acid and in alkali, their content of total and acid-labile phos muscle chromatogram, which also lacks ADP-X. phorus, their apparent nibosecontent by the orcinolmethod, Since brain is certainly not a growing tissue, at the and other tests, all of which are adequately described else present there appears to be no correlation with where (9,16). growth processes insofar as these nucleotide pat RESULTS terns per se are concerned. However, the turnover Di4,tribzdion of free nucleoti4&, in acid-soluble of the nucleotides shown in these chromatograms extracta.â€”Chart 1 shows an ion exchange chro may be greatly different, as the isotopic studies in matogram of the acid-soluble extract of Flexuer the second section of this report will show. Jobling rat carcinoma as obtained from a formic Although not shown in Chart @,the livers from acid column. Several of the peaks contain more tumor-bearing rats also contained a yellow pig ment that is probably a fiavin nucleotide (9), and aThe DNA-sac was a commercial preparation obtained from General Biochemicals, Inc., Chagrin Falls, Ohio. that follows ADP on the type I chromatogram. This yellow peak was not apparent in the tumor 4 The phosphodiesterase was a partially purified prepare.- tion according to Hurst and Butler (10) obtained from Dr. chromatograms. Kivie Moidave. The acid-soluble fraction of skeletal muscle

5 We are indebted to Dr. Mary Edmonds and Dr. G. A. (Chart4)isshowntocontainaverylargeamount LePage for assistance in the operation of this method. of ATP plus the well known AMP, ADP, and

FLEXNER-JCBUNG CAR@NOMA I 1j

GMP @A0P

CHART la.â€”Formic acid chromatogram of the acid-soluble Reservoir: 4.0 formic acid at the beginning, changed as indi fraction of Flexner-Jobling rat carcinoma. cated; Fraction.: 100 drops per tube; Rate: 7-8 drops per mm @ The pooled tumor tissue weighed 12.2 gm. and was obtained ute. The upper line gives the ratio of the to [email protected] B,,. from four rats. was determined directly on the eluates and were read with Column: Dowex-1 formate, 1X20 cm.; Mixer: 500 ml.; water in the blank cell. -â€˜p1@X275 /4

cMpURIC 5.0 @â€˜ TPN GMP wpuMp ADP UR 4.5 cop xl x2

4.0 LIVER @RA1)

5.5

2@5

â€˜.5

I.0 .5 ] @ a 50 /50 (â€”TUbe 2 CHART 2aâ€”Formic acid chromatogram of the acid-soluble the beginning, changed to 4.0 u formic acid + 0.2 ii ammoni @ fraction of liver from tumor-bearing rats. urn formate at tube 101, to 4.0 st formic acid + 0.4 am The pooled liver tissue weighed 29 gm. monium formate at tube 222, and to 4.0 u formic a@d + 1.0 U Column operation as in Chart 1 except for the points at ammonium formate at tube 275. which the reservoir was changed. Reserroir: 4.0 formic acid at 68

1.6

I.E

.8 .4

2.5

at 2.0

UR cop CTP (lOP Alp OW UTP XI X2 URX3 â€˜.5

1.0

cv 250 350 JL 47@ CHART lb.â€”(legend is given with Chart liz)

cip uop ATP GTP UTP UR@3

.5 cv 250 359

CHART 2b.â€”(legend is given with Chart 2a)

IMP. In addition, small amounts of the cytosine stead of by lyoph.ilization (9) such a result would 5'-nucleotides are present in all three stages of be expected on the basis of similar experience with phosphorylation with the major portion as the the triphosphates in other samples. In view of the triphosphate. On the other hand, even with careful extremely high ATP/ADP ratio in this sample, it rechromatography of appropriate sections from is perhaps not surprising to find that the mono-. the chromatogram shown, significant amounts of and diphosphates of uridine and guanosine were GMF, GDP, liMP, UDP, or UDP derivatives not found, since the total amount of these nucleo were not obtained. However, the peak following tides is rather small. This explanationimplies that ATP probably contained UTP and GTP, because reversible equilibria exist among the various phos when rechromatographed on the type II column it yielded uridine and guanosine nucleotides in the phorylated nucleotides. Further evidence for this positions that correspond to the 5 -monophos idea is presented in the next section in which the phates. Since the peaks from the original column mono-, di-, and triphosphates of any given nucleo had been dried in vacuo under an infra-red lamp in tideliad approximately the same specific activity.

BRMN (RAT)

Gb@w, M@ ts@alp xix2U GP CIPAIP UP G1PUTP

CHART 3.â€”Formic acid chromatogram of the acid-soluble Reservoir: 4.0 U formic acid at the beginning, changed to fraction of brain tissue from tumor-bearing rats. The pooled 4.0 U formic acid + 0.2, 0.4, and 1.0 U ammonium formats brain tissue weighed 4.8 gm. at tubes 80, 151, had 201, respectively. Fraction. and rate Column: as in Chart 1 except 1X1O cm. Mixer: 250 ml.; as in Chart 1.

CHART 4.â€”Formic acid chromatogram of the acid-soluble as noted. The ebromatogram has been condensed by omitting a fraction of skeletal muscle from tumor-bearing rats. The section that was without significant peaks. The quantity of pooled muscles from hind quarters weighed 20 gm. Al? was so great that a peak with a special scale has been Column operation: as in Chart 1 except for reservoir changes inserted.

RATESOFINCORPORATIONOFRADIOACrIVITYINTO which probably contains glucose (8), has a higher @ FREE NuciixrrmEs INTONucuac specific activity than UDP at the 1-hour point but Acm Nucz@oemi@s has essentially the same radioactivity at later Free nucleotides.â€”Ateach of the time intervals points. Since we are dealing here with radioactive after the injection of glucose-i-C'4 the acid-soluble glucose as a precursor, these data are reasonably extracts of the tumors were fractionated on the explained in terms of the more rapid turnover of formic acid columns to give chromatograms analo the glucose pool per se than some of the products gous to that shown in Chart 1. The appropriate derived from it. The radioactivity in the UDP-X1 fractions were then rechromatographed on the which probably contains glucuronic acid (8), aznmonium formate columns to obtain (16) the in would also be derived from a product from glucose dividual mono-, di-, and triphosphates of adeno in addition to the ribose of the nucleotide. These sine, guanosine, cytidine, and uridine, as well as derivatives of the uridine nucleotides may be use ful in obtaining the specific activities of the meta TABLE 1 bolic pools of glucose derivatives that participate SPECIFIC AcTIVITIES OF ACID-SOLUBLE NucLEo'rIDEs in the reactions for which these nucleotides are The 5'-mono-,-di-, and tniphosphatesof adenosine,guano presumably coenzymes (8, 1@). sine, cytidine, and uridine were obtained from the acid-soluble Table 1 aLso contains data on uric acid and fraction of Flexner.Jobllngrat carcinomasat the indicatedtime inosinic acid isolated from the acid-soluble frae intervals after the administration of glucose-i-Câ€•.The data tions at several time points. The inosinic acid cor are given as cpm/pM. responded to the specific activities of the AMP, Houss arraa owcosz nuacrics CoMPouNDs1681518AMP ADP, and ATP at the 5-hour point, but was slightly less radioactive at 1@hours. Free uric acid ADP 1,470 980 650 285 did not contain measurable radioactivity at any of AlT1,880110GMP 1,4001,000 800600 610315 80050

GDP 2,470 610 470 860 195 880CMPGTP2,260 2,500650 620500 480340 180 ,100 CDP 2,150 1.000 640 500 cTP2,000520250IJML'1,9501 950650 670485

UDP 2,600 660 470 850 175IMP245UTP2,800 2,750690 780450 470340 320140 975 UricAcid 0 0 0 a Abbreviations as in text. CHAnT 5.â€”Specffic activities of the uridine derivatives from the acid-soluble fraction of Flexner-Jobling rat carci certain uridine derivatives and inosine monophos nomas at various times after administration of glucose-i-C'4. phate, and the purified sub-fractions were plated, 0 0 = UDP-X, â€¢ S = UDPX, counted, and their absorption spectra were ob i.:@ c =. UDP-X, Ã§@ c@ = UMP and UDP tamed. From these data the specific activities were derived from UDP-X,, -X,, and â€”X,(average). calculated and are reported m Table 1. The data for the uridine derivatives are given in Chart 5. At the time points studied. This result is in agreement all time points studied (1â€”18hours) the specific with studies on the free bases obtained by hydroly activities of the 5'-nucleotides of a given base were sis of the purine nucleotides of RNA. Moreover, the same irrespective of its stage of phosphoryla the purines and pyrimidines from the acid-soluble tion, and the UMP or UDP derived from the dif nucleotides at 1, 5, and 8 hours were also shown to ferent UDP derivatives had approximately the have insignificant amounts of radioactivity in same specific activity as the lIMP, UDP, or UTP counting periods up to 20 minutes. These results obtained from the same extract. The specific ac do not mean that no glucose carbon enters the tivity of the UDP derivatives as such, however, purine or pyrimidine bases but only that under the showed distinct differences. Chart 5 reports the conditions of these experiments at least, the rate of data for the three uridine derivatives, designated entry into the ribose moieties of the nucleotides as UDP-X,, -X2, and -X3, respectively, plus the greatly exceeds the entry into the bases. specific activity of the UDP moieties. From 1â€”1@ Nucleotidesfrom the nucleic aci&.â€”Chart 6 rep hours the specific activity of the UDP-X1, which resents a typical chromatogram of the alkaline @ contains N-acetylglucosamine (8), was about X hydrolysate of a sample of tumor RNA. The @â€˜and higher than that of the IJDP moiety. The UDP-X,, 3'-monophosphates of adenosine, guanosine, and

7@ Cancer Research

cytidine were partially separated under the condi the nucleotides from non-nucleotide derivatives tions employed, while those of uridine did not sep of glucose. Further evidence on this point is arate. In the case of the first three nucleotides the the correlation between optical density and radio separations were sufficient to permit separate activity in different fractions of the same peak measurements of the radioactivity of the %â€˜and8' from a rechromatogram. The data in Chart 7 show monophosphates, and as would be expected (1) that the specific activities of the individual acid there was no difference in their specific activities. soluble nucleotides were very high relative to the specific activities of the mononucleotides obtained r RNA@MONONUcLEOflCESfrom the RNA between 1 and 5 hours, but between 8 and 18 hours the specific activities approached @ cT .U the same order of magnitude. Further studies will 4 rrs Cs Ã§ be required to determine whether the specific ac TABLE 2 SPEcIFIc AcTIvITIEs or TUE INDWIDu&L NUCLEOSIDES FROM THE DEsoxYRIBoNucLEIc ACID OF FLEXNER JOBLING RAT CARCINOMAS @ 10@@LThe compoundswere obtained at the indicated time inter vale after the administration of glucose-i-C.'4 The data are Cuaur 6.â€”Formicacid chromatogram of the mononucleo given as cpm/@tM. tides obtained by hydrolyzing the RNA from 23 mg. of the Houss AFTZRGLUCORSDUECTION mixed sodium nucleatee from Flexner-Jobling rat carcinoma. C0ILPOUND 1 6 8 18 18 Column: Dowex-1 formate, 1X1O cm.; Mixer: 250 ml.; Adeninedesoxyriboside 20 125 186 158 ReservoIr: 4.0 U formic acid throughout; FractIon.: 60 drops Guanine 25 185 145 159 per tube; Rate: 8 drops per minute. Cytosine 20 100 125 170 130 Thymine 85 160 230 180 3.0 S Some of the compounds were in the form of the deIOXy,ibOtides (see xK@ text), which would have the earns specific activitzea on the g@ basis. tivities of the nucleotides in the nuclear RNA parallel those of the corresponding acid-soluble nucleotides, as was found in concurrent studies in which the radioactive 5'-uridine nucleotides in the acid-soluble fraction of liver were rather closely paralleled by the radioactivity of the 8'-uridine monophosphate obtained from the nuclear RNA, @ I,[email protected] @L B while the total RNA showed no such correla tion (8). CHART 7.â€”Specific activities of acid-soluble nucleotides In addition to the nucleotides from RNA, the (opencircles)compared to correspondingnucleotidesobtained by hydrolysis of RNA (closed circles) from Flexner-Jobling DNA nucleotides(or nucleosides)wereobtained, rat carcinomas at various times after administration of and their specific activities are reported in Table @. glucose-i-C'4. The letters A, G, C, and U stand for adenosine, They are not plotted in Chart 7, because the latter guanosine, cytidine, and uridine phosphates, respectively. data are for ribose nucleotides, and no desoxy ribonucleotides have been found in the acid-soluble Chart 7 presents the results of the measurements fraction of the tumor or normal tissues examined of the specific activities of the @â€˜and8'-nucleotides thus far. The specific activities of the desoxyribo obtained by hydrolyzing the RNA, in relation to nucleotides are consistently lower than those of the the corresponding 5-nucleotides from the acid corresponding ribonucleotides, and the data are soluble fraction of the same tissue samples. The not incompatible with a formation of desoxyribo latter data have been averaged for the three levels tides from ribotides, a possibility recently suggest of phosphorylation of any given nucleotide at each ed by Rose and Schweigert (15). time point as given in Table 1, since the level of In the preceding paper (18) it was reported that phosphorylation did not affect the specific ac the bases of the nucleotides from RNA did not tivity at the time points studied. Since the contain significant amounts of radioactivity, and mono-, di-, and triphosphates come off the col it was assumed that the sugar moiety was the umns at widely separated points, the agreement labeled part of the molecule. This point has been between their specific activities is a good in-. tested directly with aliquots of the RNA of tumors dication that the two-column system separates taken 5 and 8 hours after the glucose injection

(compare Chart 7). The RNA was hydrolyzed in tribution of the various acid-soluble nucleotides, an 0.1 N HC1 for 80 mm. at 100Â°C.and the ribose examination of the C'4 distribution in the non phosphate from the purines was chromatographed nucleotide components of the acid-soluble fraction, on a monochioracetate Dowex-1 column according and a study of the distribution of C'4 among the to Groth et at. (5).6 The specific activity of the individual carbons of the ribose, desoxyribose, and ribose phosphates so obtained was @3Oand @90 glucose-6-phosphate molecules, following adminis cpm/pM, respectively, which is in good agreement tration of labeled glucose. with the average of the specific activities of the adenosine and guanosine monophosphates isolated SUMMARY as such from the RNA at these time points (Chart :i. At various time intervals of from 1 to 18 7). While the method (5) was described for 5'- hours following the injection of a single dose of glu ribose phosphate, the present work showed that it cose-i-C'4 into rats bearing Flexner-Jobling car is also applicable to the @â€˜-and3'-ribose phos cinomas, the tumor tissues from several rats were phates and that the latter are partially separated pooled and fractionated to give acid-soluble and from each other. Thus, the ribose was established acid-insoluble components. as the principal locus of the radioactivity under @. The acid-soluble components were subjected the conditions of these experiments, in which the to extensive chromatography and were shown to acid-soluble nucleotides as well as the nucleic include the cytidine-, uridine-, guanosine-, and acids have been shown to become radioactive after adenosine-5'-mono-, di-, and triphosphates, as well a single injection of glucose-i-C'4. as inosine monophosphate, uric acid, and three derivatives of uridinediphosphate, and the specific DISCUSSION activities of these compounds were determined at The data presented in this paper have not in the various time intervals. cluded isotopic studies on liver, mainly because the 3. The ribose- and desoxyribosenucleic acids amount of radioactivity was very low in the RNA were separated from the acid-insoluble fraction and nucleotides of liver and could not be demonstrated hydrolyzed to give the mononucleotides, and the in the DNA (18). Moreover, at 1 hour, in contrast specific activities of the latter were determined. to the high radioactivity in the adenosine-5'-phos 4. The radioactivity of the RNA nucleotides phates of tumor (Chart 7), there was no demon was shown to be located principally in the ribose strable radioactivity in these compounds in liver. moiety. The latter result may indicate that slowly growing 5. The acid-soluble ribonucleotides contained or nongrowing tissues do not have as high a rate of between 1,880 and @,800cpm/@zMat 1 hour, at pentose turnover in the acid-soluble nucleotides as which time the nucleic acid nucleotides contained do growing tissues, and may explain why Brown very little radioactivity. By about 8 hours the et at. (@)found so little incorporation of labeled values had all approached the range of about @00 adenine in the ATP of skeletal muscle. Further to 600 cpm/@. studies on the labeling of the acid-soluble nucleo 6. Since the free adenine nucleotides of the tides of nontumor tissues are in progress. livers of the tumor-bearing animals contained no Since the total acid-soluble fraction of liver was demonstrable radioactivity at 1 hour, while the as radioactive as that of tumor, but contained no total acid-soluble radioactivity of the livers was radioactivity in the adenine nucleotides at 1 hour, about the same as that of the tumors, further when the tumor nucleotides had maximal radio study of the non-nucleotide radioactivity of the activity (Chart 7) it would be of considerable in acid-soluble fraction may indicate how the flow of terest to see how the radioactivity was distributed glucose to the pentose moieties of the nucleotides among the non-nuckotide derivates of glucose that is regulated. are in the acid-soluble fractions of liver, regenerat REFERENCES ing liver, and tumor at the various time intervals. i. Baow@, D. M., and TODD,A. R. Nucleotides. X. Some Such a study might indicate how the alternative Observations on the Structure and Chemical Behavior of pathways of glucose metabolism are regulated the Nucleic Acids. J. Chem. Soc., pp. 52â€”58,1952. with respect to nucleotide synthesis. 2. Bnow@, G. B.; ROLL,P. M.; Px@mim,A. A.; and CAVA It would be premature to discuss the implica use.', L. F. The Utilization of Adenine for Nucleic Acid Synthesis and as a Precursor of Guanine. J. Biol. tions of the present work until certain additional Chem., 172:469-84,1948. studies are carried out in a variety of growing and S. C@4ai@za,C.B. Paper Chromatography of Purine and nongrowing tissues, including a survey of the dis Pyrimidine Derivatives of Yeast Ribonucleic Acid. I. Am. Chem. Soc., 72:1466-7i, 1950. S The monochioracetate column should be freshly pre 4. ConN, W. B. The Anion-Exchange Separation of Ribo pared (personal communication from the authors). nucleotides. J. Am. Chem. Soc., 72: 1471-78, 1050.

5. Gaom, D. P.; Muiiumi, G. C.; and LEPAGE,G. A. Ion W. D. Mc&aoy and B. Gi@ase (ads.), Phosphorus Exchange Purification of Ribose-5-Phosphate. J. Biol. Metabolism, 1:67â€”OS.Baltimore:The Johns Hopkins Chem., 199:880â€”91,1952. Press, 1951. 6. EDMONDS,M., and LEPAGFI,G. A. In Vivo Studies on 13. LZPAGI@, G. A. In Vitro Incorporation of Glycine-2-C'4 Acid-Soluble Precursors of Nucleic Acid Purines. Fed. into Purines and Proteins. Cancer Research, 13:178-85, Proc., 12:190, i953. 1053. 7. HUELBFIRT,R. B., and Po!rrnt, V. R. A Survey of the 14. Lim.z, J. A., and Btm@za,G. C. The Enzymatic Degrade.-. Metabolism of Orotic Acid in the Rat. J. Biol. Chem., tion of Thymonucleic Acid. I. The Preparation of the 195: 257â€”70,1952. Oligonucleotides. J. Biol. Chem., 188: 605-794, 1951. 8. . Nucleotide Metabolism. I. The Conversion of 15. ROSE,1. A., and SCHWEIGERT,B.S. Incorporation of Câ€• Orotic Acid-6-C'4 to Uridine Nucleotides. .7. Biol. Chem. Totally Labeled Nucleosidesinto Nucleic Acids. J. Biol. (inpress). Chem., 202:685â€”45,1958. 9. HURLBFIRT,R.B.; Scmin@z,H.; Bau@, A.; and Po@mt, 16. ScmsxTr, H.; HuainsmT, R. B.; and Porrza, V. H. V. R. Nucleotide Metabolism. II. Chromatographic Nucleotide Metabolism. III. Mono-, Di-, and Triphos Separation of Acid-soluble Nucleotides. J. Biol. Chem. phates of Cytidine, Guanosine, and Uridine. 3. Biol. (in press). Chem. (in press). 10. HVBST,R. 0., and Bums@a,G. C. The Chromatographic 17. Sciwm, H.; Po@rrua,V. H.; and Huiu.nmrr, R. B. Separation of Phosphatases in Snake Venoms. J. Biol. Alternative Pathways of Glucose Metabolism in Tumor Chem., 193:91â€”96,1951. Tissue. Proc. Am. Assoc. Cancer Research, 1:47-48, 11. Hune@r,R. 0.; Ln'ri@, J. A.; and BUmER, G. C. The 1958. Enzymatic Degradation of Thymonucleic Acid. U. The 18. . Alternative Pathways of Glucose Metabolism. L Hydrolysis of Oligonucleotides. J. Biol. Chem., 188: Distribution of Radioactivity from Glucose-i-C'4 in 705â€”15,1951. Acid-soluble and Acid-insoluble Fractions of Tumor and 12. Lsiwm, L F. The Metabolism of Hexose Phosphates. In Normal Tissue. Cancer Research, 14:58â€”65, 1954.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1954 American Association for Cancer Research. Alternative Pathways of Glucose Metabolism: II. Nucleotides from the Acid-soluble Fraction of Normal and Tumor Tissues and Studies on Nucleic Acid Synthesis in Tumors

Hanns Schmitz, Van R. Potter, Robert B. Hurlbert, et al.

Cancer Res 1954;14:66-74.

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