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.—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) ‘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 “c.p.m.―willbe used to mean counts @ Present address: Institut für expenimentelle Krebs per minute in the internal gas flowproportional counters used forschung, UniversitätHeidelberg. in this study. §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 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1954 American Association for Cancer Research. SCHMITZ et al.—Alternative Pathways of Glucose Metabolism. II 67 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.—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 20—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.—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.—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.