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Isolation of Acid-soluble from Rat Tissues and Measurement of Their Specific Activity Following P32Administration*

R. ÖAOusTfANDA. CANTERO

(Montreal Cancer Institute, Notre-Dame Hospital, Montreal, Canada.)

The isolation of free nucleotides from animal liver, intestinal mucosa, and liver tumors, thus tissues has recently been reported from different supporting the hypothesis that nucleic acids may laboratories (15, 16, 22-26, 28, 29). Hurlbert et al. be built up from a supply of preformed nucleotides (16) and Schmitz et al. (25, 26), in particular, have (1,14, 21). Following administration of P32,its in demonstrated that rat tissues contain several free corporation into free nucleotides was found to be nucleotides which, by extensive analyses, have relatively rapid and seemed to proceed at similar been identified as 5'-mono-, di- and tri-phosphates rates in the different tissues. In contrast, the up of , , , and (or take of P32by nucleic acids differs from one tissue derivatives). Precursors of the or pyrimi- to another and, in the case of DNA, parallels the dine moiety, glycine-2-C14 (12) and orotic acid-6- incidence of mitoses (see references in [9]and [31]). C14 (15), as well as a precursor of the Thus, if free nucleotides are considered as pre moiety, glucose-1-C14 (26), were found to be rapid cursors of , the radioactivity data ly incorporated into acid-soluble nucleotides, as would suggest that the rate-limiting step in the compared with their rate of entry into polynucleo- synthesis of nucleic acids is not the formation of tides. These studies strongly support the view that the free nucleotides from smaller precursors but acid-soluble nucleotides are intermediary com rather the building up of nucleic acids from indi pounds in the biogenesis of nucleic acids. vidual nucleotides. A similar type of work has been carried on in dependently in this laboratory. The aim of this MATERIALS AND METHODS work was twofold: (a) to determine whether a pool Preparation of tissues.—Groupsof adult, male, albino rats of nucleotides exists in tissues and (6) if present in (180-250 gm.) were sacrificed, under ether anesthesia, by appreciable amounts, to compare their rate of P32 exsanguination from the abdominal aorta. The livers were excised with scissors and placed in a beaker chilled on ice. uptake with that of nucleic acids. The method de The pooled livers were forced through a Plexiglas squeezer, vised to extract the acid-soluble nucleotides from and 45-62 gm. of liver pulp was obtained according to the tissues differed from the one of Hurlbert et al. (16), number of animals (six to eight) used in each experiment. but the results obtained by both the methods es Strands of connective tissue remaining in the squeezer were discarded. In experiments on the intestinal mucosa, groups sentially agreed. Several free nucleotides and of seven to nine animals were sacrificed. The small intestines derivatives were found to be present in normal rat were excised, cut open lengthwise, and flattened out on blotting * This work was supported by a grant from the Damon paper. The intestinal content was wiped away with absorbent Runyon Memorial Fund for Cancer Research, Inc. A pre cotton and water. The mucosa was removed by delicate liminary report was presented before the American Association scraping with a scalpel, the visible lymphatic accumulations for Cancer Research (8). and Peyer patches being excluded (SO). Twelve to 14 gm. t This investigation was conducted during tenure of a of mucosa were so collected per group of animals. The liver Damon Runyon Cancer Research Fellowship. tumors were obtained from rats fed for 150 consecutive days At present on leave at the Chester Beatty Research Insti a semisynthetic diet (réf.17,diet 3) containing 0.06 per cent of 4-dimethylaminoazobenzene, followed by a 30-day interval tute, Royal Cancer Hospital, London, as Exchange Fellow of the British Empire Cancer Campaign. of a diet of Purina Fox Chow. The whole livers, with tumor Abbreviations are as follows: P32, radiophosphorus; A, masses, were removed and placed in a beaker kept on ice. ; G, ; U, ; T, ; C, ; Small, firm tumor masses (less than 1 cm. in diameter) of a I, ; MF, monophosphate; DP, diphosphate; TP, tri- light buff color were excised with scissors. Samples were taken with a scalpel and immediately fixed in Susa fluid for histo- phosphate; DPN, diphosphopyridine ; TPN, triphosphopyridine nucleotide; RXA, ribonucleic acid; DNA logical sections. The remaining masses from three animals were deoxyribonucleic acid; TCA, trichloroacetic acid. pooled, and 17 gm. of tissue pulp was obtained by mincing with a Plexiglas squeezer. The samples fixed in Susa were de Received for publication July 14, 1955. hydrated and embedded in paraffin. Sections were prepared 734

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1955 American Association for Cancer Research. DAOUSTANDCANTERO—Acid-solubleNudeotidesin Rat Tissues 735 by the usual procedure and stained with hematoxylin and RESULTS AND DISCUSSION eosin. Microscopic examination revealed that the tumors were Chromatographie separation of known nucleo free of surrounding tissue but that inclusion of small necrotic tides.—The Chromatographie separation of ribo- regions in the analyzed material could not be avoided. The tumors were mixed hepatoma and cholangioma. (5) or (27, 32) has been pre Extraction of acid-soluble compounds from tissues.—The viously described. Since both types of nucleotides tissue pulp was homogenized with 40 cc. of ice-cold 10 per cent were expected to be present in acid-soluble frac TCA in a Potter-Elvebjem type homogenizer. The precipitate tions of tissues, the separation of complex mixtures was separated from the supernatant by centrifugation and of ribo- (-2'- and -3'-phosphates) and de washed twice with 30 cc. of ice-cold 10 per cent TCA. The washed precipitate was discarded. The supernatant fluids oxyribonucleotides (nucleoside- 5'-phosphates) has were combined (total volume: 100 cc.) and filtered. A 10-cc. been investigated. Mixtures of known nucleotides sample of this solution (acid-soluble fraction) was used for the were prepared and fractionated as described separation of inorganic and organic phosphorus (10). The TCA was removed from the remaining 90 cc. by repeated extractions above, the position of individual nucleotides on with 50-cc. volumes of ether. The ether extracts were dis chromatograms being ascertained by determining carded. The pH of the solution was determined after each step the spectral properties of several fractions of the and, after twelve extractions, was found to be about 6. At this effluent. After fractionating a few simple mixtures, stage, the solution was neutralized with 0.1 N NaOH solution. Separation of acid-soluble nucleotides by ion-exchange systems of increasing complexities were analyzed. chromatography.—The neutralized solution was passed, by A typical chromatogram of a complex system is gravity flow, through an anion-exchange column (Dowex- shown in Chart 1, to be used as reference in subse 1,200-400 mesh, Cl~ form, 1 X 12 cm.), prepared according quent studies. The position of inorganic phospho to Cohn (5). The adsorbed substances were eluted by using rus was determined by adding to the original mix HC1 solutions of increasing normalities (5, 6), the elution ture 10 fie of P32and 3 mg. of inorganic phosphorus proceeding at a rate of about 1 cc/min. The effluent was collected in 20-cc. fractions with the help of an automatic as carrier (Anachemia Standard Phosphate solu collector. To follow the elution of the nucleotides, the optical tion) and by measuring the radioactivity of the density of each fraction was determined at 260 mji with a Beck- successive fractions of the effluent. man, Model DU, spectrophotometer. Identification of the acid-soluble nucleotides.—The isolated Separation of nucleotides from acid-soluble frac tions of tissues.—The chromatograms obtained by nucleotides were identified by reference to known nucleotides, according to their position in chromatograms and their fractionation of acid-soluble fractions of normal spectral properties in ultraviolet light. Ultraviolet absorption rat liver, intestinal mucosa, and liver tumors are curves were determined for several fractions within each peak shown in Charts 2, 3, and 4, respectively. The of eluted material. Radioactivity measurements.—In experiments involving the spectrophotometric data are assembled in Table 1. use of P32, 200 pe. of the radioisotope were administered per Peaks corresponding to the 5' CMP, AMP, UMP, animal by subcutaneous injection. The animals were sacrificed and GMP (Chart 1) are evident in all chromato at given time intervals after the injection of P°, and the acid- grams, but the 2' and 3' shown in soluble nucleotides were isolated from tissues as above. To follow the elution of phosphorus compounds other than nucleo Chart 1 have no corresponding peaks in chromato tides, a 0.5-cc. sample of each fraction of the effluent was grams of tissues. The nucleoside phosphates of tis placed on a 20-mm. watch-glass, dried on a hot plate, and sues thus seem to be in the 5' form, in agreement counted with a Geiger-Müller tube and a Berkeley decimal with results of Schmitz et al. (25), and sealer (Model 2000). An elution curve of radioactive material 2' and 3' phosphates as obtained by hydrolysis of was then traced and compared with the elution curve of ultra ribonucleic acid would not exist as such in tissues. violet-absorbing material. The peaks in radioactivity which The 5' MP peaks were analyzed by the method of did not coincide with peaks of ultraviolet-absorbing substances thus represented acid-soluble phosphorus compounds other Dische (11) and Ceriotti (4) for the presence of than nucleotides. deoxyribonucleotides, but no definite proof was The specific activity of the isolated nucleotides was meas obtained. If deoxyribonucleotides are present, they ured on the fractions of the effluent previously characterized by their spectral properties. These fractions were reduced to probably make a relatively small proportion of about 1-2 cc. by boiling and were digested with 0.5 cc. of con these peaks. The latter would be chiefly composed centrated sulfuric acid. The digested solutions were completed of ribonucleotides in the 5' form which, in chro to 5 cc. with distilled water. The phosphorus content of each matograms, occupy the same positions as the solution was determined on a 2-cc. aliquot by the method of standard (5') deoxyribonucleotides (Chart 1). Fiske and Subbarow (13). For radioactivity measurements, triplicate plates were prepared with 0.5-cc. aliquots of the and DPN were eluted close to CMP, and same solutions and counted as above. The specific activity indications were obtained that small amounts of of each solution was computed as counts/100 sec//ig P. The other compounds were eluted between CMP and total activity of the injected dose of P32 was calculated from measurements made on suitable dilutions of a standard P32 AMP (nos. 4 and 5, Charts 2 and 3). The compo solution, and the results were expressed as percentages of in sition of peak no. 8, eluted next to UMP, has not jected dose/Mg P X IO-4, the figure for the injected dose being yet been clarified. This peak was constant in ap adjusted for 200 gm. of body weight. pearance, but its spectral properties showed impor-

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1955 American Association for Cancer Research. 736 Cancer Research

tant variations in different experiments (Table 1, pounds, not yet identified, were eluted after UX4. no. 8). Part of this peak may consist of a cytosine In normal liver, one of these peaks showed charac derivative—probably CDP (25)—mixed with a teristics of a guanine derivative (GX4, Chart 2) guanine derivative. A constant, and not hitherto and is presumably GTP (25). reported, feature of these fractions was a maxi The nucleotide patterns obtained from normal mum at 230-232 m/u in their ultraviolet spectrum. rat liver, intestinal mucosa, and liver tumors This feature may indicate the presence of an addi showed several common features which suggest tional compound or a special structure in the nu- that, qualitatively, the free nucleotide composition cleotides already mentioned. Following the elution among these tissues is much alike. Intestinal mu of compound no. 10 (Chart 2), which was not con cosa showed only minor differences from normal stant in appearance, guanine derivatives were iso liver, and the fact that compounds nos. 10, GXi, lated from normal rat liver (GXi, Chart 2) and and 6X4 were not evident in chromatograms of liver tumors (GXi and GX2, Chart 4). Whether intestinal mucosa may be owing to the smaller

CHROMATOGRAM OF KNOWN NUCLEOTIDES

a. i O IO CM

250 300 350 4OO FRACTION No.

CHAKT1.—Ion-exchange separation of known nucleotides. in another series. The peak shown in broken lines indicates The peaks shown in heavy lines were obtained in the same tïïëpositionof inorganic phosphorus as determined by radio experimental series; those shown in lighter lines were obtained activity measurements (see text).

these compounds represent derivatives of amounts of starting material used in these experi guanosine phosphates (2) was not determined. ments. In liver tumors, uracil derivatives, UXi, Uracil derivatives UXi and UXz, eluted from nor UXj, and UX3, were not found, while the presence mal liver and intestine (Charts 2 and 3), probably of additional guanine derivatives, GXs and GXs, correspond to UDP sugar derivatives (3, 18-20, was evident. Further experimentation is necessary 25). The compound AX i is possibly an ADP deriva to determine whether these differences are con tive (26) eluted before standard ADP. Additional stant in liver tumors and whether they are related uracil derivatives, TJXs and UX4, eluted close to to parenchymal cells or other tissue elements (7). ATP, may correspond to UDP and UTP, respec Specific activity of acid-soluble nucleotides follow tively (25). The latter peaks showed much over ing administration of P32.—In all series of experi lapping, however, and their spectral properties ments, the elution of radioactive material was fol (Table 1, nos. 17,19, 20) gave intermediary values lowed by measuring the radioactivity of the suc between those of adenylic and uridylic acids. The cessive fractions. The radioactivity curve paral same peaks might also contain small amounts of leled the absorption curve in the major part of CTP and GDP (25). Actually, a guanine deriva each chromatogram. In the region of AMP and tive (GX3, Chart 4) occupied the position of UX3 UMP, however, peaks of radioactivity were pres in chromatograms of liver tumors. A few corn- ent which did not coincide with those of sub-

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•s -& '—I—"~ too 150 2OO 25O 300 4OO 45O soo 55O FRACTION No. CHART2.—Ion-exchangeseparation of acid-soluble nucleo- were obtained in the same experimental series; those shown in tides from normal rat liver. The peaks shown in heavy lines lighter lines were obtained in other series.

ACID-SOLUBLE NUCLEOTIDES OF RAT INTESTINAL MUCOSA

T IOO 150 2OO 25O 3OO 350 4OO 45O FRACTION No. CHAHT3.—Ion-exchangeseparation of acid-soluble nucleotides from rat intestinal mucosa. Light and heavy lines as in Chart 2

ACID-SOLUBLE NUCLEOTIDES OF RAT LIVER TUMORS

100 I5C 200 260 300 FRACTION No. CHART4.—Ion-exchangeseparation of acid-soluble nucleotides from rat liver tumors. Light and heavy lines as in Chart 2

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1955 American Association for Cancer Research. DAOUSTANDCANTERO—Acid-solubleNucleotidesin Rat Tissues 739 stances absorbing ultraviolet light, thus indicating lower than those of the whole fraction at corre that phosphorus compounds other than nucleo- sponding time intervals (10.78 and 11.81). These tides were eluted in that region. These substances differences diminish after 6 hours, and, by 12 probably include organic phosphorus compounds hours, free nucleotides, like other acid-soluble or as well as inorganic phosphorus, since they con ganic compounds, reach an equilibrium with in stantly spread over a larger number of fractions organic phosphorus. By that time, their average than inorganic phosphorus alone (Chart 1). Their specific activity was similar to that of the whole elution curve generally overlapped the AMP and acid-soluble fraction (Table 2), that is close to that UMP peaks, thus contaminating the latter sub of either organic or inorganic phosphorus (30). Al stances. The other nucleotides, however, appeared though the free nucleotides showed a slower rate of free of nonabsorbing phosphorus compounds. P32 uptake than other acid-soluble organic com In rat liver, the specific activity-time curve of pounds, they may be considered as being rapidly the whole acid-soluble fraction was similar to the labeled also, if comparison is made with nucleic

TABLE 2 SPECIFICACTIVITIES*OFACID-SOLUBLENUCLEOTIDESATVARIOUSTIME INTERVALSAFTERINJECTIONOFRADIOPHOSPHOHUS

LIVER1 nimShr.3.646.10t COMPOUND hr.6. hr.5.296.67t hr.7.345.20t CMP Sit DPN 6.40J 6.20t AMP 13.61J 11.19t UMP 14.11t 12.44J 10.32t 5.22J 7.07t 14.12J 7.00J 6.83J 8 5.05 7.40 10.37t 9.59 9.23t 8.29t 4.51 4.35 GMP 2.808.856.834.805.086.215.758.444.126.02Shr.11.37t16.53t4.716.885.527.5711.767.658.497.007.8810.007.38«hr.5.615546.768.767.879.9111.354.959.635.94125.476.917.56Ihr.2.799.66t3.924.484.703.571.315.341.34INTESTINALMI5.704.387.937.375.851*2.915.512.394.946.985.20LlVEBTUMORSShr.2.257.12t5.545.508.285.005.044.895.755.404.94 GX, GX, UX, UX, AX, ADP UX, GX3 ATP UX4 21 22 23 GX« 25 4.38 5.81 7.68 7.41 6.98 3.43 5.81 4.97 4.69 Whole acid-sol. 13.18 14.13 12.93 6.64 4.70 5.73 5.82 7.72 * Expressed as percentage of the injected dose per microgram ol P«X 10"', the figure (or the injected dose being adjusted for ÕOOgm.of body weight. t Contaminated by phosphorus compounds other than nudeotides. ÕExcluding the values for contaminated nucleotides.

one previously reported (30), although the value acids, which, in normal liver, reach activities of at the 3-hour interval was somewhat lower in the only 0.11 (DNA [30]) and 1.83 (RNA)1 after 24 present experiments (Table 2). Fractionation of hours. the whole acid-soluble fraction into inorganic and In the intestinal mucosa, the entire acid-soluble organic phosphorus gave specific activity values of fraction showed a specific activity of 4.70 (Table 17.95 and 19.24 for inorganic phosphorus at 1 and 2) at 1 hour after injection, and its inorganic and 3 hours, respectively, and corresponding values of organic parts had activities of 9.05 and 2.23, re 10.78 and 11.81 for organic phosphorus. This indi spectively. At the same time interval, the acid- cates a rapid labeling of the acid-soluble organic soluble nucleotides showed an average specific ac compounds which were shown to reach an equi tivity of 3.43 (Table 2), indicating that their rate librium with inorganic phosphorus by 12 hours of labeling is comparable to that of other acid- after injection (30). The acid-soluble nucleotides soluble organic compounds. At 3 and 12 hours, the are part of the organic fraction but seem to be less values obtained for the free nucleotides were close rapidly labeled than other organic phosphorus to those of the whole acid-soluble fraction (Table compounds, since their average specific activities 2). The acid-soluble nucleotides thus behave as do at 1 and 3 hours (5.81 and 7.68, Table 2) were 1R. Daoust and A. Cantero, unpublished data.

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1955 American Association for Cancer Research. 740 Cancer Research the whole acid-soluble organic fraction (30) and sues and that this pool rapidly equilibrates with in rapidly become equilibrated with inorganic phos organic phosphorus and other precursors favors phorus. A comparison of the intestine and liver the hypothesis that nucleic acids are built up from data reveals that the free nucleotides were labeled a supply of preformed nucleotides (1, 14, 21). Fur at similar rates in both tissues, as indicated by thermore, the finding that acid-soluble nucleotides their relative specific activities at 1 hour (ratio of incorporate P32 at comparable rates in normal the specific activity of the nucleotides over the liver, intestinal mucosa, and liver tumors, by con specific activity of inorganic phosphorus), which trast with the fact that P32enters nucleic acids at were of the same order of magnitude, namely, 0.38 rates which differ from one tissue to another, sug in intestinal mucosa and 0.32 in normal liver. In gests that, if the free nucleotides are actually pre contrast with liver nucleic acids, however, the in cursors of polynucleotides, the rate-limiting step in testinal DNA is known to incorporate P32rapidly, the biogenesis of tissue nucleic acids would not be owing to the high rate of cell formation in that the formation of free nucleotides from smaller pre tissue (30). cursors but rather the building up of polynucleo In liver tumors, the whole acid-soluble phos tides from individual nucleotides. Hence, the fac phorus had a specific activity of 7.72 at 3 hours tors which control synthesis in the after injection (Table 2). A possible explanation cells would be primarily those concerned with the for this value's being lower than the corresponding system, free nucleotides —»polynucleotides.This value in normal liver might be that the tumors system thus appears of particular interest in re show a slower exchange of inorganic phosphorus gard to the regulation of normal and abnormal with the plasma than does the normal liver. Simi growth processes (9). larly, the average activity of the acid-soluble nu cleotides was lower in the tumors than in normal SUMMARY liver, but, relative to the whole acid-soluble frac 1. A method was described to isolate free nucle tion in each tissue (Table 2), free nucleotides ap otides from acid-soluble fractions of tissues. peared to be labeled at comparable rates in both 2. This method was applied to normal rat liver, tissues. The nucleic acids, by contrast, incorporate intestinal mucosa, and liver tumors, and several P32 much more rapidly in tumors than in normal free nucleotides and derivatives were found to be liver, specific activities of 1.48 (DNA) and 3.58 present in appreciable amounts in these tissues. (RNA) having been obtained in tumors at 24 3. The incorporation of radiophosphorus into hours after injection.1 free nucleotides was observed to be rapid and to Some differences between the specific activities proceed at similar rates in normal liver, intestinal of the individual nucleotides were observed at each mucosa and liver tumors. interval studied (Table 2). As was expected, the These findings support the hypothesis that nu nucleotides contaminated by inorganic phosphorus cleic acids may be built up from a supply of pre constantly showed relatively high activities, espe formed nucleotides. Furthermore, since the nucleic cially at early intervals after injection, and were acids are known to incorporate radiophosphorus at excluded from the means. Whether the differences different rates in these tissues, the radioactivity between the other nucleotides were significant data suggest that the rate-limiting step in the bio could not be decided. Since these compounds were genesis of nucleic acids is the formation of poly found to be rapidly labeled with P32,as with pre nucleotides from individual nucleotides. cursors of the purine (12), (15), and sugar moieties (26), we feel that individual varia ACKNOWLEDGMENTS tions should be investigated at still shorter inter The authors wish to thank Mr. RenéLemayfor valuable technical assistance. vals (12) than the ones used in the present work. Moreover, such a study would necessitate further REFERENCES resolution of the individual peaks (16) to eliminate 1. ABRAMS,R.,and GOLDINGER,J.M. Utilization of cross-contamination of nucleotides. 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cleotides. J. Am. Chem. Soc., 72:1471-78, 1951. 19. . Uridine-5'- Derivatives. II. A 6. . Some Results of the Applications of Ion-exchange Structure Common to Three Derivatives. Ibid., pp. 885-95. Chromatography to Nucleic Acid Chemistry. J. Cell. & 20. . Uridine-S'-Pyrophosphate Derivatives. III. Amino Comp. Physiol., 38 (Suppl. 1): 21-40, 1951. Acid-containing Derivatives. Ibid., pp. 897-904. 7. DAODST,R. Quantitative Histology of Rat Liver during 21. PAYNE,A. H.; KELLY,L. S.; and JONES, H. B. The In Carcinogenesis by p-Dimethylaminoazobenzene (DAB). corporation of Formate-C14, Glycine-2-C14, Adenine- J. Nat. Cancer Inst., 16:1447-49, 1955. 4,6-C14, and Phosphate-P32 into Nucleic Acids. Cancer 8. DAOUST,R., and CANTERO,A. Isolation of Acid-soluble Research, 12:666-70, 1952. Mononucleotides from Rat Tissues. Proc. Am. Assoc. 22. SACKS,J.; LUTWAK,L.;and HURLEY,P. D. On the Soluble Cancer Research, 1:10, 1954. Nucleotides of Liver and Muscle. J. Am. Chem. Soc., 76: 9. DAOUST,R., and LAMIKANDE,G. DE. Deoxyribonucleic 424-26, 1954. Acid Metabolism and Growth Processes. In: Canadian 23. SCHMITZ,H. Isolierung von freien Nucleotiden aus ver Cancer Conference, 1:365-73. New York: Academic schiedenen Geweben. II. Isolierung der 5'-Mono-, Di- und Press, Inc., 1955. Triphosphate von Adenosin, Guanosin, Cytidin und 10. DELOKY,G.E. A Note on the Determination of Phosphate Uridin aus dem Ascitestumor der Maus. Biochim. et in the Presence of Interfering Substances. Biochem. J., Biophys. Acta, 14:160-61,1954. 31:1161-62, 1938. 24. SCHMITZ,H.; HART, W.; and RIED, H. Isolierung von 11. Ilisi UK. Z. Some New Characteristic Color Tests for freien Nucleotiden aus verschiedenen Geweben IV. Thymonucleic Acid and a Microchemical Method for Mitteilung. Überden Nucleotidgehalt des Sarkom 37 Determining the Same in Animal Organs by Means of (Ascitesform). Ztschr. f. Krebsforsch., 60:301-6, 1955. These Tests. Mikrochemie, 8:4-32, 1930. 25. SCHMITZ,H.; HURLBERT,R. B.; and POTTER, V. R. 12. EDMONDS,M. P., and LEPAGE,G. A. The Incorporation Nucleotide Metabolism. III. Isolation of 5'-Mono-, Di-, of Glycine-2-C14 into Acid-soluble Nucleotide Purines. and Triphosphates of Cytidine, Guanosine and Uridine. Cancer Research, 15:93-99, 1955. J. Biol. Chem., 209:41-54,1954. 18. FISKE, C. H., and SUBBAROW,Y.The Colorimetrie De 26. SCHMITZ,H.; POTTER, V. R.; HURLBERT,R. B.; and termination of Phosphorus. J. Biol. Chem., 66:375-400, WHITE,D. M. Alternative Pathways of Glucose Metabo 1925. lism. II. Nucleotides from the Acid-soluble Fraction of 14. HDLTIN,T.; SLAUTTEHBACK,D.B.; and WESSEL,G. In Normal and Tumor Tissues and Studies on Nucleic Acid corporation in vivo of Labeled Nitrogen and Phosphorus Synthesis in Tumors. Cancer Research, 14:66—74,1954. into the Ribonucleic Acid of Chick-Liver Cytoplasm 27. SINSREIMEK,R. L., and KOERNER,J. F. Ion Exchange Fractions. Exper. Cell Research, 2:696-99, 1951. Separation of Desoxyribonucleotides. Science, 114:42-43, 15. HDRLBERT,R.B., and POTTER,V. R. Nucleotide Metabo 1951. lism. I. The Conversion of Orotic Acid-6-C14to Uridine 28. SMITH,E. E. B., and MILLS, G. T. Uridine Nucleotide Nucleotides. J. Biol. Chem., 209:1-21, 1954. Compounds of Liver. Biochim. et Biophys. Acta, 13: 16. HUHLBERT,R. B.; SCHMITZ,H.;BRUMM,A.;and POTTER, 386-400, 1954. V. R. Nucleotide Metabolism. II. Chromatographie 29. . Nucleotide Components of the Mammary Gland. Separation of Acid-soluble Nucleotides. J. Biol. Chem., Ibid., pp. 587-88. 209:23-39, 1954. SO. STEVENS, C. E.; DAOUST,R.; and LEBLOND,C. P. Rate 17. MILLER,E. C.; MILLER,J. A.; KLINE, B. E.; and RUSCH, of Synthesis of Desoxyribonucleic Acid and Mitotic Rate H. P. Correlation of the Level of Hepatic Riboflavin with in Liver and Intestine. J. Biol. Chem., 202:177-86, 1953. the Appearance of Liver Tumours in Rats Fed Aminoazo 31. . The Desoxyribonucleic Acid of Interphase and Dyes. J. Exper. Med., 88:89-98, 1948. Dividing Nuclei. Can. J. Med. Sc., 31:263-78, 1953. 18. PARK, J. T. Uridine-5'-Pyrophosphate Derivatives. I. 32. VOLKIN,E.; KHYM,J. X.; and COHN,W. E. The Prepara- Isolation from S. aiireus. J. Biol. Chem., 194:877-84, ration of Desoxynucleotides. J. Am. Chem. Soc., 73: 1952. 1533-36, 1951.

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1955 American Association for Cancer Research. Isolation of Acid-soluble Nucleotides from Rat Tissues and Measurement of Their Specific Activity Following P 32 Administration

R. Daoust and A. Cantero

Cancer Res 1955;15:734-741.

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