Purine Synthesis in Mouse Tissues*

ABRAHAM M. STEIN,~ ~LLIAM T. MURAKAMI,~ AND DONALD W. VISSER

(Department of Biochemistry and Nutrition, University of Southern California Medical School, Los Angeles 7, Calif.)

LePage (9) and Edmonds and LePage (5) have caecum, stripped so as to remove the mesenteries, documented an anomalous incorporation of gly- washed by infusion of 20 ml. of ice-cold saline, cine-2-C 14 into purines of the acid-soluble fractions frozen, and crushed as above. Tissues of four of Ehrlich ascites carcinoma cells and Flexner- to five mice were pooled in one group. The frozen Jobling carcinoma, wherein the specific activity tissues were pulverized, homogenized with 20 ml. of guanine was reported to be greater than that of cold 0.6 ~ perchloric acid, and centrifuged of hypoxanthine. These findings cannot be ex- in the cold. The precipitate was re-extracted with plained easily on the basis of the purine synthetic 15 ml. of cold 0.2 rr perchloric acid and centrifuged; pathway obtaining in avian liver, whereby hypo- the supernatants were combined and neutralized xanthine is the first purine formed (7, 10), or to the red color of phenol red with 5 N potassium the pathway of guanine synthesis of Abrams (1), hydroxide. The suspension was kept at 5 ~ C. over- Magasanik (12, 14), and Lagerkvist (8), unless night and centrifuged to remove insoluble potas- complex assumptions are made regarding pool sium perchlorate. size and compartmentalization in the cell. Ed- Isolation of purines.--The supernatant was brought to room monds and LePage (5) have suggested that ade- temperature and passed through a 2.3 em. (diam.) X 1.6 cm. col- nine, guanine, and hypoxanthine nucleotides are umn of Dowex-1 in the chloride form, and the column was formed from a common nucleotide precursor aris- washed with 50 ml. of water. Acidic compounds were eluted with 20 ml. of 6 rr hydrochloric acid and taken to dryness in a ing early in synthesis. Welch (19) has discussed vacuum desiccator over calcium chloride and sodium hydrox- possible synthetic pathways of adenine and gua- ide. The dried fractious were transferred to pyrex test tubes nine nucleotides not involving inosinic acid. with three 0.5-ml. portions of 1 ~r sulfuric acid and hydrolyzed This study was designed to gain information in a boiling water bath for 1 hour. The hydrolysates were dilut- ed with 5 ml. of distilled water and placed in a water bath at concerning possible differences in patterns of pu- 500-60~ C. (11). One ml. of 1 M silver nitrate was added, and rine synthesis in mouse tissues and in tumor, the samples were allowed to stand overnight at 5~ C. The pre- by following the specific activities of the total cipitate was centrifuged and washed with three 8-ml. portions acid-soluble purine nucleotide bases with time. of ice-cold 0.05 Msilver nitrate in 0.1 N sulfuric acid. The silver salts were decomposed with 4 ml. of 1.5 rr hydrochloric acid in MATERIALS AND METHODS a boiling water bath, and the precipitate was washed on a sintered glass funnel with 2 ml. of 1.5 N hydrochloric acid. Male mice of the Webster strain, 6-8 weeks Separation and purification of purines.--The purines were after weaning, were used. Ehrlich ascites carcino- separated on 50-cm. columns of Dowex-50-X8 in the hydro- ma was implanted subcutaneously in two sites gen form, by a modification of a method of Wall (18). Up to four columns were operated in parallel on a Technicon fraction to yield solid tumors. Mice that developed ascitic collector. The mixer contained 400 ml. of 1.5 rr hydrochloric tumors were discarded. Each mouse received, by acid per column operated; the reservoir contained 7 ~ hydro- intraperitoneal injection, a single dose of either chloric acid. Fractions were collected at 20-minute intervals. 7.5 tLc. of formate-C 14 (specific activity, 1.0 ~c/ At a pressure of 5-7 cm. of mercury, flow rates averaged 18 #mole) or 5 uc. of glycine-l-C ~4 (specific activity, ml/hour at the beginning of the chromatographic separation and increased to 80 ml/hour at the adenine peak. 0.83 ~uc/#mole) in 0.25 ml. of distilled water. Chart 1 shows the separation of hypoxanthine, guanine, The mice were killed by cervical dislocation, and and adenine from the hydrolyzed acid-soluble nucleotide frac- the livers and tumors were rapidly removed and tion of 15 gm. of mouse liver without prior isolation of the crushed between two slabs of dry ice. The small silver purines. Chart 2 shows isolated purine peaks from the chromatograms of the livers and small intestines of three mice intestine was separated from the pylorus to the injected with 5 gc. each of glycine-2-C 14 and sacrificed 20 min- * This research was supported in part by the American utes later. Although these chromatograms show considerable Cancer Society, California Division, and in part by the U.S. purification of purines from extraneous radioactivity when Public Health Service, grants number CY-~78(C) and compared with similar chromatograms in which preliminary B-512(C8). silver purification had not been effected, it is apparent that af Present address: Graduate Department of Biochemistry, liver hypoxanthine was still highly contaminated. Brandeis University, Waltham 54, Massachusetts. The purine-containing fractions were taken to dryness in a vacuum desiccator as described above. Hypoxanthine was Received for publication August 4, 1958. ehromatographed on Whatman-1 strips in butanol-diethylene 84

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. STEIN et al.~Purine Synthesis in Mouse Tissues 85 glycol-water (4:1:1) in an ammonia atmosphere (17), descend- corporation of purine precursors into liver RNA ing. Hypoxanthine, obtained from the previous paper chroma- and DNA of tumor-bearing mice. Similar effects tography, guanine, and adenine were chromatographed on What- man-1 strips in 65 per cent isopropanol-2 ~r hydrochloric acid were found by Conzelman et al. (4) with 4-amino- (20), descending. The purine spots were visualized under a 5-imidazolecarboxamide-C 14. Furlong et a/. (6) Mineralite lamp, cut out, and eluted with 4 ml. or more of 0.1 have shown an increase in total radioactivity zr hydrochloric acid and read in a Beckman model DU speetro- of the liver and spleen DNA of tumor-bearing photometer at 250, 260, 280, and 290 m/~. The values were cor- mice. The present data indicate that at least rected for the absorption of the eluate of a corresponding blank spot on the same paper strip. The ratios obtained were in good part of this effect was due to stimulation of purine agreement with literature values; concentrations were esti- synthesis per se in tissues of tumor-bearing mice. mated from the absorbance at 260 mg (2). Aliquots of the By contrast, there was no effect by tumor upon purine chafe were dried under an infrared lamp in Pacific incorporation of formate into acid-soluble purines Nuclear concave glass planchets (Braun Corporation). The samples were counted under a Nuclear Chicago D-47 micromil in the small intestine. end-window. At least 10,240 counts were taken. Tables 1 and Chart 3 summarizes the data on incorporation 2 show the purification of purines at various stages, including of formate-C 14 and glycine-l-C ~4 into hypoxan- paper chromatography in butanol-acetic acid-water (40:20: 20) thine, adenine, and guanine from acid-soluble nu- not employed in routine estimation. Mtrlerials.--Dowex-50-X8 was washed by the procedure of cleotides at 10-80 minutes in the three tissues Moore and Stein (lS). Arbitrarily, the resin was then treated sampled. twice with 2 rr sodium hydroxide followed by 4 N hydrochloric acid, and finally washed with 1.5 ~r hydrochloric acid. The columns were poured in 5-cm. sections in 0.8-cm. diameter 3O Ad 20 tubes. A continuous flow of 1.5 z~ hydrochloric acid was main- 2O ~ .... :~ ! .....: i[..,.U.~...... ,( ~,..~:-:~ ~.:.... ]~176 tained through the columns when they were not in operation. Background absorbance of the chafe was less than 0.1 optical 0 "~""~ :": 1 density unit at 260 m~. Glycine-l-C 1~ and glycine-2-C 14 were obtained from Isotopes Specialties, Glendale, California. Sodium formate-C 14 was obtained from Tracerlab, Inc. The Ehrlich ascites carcinoma strain was obtained from Dr. K. Sugiura of the Sloan-Kettering Foundation. RESULTS Table 3 shows the effect of tumor on the in- t0 20 30 40 50 corporation of formate-C 14 into the acid-soluble TUBE NO. nucleotide purines of liver and intestine of mice CHART l.--Gradient elution ion exchange chromatography bearing the Ehrlich ascites carcinoma. The data of the hydrolyzed acidic fraction of 15 gin. of mouse liver per- show a relatively uniform stimulation of incor- chloric acid extract. The solid line represents absorbance at 260 m/~; the dashed line represents the ratio of absorbances at poration into the purines of liver. A similar tumor 260-280 m~. Hx, Gu, and Ad are abbreviations for hypoxan- effect was shown by Payne et al. (16) upon in- thine, guanine, and adenine, respectively.

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CHART s sections of ion exchange chromatograms sorbanee at s m/~; the hatched areas represent counts/rain/ of decomposed silver purines o[ the livers and small intestines /ml. Hx, Gu, and Ad are abbreviations for hypoxanthine, of three mice each injected with 5/~c. of glycine-2-C14. The tis- guanine, and adenine, respectively. sues were obtained at 20 minutes. The solid line indicates ab-

The hypoxanthine samples of the livers of mice The rapid drop in specific activity of the purines injected with glycine-l-C 14 were lost, and a de- labeled from formate probably reflects the rapid tailed comparison of formate and glycine incor- oxidation of formate in the organism and corre- poration in liver hypoxanthine cannot be made. lates well with the active formate oxidation which However, preliminary experiments at 20 minutes obtains in liver (15). (Tables 1 and 2) show no appreciable difference In tumor tissue, guanine specific activity was in the specific activity of hypoxanthine and gua- always equal to or lower than hypoxanthine when nine with either label. The specific activity of formate was used. With glycine-l-C 14, the initial hypoxanthine of liver with formate-C 14 is higher specific activity of hypoxanthine was equal to than that of guanine in the period under study. or slightly higher than that of guanine. This

TABLE 1 PURIFICATION OF PURINE BASES* GLI,CIN~.- I-C14 GLYCINE-$-C 14 COMPOUND PURIFZCA- Liver Small intestine Liver Small intestine I~OLATF-,D TION SYSTEM (eounts/min/~mole) Hypoxanthine 1 61 2030 84 188() 2 62 2510 98 2490 3 55 2270 87 ~510 Guanine 4 52 2340 63 2450 2 51 3070 71 3000 8 82 2600 48 2745 Adenine 4 ~5.5 951 44.7 1010 2 24.8 949 50.0 1070 3 21.7 794 38.1 962 * Purification without prior isolation as the silver salts. t Purification systems 1, 2, and 3 refer to paper chromatography in n-butanoh diethylene glycol: water (17), isopropanol: HC1 (20), and n-butanol: acetic acid: water (40: 20: 20), respectively. Purification system 4 refers to gradient elution chromatography on Dowex-50-H + as described under "Materials and Methods."

TABLE 2 PURIFICATION OF PURINE BASES* HC"OONA GLrOINF.-1-C14 COMPOUND PUmFXCA- Liver Small intestine Liver Small intestine I80LA'rF~ TION SYSTEMt ~ounts/min/#mole) Hypoxanthine 1 456 5320 339 959 2 412 5900 96 1080 S 394 5600 73 934 Guanine 4 538 11,900 172 1400 2 470 12,600 97 1260 3 381 12,100 64 1180 Adenine 4 285 5000 44 530 2 288 5140 40 520 * Purification without prior isolation as the silver salts. t See footnot~Table 1.

TABLE 3 EFFECT OF TUMOR ON INCORPORATION OF HCt4OONA IN LIVER AND INTESTINE BASES* CONTROL EHRLICH ASCITES CARCINOMAt COMPOUND Liver Small intestine Liver Small intestine ISOLATED (eounts/mln//~mole) $ Hypoxanthine 412 5900 1210 6830 Guanine 470 12,600 1580 13,300 Adenine 288 5000 631 4640 * 10 #e. HCX4OONa/mouse, five mice pooled, sacrificed at 20 minutes. t Ascites form, 11-day implant. After chromatography in 65 per cent isopropanol-2 N HC1 (20); without prior pre- cipitation as silver salts.

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. STEIN et al.--Purine Synthesis in Mouse Tissues 87 trend was reversed at 80 minutes, when the spe- and adenylic acid of Flexner-Jobling carcinoma cific activity of guanine was about 30 per cent with glycine-2-C 14 as a precursor. greater than that of hypoxanthine. The data are complicated by differences in The rate of labeling of the intestinal purines the over-all metabolism of the precursors by the was of particular interest and markedly different individual tissues. For example, in liver with for- from that of tumor purines. When formic acid mate-C 14, there is a rapid decrease in the specific was used, the specific activity of guanine rose activity of hypoxanthine and guanine which is to a peak value twice as great as that of hypo- probably attributable to the rapid oxidation of xanthine at ~0 minutes, then rapidly declined formate in the liver (15). In contrast, there is to values approximating those of hypoxanthine. a continuous increase in specific activity of purine With glycine-l-C ~4, the guanine values were equal bases of tumor when glyciue-l-C ~4 is the precursor. to or lower than those of hypoxanthine. This is probably related to the rapid uptake of

LIVER INTESTINE TUMOR

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l, I I I 20 80 20 40 80 20 40 80 MINUTES CHART 3.--Rate of synthesis in vivo of labeled hypoxan- formate-C14; the lower three graphs, the data obtained with thine, guanine, and adenine in mouse organs and tumor. Circles glycine-l-C14; the vertical pairs present the data for liver, in- denote hypoxanthine, crosses guanine, and solid circles ade- testine, and tumor. nine. The upper three graphs represent the data obtained with

The purine base ratios of hypoxanthine, gua- glycine by the Ehrlich ascites tumor (3). The nine, and adenine were 1:3:30 for liver; 1:7:53 low initial specific activity of the bases may reflect for small intestine; 1:4: ~6 for tumor, respectively. a large pool of free glycine in tumor, consequent upon the concentrative uptake of glycine. DISCUSSION The data obtained with formate-C 14 and gly- Preliminary degradation of the acid-soluble pu- cine-l-C 14 in tumor appear to be consistent with rine compounds to the free bases was chosen a scheme of synthesis according to which hypo- as a convenient analytical procedure to obtain xanthine is the precursor of adenine and guanine. the gross pattern of labeling of purines from The initial rate of change of specific activity different precursors. It is of interest to note was more rapid for hypoxanthine than for adenine that trends have been obtained in some of the or guanine. Furthermore, the specific activity of time curves (intestine-formate-C 14, tumor-glycine- hypoxanthine began to decline at a time when 1-C ~4) which are similar to those reported by the specific activities of adenine and guanine were Edmonds and LePage (5) for inosinic, guanylic, still increasing. The same arguments can be ad-

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1959 American Association for Cancer Research. 88 Cancer Research Vol. 19, January, 1959 vanced in the case of liver and formate-C 14, where 3. CHRISTENSEN, I-I. N. In: W. D. McELRoY and B. GLAss only the decaying phase of hypoxanthine specific (eds.), Symposium on Amino Acid Metabolism, pp. 63-106. Baltimore: 1955. activity was detected. In this case, loss of hypo- 4. CONZE~N, G. M., JR.; M~EL, H. G.; and SMITH, P. K. xanthine radioactivity was initially more rapid The Effects of Tumor Growth and X-Radiation on the than that of guanine, while the specific activity Incorporation of Radiocarbon from 4-Amino-5-Imidazole- of adenine increased in the initial part of the curve. carboxamide-C j4 into Nucleic Acids. Cancer Research, 14: The results with the purines of small intestine 100-102, 1954. 5. EDMONDS, M., and LEPAGE, G. The Incorporation of Gly- are more difficult to interpret. With formate-C ~4 cine-2-C 14 into Acid-soluble Nucleotide Purines. Cancer the specific activity of guanine was initially higher Research, 15:93-99, 1955. than that of hypoxanthine, a result not obtained 6. FURLONG, N. B.; WATSON, ]~. J. P.; DAvis, W. E.; and with liver or tumor even though the pool of gua- GRIF~N, A. C. The Effect of Tumor Fractions on the Up- take of Carbon-14-Labeled Adenine into the Deoxyribo- nine was found to be, relative to hypoxanthine, nucleic Acids of Mouse Tissues. Cancer Research, 15:710- twice as large in intestine as in the other tissues. 14, 1955. Furthermore, the initial rate of increase of specific 7. GREENBERG, G. R. De Novo Synthesis of I-Iypoxanthine activity of guanine was about 10 times that of via Inosine-5-Phosphate and Inosine. J. Biol. Chem., 190: 611-30, 1951. hypoxanthine and was followed by an equally 8. LAGERKVIST, U. Enzymic Synthesis of Xanthosine and rapid loss of label. The fact that this unusual Guanosine-5-Phosphate from Inosine-5-Phosphate. Acta relationship was not obtained with glycine-l-C ~4 Chem. Scandinav., 9:1028-29, 1955. in small intestine suggests that the formate effect 9. LEPAGv., G. In Vitro Incorporation of Glycine-2-C14 into reflects a different route of incorporation of one Purines and Proteins. Cancer Research, 13:178-85, 1953. 10. LEVENBERG,B., and BUCH~AN, J. M. Biosynthesis of the or both of the amidine carbons or, alternately, Purines. XIII. Structure, Enzymatic Synthesis and an unusual exchange reaction of one or both of Metabolism of (Alpha-N-Formyl)-Glycinamidine Ribo- these carbons with radioactive formate or a de- tide. J. Biol. Chem., 9.24:1019-27, 1957. rivative thereof. A study of the mechanism of 11. LORING, H. S.; FAIRLEY, J. L.; and SEAGRAM, H. L. A Spectrophotometric Method for the Analysis of the Purine incorporation of one-carbon compounds into in- and Pyrimidine Components of Ribonucleie Acid. J. Biol. testinal guanylic acid is suggested by these data. Chem., 197: 809-21, 1952. 12. MAGASAZ~K,B.; MOYV.D, H. S.; and GEHRING, H. B. En- SUMMARY zymes Essential for the Biosynthesis of Nucleic Acid Guanine; Inosine 5'-Phosphate Dehydrogenase of Acro- 1. The rate of incorporation of formate-C 14 batter aerogenes. J. Biol. Chem., 226:339-50, 1957. and glycine-l-C 14 into purine bases of the hy- 13. MOORE, S., and ST~.n% W. H. Chromatography of Amino drolyzed acid-soluble fractions of mouse liver, Acids on Sulfonated Polystyrene Resins. J. Biol. Chem., small intestine, and Ehrlich ascites carcinoma 192: 663-81, 1951. 14. MOYED, H. S., and M~GAS~CIK, B. Enzymes Essential for grown in the solid phase has been studied. the Biosynthesis of Nucleic Acid Guanine; Xanthosine 2. The incorporation of both precursors into 5'-Phosphate Aminase of Aerobacter Aerogenes. J. Biol. purines of liver and tumor, and of glycine-l-C ~4 Chem., 226:351--63, 1957. into intestine, appears to be consistent with cur- 15. NAKADA, H. I., and Wv.n~HOUSE, S. Studies of Glycine rently accepted pathways of biosynthesis of hypo- Oxidation in Rat Tissues. Arch. Biochem. & Biophys., 49.: 257-70, 1953. xanthine, guanine, and adenine, although these 16. PA~v., A. H.; KELLH, L. S.; BEACH, G.; and JONES, H. B. data do not constitute a critical test. The Effect of Neoplasia on the Turnover of Nucleic Acids 3. In small intestine, formate-C ~4 labels guanine Studied with Formate-C 14 and Glycine-2-C 14. Cancer Re- to a greater extent than hypoxanthine or adenine. search, 12:426-28, 1952. 17. u E., and CHXRGAFF, E. The Separation and Quan- This is discussed in terms of an atypical route titative Estimation of Purines and Pyrimidines in Minute of incorporation or of a possible exchange mech- Amounts. J. Biol. Chem., 176:703-14, 1948. anism. 18. Wxu~, J. S. Simultaneous Separation of Purines, Pyrimi- dines, Amino Acids and Other Nitrogenous Compounds. REFERENCES Anal. Chem., 2w 1953. 1. ABR~S, R., and BENTLEY, M. Biosynthesis of Nucleic 19. WELCH, A. D. In: Enzymes: Units of Biological Structure Acid Purines. II. Role of Hypoxanthine and Xanthine and Function, pp. 547-72. New York: O. H. Gaebler, 1956. Compounds. Arch. Biochem. & Biophys., 58:109-18, 1955. ~0. WYATT, G. R. The Purine and Pyrimidine Composition of 2. CHA~GAFF, E., and DAVIDSON, J. N. The Nucleic Acids, Deoxypentose Nucleic Acids. Biochem. J., 48:584-90, 1: 502, New York, 1955. 1951.

Abraham M. Stein, William T. Murakami and Donald W. Visser

Cancer Res 1959;19:84-88.

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