The Biosynthesis of Free Glycine and Serine by Tumors*

SAULKIT

(University of Texas M. D. Anderson Hospital and Tumor Institute, Department of Biochemistry, Houston, Texas) When cell suspensions of the Gardner lympho- Tissues and incubation procedure.â€”TheGardnerand Mecca sarcoma were incubated with acetate-2-C14, ap lymphosarcomas, previously transplanted to grow as solid tumors, were transformed into ascites tumors. It was observed preciable radioactivity was observed in the alpha that less total radioactivity was found in Gardner ascites carbon of free glycine (4). There are described be tumor glycine after incubations with labeled acetate than in low experiments showing that the methyl carbon the earlier experiments (4), which were carried out with cell of acetate may also be utilized in the formation of suspensions made from the solid tumors. The preparation of free serine. The incorporation of radioactivity cellular suspensions from the solid tumors involves a rather thorough extraction of soluble proteins and endogenous from labeled glucose into both amino acids and of metabolites. Possibly, the difference is partly attributable labeled ribose into glycine is also demonstrated to this factor. (Note, in this connection, Table 4 and Table 5, (5). The latter conversions take place in tumors experiment 1.) However, the observed conversion by the other than the Gardner lymphosarcoma. ascites cells was deemed adequate for our purposes, so that ascites cells were used thereafter. The author proposes the following scheme as a Tumor-bearing animals were sacrificed 7-10 days after basis for discussion : the intraperitoneal inoculation of 0.1-0.2 cc. of ascites fluid. Acetate â€”Â»oxalacetate<->pyruvate I ? Glucoseâ€”>hexosephosphate <->phosphoglycerate â€”>(phosphohydroxypyruvate)

Ribose glycine Â«-Â»serine<â€” (phosphoserine)

It will be noted that serine is formed prior to In the acetate, ribose, and formate experiments, tumor cells glycine and that phosphoglycerate is suggested as were twice washed with modified Krebs-Ringer-phosphate buffer (6), a refrigerated centrifuge being used to sediment an intermediate common to all three substrates. the cells between washes. In the experiments with labeled The results presented in this paper are consistent glucose, the ascites plasma was decanted after the tumor cells with the above scheme. A number of other hypo were sedimented. The cells were then resuspended in 2 volumes thetical pathways were also considered, but in of Krebs-Ringer-phosphate buffer and used without further most instances were at variance with the data. washing. To obviate possible bacterial contamination, streptomycin Examples are to be found in the discussion. The sulfate and potassium penicillinate were incorporated into the inclusion of phosphohydroxypyruvate and phos incubation medium in some experiments. No effect upon phoserine is as yet purely speculative. amino acid biosynthesis resulted from these additions. The absence of bacterial contamination from the Gardner ascites EXPERIMENTAL tumor was also verified through the kindness of Dr. E. Staten Labeled substrates.â€”Acetate-2-C14and formate-C14 were Wynne, Department of Pathology of this Institution. purchased from Tracerlab Inc.; glucose-1-C14 from Nuclear The general methods were those described previously (8). Instrument and Chemical Co.; glucose-6-C14, glucose-2-C14, Details concerning protocols are to be found with the tables. and ribose-1-C14 from the National Bureau of Standards; all RESULTS AND DISCUSSION on allocation from the Atomic Energy Commission. Uniformly labeled D-ribose, as supplied by Schwartz Laboratories, was Formation of labeled serine and glycine from glu dissolved in 75 per cent ethanol; the alcohol was evaporated in coselabeledwith C14.â€”-Whencellsuspensions of the tacuo before the cells were added to the Warburg vessels. Gardner, Mecca, or Ehrlich ascites tumors were * Aided in part by grants from the National Cancer Insti incubated with glucose-1-C14 or glucose-6-C14, ap tute, Department of Health, Education and Welfare, and by preciable radioactivity was found in free alanine the American Cancer Society. The author is indebted to and serine (Table 1). Somewhat less radioactivity Odette Graham, with whose assistance the experiments de was found in aspartic acid, glutamic acid, and pro- scribed below were performed. line, but none was detected in glycine. By the re Received for publication June 21, 1955. actions of glycolysis, glucose-1-C14or glucose-6-C14 715

is metabolized to phosphoglyceric acid-3-C14. The CC>2when glucose is metabolized via the shunt. latter would presumably give rise to serine-3-Cu The results also indicate that labeled serine may and unlabeled glycine. No amino acid biosynthesis be formed from glucose- 1-C14 by a mechanism not was observed when trichloroacetie acid was added involving glyoxalate as an intermediate, since no at the start of the experiment. radioactivity was found in glycine. It has been The addition of nonlabeled serine to the incuba shown that glyoxalate is a glycine precursor (12). tion medium increased serine radioactivity. In the When glucose-2-C14 was the substrate, the label absence of oxygen, there was a marked reduction ing of glycine as well as serine was observed of radioactivity in serine, glutamic acid, or aspar- (Table 2). As alpha-labeled phosphoglyceric acid tic acid, but that of alanine was reduced to a lesser would be formed in this case, the radioactivity extent. Anaerobically, the reoxidation of Coen- found in glycine is in accord with expectation. zyme I, which is reduced by triÃ³se phosphate de- Although the incorporation of carbon from la hydrogenase, takes place at the level of lactate. beled glucose into free serine by various normal The inhibition of serine labeling probably indicates tissues of adult rodents was also studied, relatively that the serine precursor does not effectively com little radioactivity was observed in the serine. A pete for oxidized Coenzyme I. comparison of free amino acid biosynthesis of nor- TABLE1 AMINOACIDBIOSYNTHESISFROMGuicosE-6-C"ORGLUCOSE-I-C"BYTUMORS Flask contents: 2.45 cc. including 0.1 mg. potassium penicillinate, 0.1 mg. streptomycin sulfate, unlabeled serine as indicated, Krebs-Ringer-phosphate buffer, pH 7.4, 0.01 M. Glucose-1-C14:10 yuM/flask(216,000 counts/min/juM). Glu- cose-6-C14:5 ^M/flask (424,000 counts/min/juM). Krebs-Ringer-bicarbonate buffer (0.028 M) used in anaerobic experi ments. Exp. 2: 10 jiM fluoride, 5 /aMpyruvate/flask. Exp. 8: 15 juMfluoride, 15 /Â¿Mpyruvate.Exp. 4: 30 /iM pyruvate, 15 Â¡Mfluoride. Time: 60 minutes. Temp.: 38Â°C.Atmosphere: air, except where indicated by asterisk. TOTAL CODNTB/MINX10~* Aspartic Glutamic EXP. TUMORGardnerGardnerMeccaGardnerGardner*EhrlichEhrlich*EhrlichGardnerGardnerEhrlichEhrlichGLUCOSE-C"666666661111SERINE(CM)none33111115622TUMOR C14 (>IM) ADDITIONS acid acid Serine Alanine none 2.35 14.2 38.2 80.5 1 Gardner 6 3 none 3.61 18.6 96.0 90.5 none 2.45 9.4 94.2 94.4 none 2.17 19.3 35.7 70.5 none 0.49 5.8 5.6 42.8 none 1.71 17.6 10.9 102 none 4.3 1.4 83.0 F-Pyruvate 34.2 36.7 none 1.46 7.08 43.3 29.5 F-Pyruvate 1.34 5.00 90.5 11.2 none 3.91 5.70 14.1 46.5 F-Pyruvate 1.36 3.95 42.6 18.3 * Atmosphere: 95 per cent Nt-5 per cent COi.

To establish the nature of the proximal serine mal tissues with that of tumors will be dealt with precursor, tumor cells were incubated in the pres in a separate communication. ence of potassium fluoride, an enolase inhibitor. A Formation of serine from formate and glycine.â€” pool of nonlabeled pyruvate was added to dilute Evidence that serine and glycine are intercon any radioactive pyruvate formed despite the vertible in the tumors as in other tissues (9) was block. The radioactivity of alanine, glutamic, and obtained by experiments with formate-C14 and aspartic acids was markedly reduced, demonstrat nonlabeled glycine. The free serine which was ing that the metabolic block was effective. Never isolated contained appreciable radioactivity (Table theless, the radioactivity of serine was increased 3). (Table 1). It must, therefore, be concluded that Experiments with ribose labeled with C'14.â€”-Ho- serine is not formed from pyruvate by a reversal recker et al. (2) have observed the transformation of the serine dehydrase reaction (10) but that of l-C14-ribose-5-phosphate to l,3-C14-hexose phos phosphoglyceric acid is a more likely precursor. phate by rat liver enzymes. "Active glycolalde- It should be noted that the results with glucose- hyde," derived from the first two carbons, and 1-C14 (Table 1, Exps. 3 and 4) indicate that appre triÃ³se phosphate, derived from carbons 3-5 of the ciable labeled serine may be formed by tumors pentose, are presumed intermediates (1, 7). En without the necessary participation of the hexose zymes capable of effecting this transformation are monophosphate shunt. The first carbon is lost as widely distributed in mammalian tissues (7), in-

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1955 American Association for Cancer Research. KITâ€”Glycine and Serine Biosynthesis by Tumors 717 eluding the Gardner lymphosarcoma (11). It is, found in serine was one-third that of glycine, the therefore, apparent that all five pentose carbons ratio could be reversed by adding nonlabeled serine are potential sources of glycine and serine. To test to the medium and thus increasing the size of the this possibility, cell suspensions were incubated serine pool. As a result, radioactivity of glycine with uniformly labeled ribose-C14 and also with was reduced, although that of glutamic acid was ribose-1-C14. Relatively little exogenous ribose was not affected. oxidized to C14O2or converted to amino acids The labeling of glycine and of aspartic acid from (Table 3). The labeling of glycine was, however, acetate-2-C14 was also markedly reduced when the readily detectable. Radioactivity was also found in incubations were conducted in the presence of glutamic acid, and somewhat less in alanine and glucose (Table 5). The latter cannot be attributed aspartic acid. to dilution of acetate-2-Cu by glucose at the sub All the radioactivity of the glycine may have re strate level, since the radioactivity of glutamic sulted from the indirect transformation of ribose to acid was not reduced. The results are, however, hexose phosphate and the subsequent metabolism consistent with the metabolism of acetate by way of the latter to phosphoglycerate and serine. Al- of the tricarboxylic acid cycle to oxalacetate.'de- TABLE2 AMINOACIDBIOSYNTHESISFROMGLUCOSE-Ã•-C" Each flask contained 20 /Â¿Mglucose-2-C14(63,800 countÂ«/min//Â¿M),1nii L-serine, 0.1 mg. streptomycin sulfate, 0.1 mg. potassium penicillinate, about 20 mg. dry weight of tissue, and buffer to a total volume of 2.45 cc. Incuba tion time: 2 hours at 88Â°C.Atmosphere: air. Values are for separate experiments performed in duplicate.

TOTALCODNTS/MIN COUNT8/MIN//Â»M GlutamicAcid15001800130019501810Alanine19,80017,00019,30012,20018,300Serine38604200420070505370Glycine47504760479092908710GlutamicAcid60004700670066005800Alanine17,00011,00018,00011,00016,000Serine54003700510072007400Glycine38002400300034003900 TuMOB Ehrlich Ehrlich Ehrlich Gardner Gardner

TABLE 3 UTILIZATIONOFLABELEDRIBOSEANDFORMATE Each flask contained the following: (a) ribose experiments: 2.8 juMuniformly labeled ribose (86,400 counts/mm/ |tM) and Krebs-Ringer-phosphate, 0.01 M, pH 7.4 to make a total volume of 2.45 cc. or (6) Formate experiments: 3 fiM glycine, 7.5 /Â¿Mformate(67,000 counts/min/^M) and buffer solution to a total of 2.45 cc. Time: 2 hours. Temp.: 38Â°C.Atmosphere: air. RlBOBZ-C" FORMATI-C"PLUSOLTCINE Total Counts/ C"0. PMC"g VMGlutamic'mintÂ» 'min xioo counta/minGlutamic TISSUEGardner*GardnerfGardnerGardnerEhrlichThymus,prot.0.110.150.250.310.230.22TotalAcidGlycine3501010700635115425240160315150014401540295810430126

ratSpleen (C3H)Spleen* "0,0.090.260.080.130.030.810.140.14 * Ribose-1-C", 7.5 Â¡at(ISZ.OOOcounti/min/jiM),incubated 75 minutes. t 5.6 Â«IMribose-C". ternatively, some "active glycolaldehyde," formed TABLE4 from ribose-1-C14, may have been oxidized to gly- EFFECTOFSERINEONCONVERSIONOFACETATETO oxylic acid, a glycine precursor (12). The latter GLYCINEBYGARDNERLYMPHOSAHCOMA metabolic sequence apparently occurs in spinach- CODNTB/MIN/TOTAL leaf preparations (13). 10-Â«EXPEH.ControlSerineControlSerineGlutamicAcid1341037775.5GlyCOUNTS/MINX 10-Â»GlutamicAcid407344587456Gly Experiments with acetate-2-C1^.â€”Byemploying acetate-2-C14 having 10 times the specific activity cine26.79.28.82.6Serine10.326.82.57.9Pro-line72.560.014.712.0MÂ«Xcine11.43.015.91.8 of the substrate previously utilized (4), it was shown that acetate could be transformed to serine as well as to glycine by the Gardner lymphosar coma (Table 4). Although the total radioactivity Conditions were the same as (or Exp. 3, Table 6. Serine concentration:

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1955 American Association for Cancer Research. 718 Cancer Research carboxylation of the latter to pyruvate, and sub studied, with carbon-14-labeled acetate, glucose, sequent transformation of the pyruvate to serine ribose, and formate as substrates. The conversion and to glycine via the same intermediates as those of acetate-2-C14 to glycine by the Gardner ascites generated from glucose. Also consistent with this tumor was markedly inhibited by glucose, serine, formulation was the observation that malonate re fluoride, and malonate, but not by hydroxypyru- duced glycine radioactivity to one-sixth, while vate or glycollic acid. Radioactivity was also ob aspartic acid radioactivity was reduced to one- served in free serine. fourth to one-fifth and glutamic acid radioactivity After incubating tumor cells with glucose-6-C14 to one-half that of the controls. Moreover, the or glucose-1-C14, serine, but not glycine, was la conversion of radioactivity from acetate to glu beled. Serine and glycine were both labeled when tamic acid and aspartic acid was diminished by glucose-2-C14was used. In the presence of fluoride fluoride to one-fifth, attendant upon a reduction of and pyruvate, the radioactivity of serine was in respiration of 50 per cent. Glycine radioactivity, creased but that of alanine was reduced. however, was reduced to one-eighth that of the Some labeled glycine was formed by lymphatic controls. Neither glycollate nor sodium hydroxy- tissues and by tumors from ribose-C14. The same pyruvate (0.012 M) influenced the labeling of gly tissues formed labeled serine from formate-C14 and cine from acetate-2-C14. glycine. TABLE5 REFERENCES 1. HOHBCKEH,B. L. Symposium on the Metabolic Role of EFFECTOFGLUCOSE,MALONATE,ANDFLUORIDEON Lipoic (Thioctic Acid), Discussion. Fed. Proc., 13:711-12, THECONVERSIONOFACETATETOGLYCINE 1954. Volume: 2.45 cc. Glucose, malonate, or fluoride: 30 pM./ 2. HORECKER,B.L.; GIBBS,M.; KLENOW,H.; and SMYHNI- flask. Acetate-2-C": Exps. 1and 2,15 /iM/flask (76,000 counts/ OTIS,P. Z. The Mechanism of Pentose Phosphate Con min//jM), Exp. 8 (6.6 X IO6 counts/min//iM), 5 /iM/flask. version to Hexose Monophosphate. I. With a Liver En Temp.: 88Â°C.Atmosphere: air. Time: 90 minutes. Experiments zyme Preparation. J. Biol. Chem., 207:393-403, 1954. were performed in duplicate. Values are means. 3. KIT, S. The Utilization and Formation of Dicarboxylic Amino Acids by Cell Suspensions of Normal and Malignant COUNTS/MIN/VMX10"' Lymphatic Tissues. Cancer Research, 14:397-402, 1954. Aspartic Glutamic 4. . The Conversion of Acetate-2-C14 to Glycine by acid Acid Glycine EXP. CONDITION Cell Suspensions of the Gardner Lymphosarcoma. J. Biol. 1 Control 16.5 13.3 1.35 Chem., 212:311-17, 1955. Glucose + 22.0 0.08 5. . Glycine-Serine Formation from Nonamino Acid 2 Control 18.8 16.5 0.83 Precursors by Tumors. Proc. Am. Assoc. Cancer Re Glucose 5.6 26.4 0.11 search, 2:29, 1955. Malonate 3.9 7.7 0.14 6. KIT, S., and GREENBEKG,D. M. Tracer Studies on the 3 Control 435 303 16.4 Metabolism of the Gardner Lymphosarcoma. II. Energy- KF 81 62 2.2 yielding Reactions and Amino Acid Uptake into Protein of the Tumor Cell. Cancer Research, 11:495-99, 1951. Some serine which had been derived from ace- 7. RACKER,E. Alleniate Pathways of Glucose and Fructose tate-2-C14 was degraded with periodate (8), and Metabolism. Adv. Enzymol., 15:141-82, 1954. 8. SAKAMI,W. Formation of Formate and Labile Methyl the radioactivity of the beta carbon measured as Groups from Acetone in the Intact Rat. J. Biol. Chem., the dimedon derivative. Twenty-five and 49 per 187:369-78, 1950. cent of the radioactivity was found in the beta car 9. SIEKEVITZ,P., and GREENBERG,D. M. The Biological bon in two experiments. After degradation with Formation of Serine from Glycine. J. Biol. Chem., 180: 845-56, 1949. ninhydrin, 7 per cent of the radioactivity was 10. SPRINSON,D., and CHARGAFF,E.Studies on the Mecha found in the carboxyl group. nism of Deamination of Serine and Threonine in Bio Radioactive glycine from the same experiment logical Systems. J. Biol. Chem., 151:273-80, 1943. had 16 per cent of the radioactivity in the carboxyl 11. VILLAVICENCIO,M.,and GUZMANBAHRON,E. S. Path ways of Hexose Metabolism in Lymphatic Cells (Rabbit carbon and 84 per cent in the alpha carbon. The Appendix) and in Lymphosarcoma (Gardner's Mouse results further support the concept that glycine Lymphosarcoma). Fed. Proc., 14:297, 1955. was formed from serine and that the serine radio 12. WEINHOUSE,S., and FHIEDMANN,B. Metabolism of activity had been derived from acetate which had Labeled 2-Carbon Acids in the Intact Rat. J. Biol. Chem., traversed the citric acid cycle. 191:707-17, 1951. 13. WEISSBACH,A.,and HORECKEH,B. L. The Formation of SUMMARY Glycine from Ribose-5-phosphate. In: W. D. MCELROY and B. GLASS (eds.), A Symposium on Amino Acid The mechanism of tumor glycine and serine bio Metabolism, pp. 741-42. Baltimore: The Johns Hopkins synthesis from nonamino acid precursors was Press, 1955.

Saul Kit

Cancer Res 1955;15:715-718.

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