Incorporation of Canavanine Into Protein of Vvalker Carcinosarcoma 256 Cells Cultured in Vitro

Incorporation of Canavanine into Protein of VValker Carcinosarcoma 256 Cells Cultured in Vitro

PAUL F. KRUSE, JR., PAT B. WHITE, HENRY A. CARTER, AND THOMAS A. McCoY

(Biomedical Ds~ision, The Samuel Roberts Noble Foundation, Inc., Ardraore, Okla.)

The inhibitory effect of canavanine on the exposure to the arginine-deficient medium for 4~) growth of Walker carcinosarcoma ~56 cells in minutes, sufficient L-canavanine .H2SO41 (~0.9 vitro was described in a previous report (9). Ap- mg. dissolved in Earle's balanced salt solution) parently, the inhibition rested in a competitive was added to produce a final concentration of 0.~0 interference of canavanine with the utilization of mM (10:1 ratio of canavanine to arginine). The arginine. inhibited cultures were incubated for ~4 hours and A number of recent reports have demonstrated allowed to stand at 4 ~ C. for ~ hours to free the that certain amino acid analogs--e.g., ethionine cells from the glass (1~); cell counts indicated a (6, 10, ~, ~6), p-fluorophenylalanine (2, 17, 9.~5), total of 104 X 106 cells. The contents of the flasks fl-~-thienylalanine (17), selenomethionine (4), 7- were combined and centrifuged in ~50-ml. bottles azatryptophan (18, 19), and tryptazan (3)--can for 6 minutes at 2750Xg. The cell buttons were be utilized in place of their respective metabolic transferred ~u a 15-ml. centrifuge tube and counterparts for protein synthesis. Accordingly, washed 3 times with 5-10-ml. portions of Earle's it was of interest to determine whether the tumor solution minus CaCl~. The cells were then ex- cells could utilize canavanine in this manner as a tracted successively with 80 per cent ethanol, substitute for arginine. If so, a contributing factor ethanol-ether (1:1), and cold 10 per cent tri- in the inhibition of growth could have been a sub- chloroacetic acid (TCA). The residue was sus- sequent malfunction of the resultant protein. pended in s ml. of 5 per cent TCA, heated for 15 Canavanine has not been shown to participate in minutes in an oil bath at 90 ~ C., allowed to cool, protein synthesis and has been reported (7) to and washed twice with water. It was next heated exist only in the free state in the Jack bean, its for 15 minutes in s ml. of 0.1 ~r NaOH in an oil principal source. bath at 60 ~ C., reprecipitated with 2 ml. of s per In the present investigation it was interesting, cent TCA, and washed with water. Next, the therefore, to find an appreciable incorporation of residue was washed with absolute ethanol, etha- canavanine in the tumor cell protein. nol-ether (1:~), ether, dried in vacuo for 7 hours (37.1 rag.), and hydrolyzed by being refluxed in MATERIALS AND METHODS 3.0 ml. of 6 N HC1 for 18 hours. The hydrolysate Tissue cultures.--Suspensions of freshly excised was taken to dryness twice in vacuo, and the Walker tumor cells were cultured in T-60 flasks. residue was dissolved in pH 3.1 citrate buffer (14) All operations were performed aseptically, solu- and clarified by centrifugation. Eighty per cent of tions were sterilized by passage through Selas the solution was chromatographed. filters, and growth was determined by cell counts. A "control" protein hydrolysate was obtained An initial inoculum of ~00,000 cells/ml was used, as follows: After 48 hours' incubation, cell counts and the cells were established in medium, 20 ml/ indicated a total of 157 X 106 cells in 16 T-60 flask culture, containing fourteen essential amino acids, cultures (minus canavanine). Two milliliters of a serine, glycine, vitamins, antibiotics, salts, glucose, solution of L-canavanine .H2SO4 in Earle's solu- and dialyzed human serum (11). tion, 1 mg/ml, were added to each culture. After Incorporation of canavanine.--After incubation standing at room temperature for 15 minutes, the at 87~ C. for $4 hours, the medium was withdrawn cells were separated from the medium and washed from nineteen T-60 flask cultures and replaced as described above. The cell button was suspended with an identical medium, except that the arginine in 1 ml. of the canavanine-Earle's solution, and content was reduced from 0.s to 0.0~ inM. After 1Purchased from California Foundation for Biochemical Received for publication August 28, 1958. Research. 12~

6.7 ml. of 90 per cent ethanol was added. After The calculations were made from a standard thorough mixing, the cell residue was centrifuged curve of ninhydrin analysis of L-leucine;2 the color and treated with the series of extractions outlined yield of canavanine relative to leucine was found above. The amounts of extractants were increased to be 1.01. The quantities of histidine, canavanine, in proportion to the number of cells. The protein and arginine were corrected for the recoveries-- residue, 45.7 rag., was hydrolyzed by being re- 91.0, 82.5, and 98.8 per cent, respectively--which fluxed in 8.7 ml. of 6 N HC1 and treated as above. were obtained from chromatography of the "hy- Seventy per cent of the hydrolysate was chro- drolyzed" mixture of nineteen amino acids) matographed. The chromatographic conditions caused lysine Chromatography of canavine and protein hydroly- and ammonia to emerge together in the eluate, sates.--Ion exchange chromatography was per- permitting canavanine to emerge shortly there- formed as described by Moore and Stein (12, after as an isolated substance. Two coincident 13), with a 1.2 X 20-cm. column of 200-mesh 80-20 mixture of Dowex 50-x4,-x5. The column P.o was operated at room temperature, at a flow rate of 9-10 ml/hr, and 0.95 (control) and 0.91 1.5 i A (canavanine) ml. fractions were collected. Prior I.o to chromatography of the protein hydrolysates, a mixture of nineteen amino acids (including canavanine), which had been subjected to hydroly- >- sis conditions, was chromatographed. Location and o~-o.2t- quantities of amino acids in the effluent were determined by ninhydrin analysis (15); alternate t~ _j 0 ~L2:::2:2:~ fractions in the region of canavanine elution were <~.o also analyzed by the colorimetric procedure of ~_o Archibald (1). a. Z 0 1.5 0 RESULTS 3E In preliminary experiments with 1 : 1 and 2:1 1.0 4" w z exogenous canavanine-arginine ratios and 20-48 • z < r.~ 106 cells, the antimetabolite was not readily de- 0.5 tectable in the protein hydrolysates. It appeared likely, however, that interference with arginine i 0 --= incorporation had occurred, since the molar ar- I00 150 200 25O ginine/histidine ratio was always uniformly lower FRACTION NUMBER than that in protein from uninhibited cells. When CHART 1.--Chromatography of protein hydrolysates from the number of cells and the canavanine concen- Walker carcinosarcoma 256 cells cultured without (A) and tration were increased approximately two- and with (B) eanavanine. For culture and chromatographic condi- tenfold, respectively, and the cells were treated tions, see text. for a short time in arginine-deficient medium prior to introduction of the antimetabolite, canav- color peaks were obtained when alternate fractions anine was readily detectable in the protein hy- in the canavanine region of the eluate were ana- drolysate, as illustrated in Chart 1. Here the lyzed with ninhydrin (14) and alkaline nitroprus- histidine, canavanine, and arginine peaks were cal- side (1). None of the fractions in adjacent regions culated to represent 6.11, 1.60, and 13.42 gmoles, gave the nitroprusside test. respectively, in the total protein hydrolysate; in DISCUSSION the control hydrolysate (from Chart 1), the quantities of histidine and arginine were 7.82 and The most likely explanation of the above results 19.18 /zmoles in the total hydrolysate. Thus in is that canavanine participated in protein syn- the presence of canavanine the arginine/histidine thesis and became a part of the polypeptide chain. ratio was 2.20 in contrast to 2.45 for the con- The possibility that canavanine was merely ab- trol. However, the combined canavanine-arginine/ Purchased from H and M Chemical Co., AP grade. histidine ratio, 2.46, matched that of the con- s The assumption that these recoveries would be the same trol. The ratio of the amount of antimetabolite/ for amino acids released from peptide linkage during hydrolysis may not be valid, but the figures do agree with the metabolite in the protein under these conditions report (8) that canavanine is more labile to acid hydrolysis was 0.12. conditions than is arginine.

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1959 American Association for Cancer Research. 1~4 Cancer Research Vol. 19, January, 1959 sorbed on the precipitated, amorphous protein nine permitted an approximate doubling of the pro- was virtually eliminated in the control experi- tein of a strain of E. coli, whereas canavanine ment. Additional evidence of incorporation was was incapable of substituting for arginine in that seen in the change in arginine content, as meas- respect. The degree of incorporation into protein ured by its relationship to histidine. In the pres- of the two analogs was not measured. Recently, ence of canavanine the ratio of these two amino Yoshida (26) found that one third of the methio- acids was always lowered. The combined arginine- nine in a-amylase produced during culture of B. canavanine content, however, was of the same 8ubtilis could be replaced with ethionine. Whether order of magnitude with respect to histidine as the different degrees of incorporation of analogs arginine alone in the protein of uninhibited cells. are primarily a reflection of experimental condi- From this, it appeared that a portion of the tions, a measure of differences in the specificities protein normally occupied by arginine was taken of protein synthesizing systems, and/or the degree up by canavanine. The results of measurements of similarity between analog and metabolite can- of these ratios also preclude the possibility that not be determined at present. Probably many canavanine was bound to the protein in some such factors are involved in any comparison of manner similar to the reported (23, 24) binding the tendency of two or more analogs to be caught of lysine to glutamic acid residues through the up in protein synthesis. It appears likely, from e-amino group, unless it is normal for a certain studies with tryptophan analogs (18), that protein amount of arginine to be bound in this manner incorporation is more apt to take place with through the guanidino group. analogs whose structural change is in the ring In all probability, the previously noted (9) structure of the molecule in contrast to those competitive inhibition of tumor cell growth in having substituents attached to the ring. On the vitro by canavanine can be attributed in part other hand, the incorporation into protein of p- to the aberrant protein synthesis phenomena found fluorophenylalanine involves an analog with a in the present study. It would be of interest to substituent on the ring. Here, the stability and determine whether incorporation of canavanine small size of the substituent may be of importance. into protein occurred in cultures of chick em- Molecular models indicate an extremely close bryonic heart cells, in which Morgan et al. (16) structural similarity between arginine and canav- recently determined canavanine to be a competi- anine, whose structural change is in the chain tive antagonist of arginine. Perhaps some of the of the molecule, and it seems reasonable that striking examples of antagonism between cana- this was responsible in large part for the incor- vanine and arginine in microbiological systems 4 poration of the analog into protein in the present might be explained on the basis of incorporation study. of canavanine into the protein. Also, the ability In any event, attempts at correlation of struc- of canavanine to inhibit multiplication of certain ture with biological activity provide fuel for future viruses (20, 21) could conceivably have been the considerations. The elucidation of the mechanism result of abnormal protein synthesis. As suggested of action of amino acid analogs may also serve by Brawerman and Ycas (3), in a study of tryp- to determine better their eventual place in the tazan incorporation, a disorganizing action of chemotherapy of disseminated cancer. bogus protein formation might result in changes in nucleic acid formed under its influence. SUMMARY It is increasingly apparent that protein- Canavanine was found to be incorporated into synthesizing systems possess considerable flex- the proteins of Walker carcinosarcoma 256 cells ibility when presented an unnatural amino acid under tissue culture conditions. The incorporation substrate. In this regard, canavanine can be con- of the analog appeared to occur at the expense sidered as an unnatural amino acid, since it is of arginine, since the molar ratio of the latter of natural occurrence only in several plant tissues to histidine was reduced from 2.45 in the protein (5, 7) and has never been found to occur in of uninhibited cells to 2.20 in the protein of mammalian tissue. canavanine-inhibited cells. The combined ratio In the present system the ratio of the amount of canavanine q- arginine/histidine was 2.46. The of canavanine:arginine was 1:8, whereas Rabino- probability that abnormal protein synthesis in- vitz et a/. (22) found the ratio of ethionine: volving canavanine accounts in part for its un- methionine in Ehrlich ascites cells to be 1:600. usual growth-inhibitory properties in microbiologi- On the other hand, Pardee and Prestidge (18) cal and mammalian systems was discussed, as found that substitution of ethionine for methio- well as some structure-activity correlations in 4 For a discussion of these, see reference (9). amino acid antagonists.

REFERENCES 14. ~. Procedures for the Chromatographic Determina- 1. ARCHIBALD,R. M. Colorimetric Determination of Canav- tion of Amino Acids on Four Percent Cross-Linked anine. J. Biol. Chem., 165:169-78, 1946. Sulfonated Polystyrene Resins. Ibid., 211:893-906, 1954. 2. BAKER, R. S.; JOHNSON, J. E.; and Fox, S. W. Incorpora- 15. --. A Modified Ninhydrin Reagent for the Photo- tion of T-Fluorophenylalanine into Proteins of Lacto- metric Determination of Amino Acids and Related Com- bacillus arabinosus. Biochim. et Biophys. Acta, 28:318-27, pounds. Ibid., pp. 907-13. 1958. 16. MORGAN, J. F.; MORTON, H. J.; and PASIEKA, A. E. The 3. BRAWERM~, G., and YcAs, M. Incorporation of the Arginine Requirements of Tissue Culture. I. Interrelation- Amino Acid Analog Tryptazan into the Protein of Escheri- ships between Arginine and Related Compounds. J. Biol. chia coli. Arch. Biochem. & Biophys., 68:112-17, 1957. Chem. (in press). 4. Cowi~, D. B., and COHEN, G. N. Biosynthesis by Es- 17. MUNIER, R., and COHEN, G. N. Incorporation of Struc- cherichia coli of Active Altered Proteins Containing Seleni- tural Analogs of Amino Acids into Bacterial Protein. Bio- tun instead of Sulfur. Biochim. et Biophys. Acta, 26:252- chim. et Biophys. Acta, 21:592-93, 1956. 61, 1957. 18. PARDEE, A. B., and PRESTIDGE, L. S. Effects of Azatrypto- 5. FEARON, W. R., and BELL, E. A. Canavanine: Detection phan on Bacterial Enzymes and Bacteriophage. Biochim. and Occurrence in Colutea arborescens. Biochem. J., 59: et Biophys. Acta, 27:330-44, 1958. 221-24, 1955. 19. PARDEE, A. B.; SHORE, V. G.; and PRESTIDGE, L. S. In- 6. GRoss, D., and T~RVER, H. Studies on Ethionine. IV. The corporation of Azatryptophan into Proteins of Bacteria Incorporation of Ethiouine into the Proteins of Tetra- and Bacteriophage. Biochim. et Biophys. Acta, 21:406-7, hymena. J. Biol. Chem., 217:169-82, 1955. 1956. 7. KITAGAWA,M. A Diamino Acid, Canavanine, and a Mono- 20. PEARSON, H. E.; LAGERBORG, D. L.; and WI~ZLER, R. J. amino Acid, Canaline. J. Biochem. (Japan), 25:23-41, Effects of Certain Amino Acids and Related Compounds 1937. on Propagation of Mouse Encephalomyelitis Virus. Proc. 8. KITAGAWA,M., and TSUKAMOTO, J. Studies on a Diamino Soc. Exper.Biol & Med., 79:409-11, 1952. Acid, Canavanine. u The Formation of Desamino 21. PILCHER, K. S.; SOIKE, K. F.; SMITH, V. H.; TROSPER, F.; Canavanine from Canavauine. J. Biochem. (Japan), 26: and FOLSTON, B. Inhibition of Multiplication of Lee In- 373-85, 1937. fluenza Virus by Canavanine. Proc. Soc. Exper. Biol. & 9. KRus~., P. F., JR., and McCoY, T. A. The Competitive Med., 88: 79-86, 1955. Effect of Canavanine on Utilization of Arginine in Growth 22. Ri~BINOWTZ, M.; OLSON M. E.; and GREENBERO, D. M. of Walker Carcinosarcoma 256 Cells in Vitro. Cancer Re- Characteristics of the Inhibition by Ethionine of the In- search, 18: 279-82, 1958. corporation of Methionine into Proteins of the Ehrlich 10. LEvr~s, M., and TARVV,R, H. Studies on Ethionine. III. Ascites Carcinoma in Vitro. J. Biol. Chem., 9-27:217-24, Incorporation of Ethionine into Rat Proteins. J. Biol. 1957. Chem., 192: 835-50, 1951. 23. SCHWEET, R. Incorporation of Radioactive Lysine into 11. McCoY, T. A.; MAxwv.LL, M.; and NEUMXN, R. E. The Protein. Fed. Proc., 14: 277-78, 1955. Amino Acid Requirements of the Walker Carcinosarcoma 24. --. Incorporation of Radioactive Lysine into Protein. 256 in Vitro. Cancer Research, 16:979-84, 1956. Ibid., 15:850-51, 1956. 12. McCoY, T. A., and NEUMAN, R. E. The Cultivation of 25. VAUGHAN, M., and STEINBERG, D. Incorporation of p- Walker Carcinosarcoma 256 in Vitro from Cell Suspensions. Fluorophenylalanine into Crystalline Proteins. Fed. Proc., J. Nat. Cancer Inst., 16:1221-29, 1956. 17:328, 1958. 18. MOORE, S., and STEIN, W. H. Chromatography of Amino 26. YOSHIDA, A. Studies on the Mechanism of Protein Syn- Acids on Sulfonated Polystyrene Resins. J. Biol. Chem., thesis: Bacterial ~-Amylase Containing Ethionine. Bio- 192: 663-81, 1951. chim. et Biophys. Acta, 29:213-14, 1958.

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1959 American Association for Cancer Research. Incorporation of Canavanine into Protein of Walker Carcinosarcoma 256 Cells Cultured in Vitro

Paul F. Kruse, Jr., Pat B. White, Henry A. Carter, et al.

Cancer Res 1959;19:122-125.

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