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JOURNAL OF BACTERIOLOGY, Dec. 1968, p. 2043-2048 Vol. 96, No. 6 Copyright @ 1968 American Society for Printed in U.S.A.

Production of L- II by HOWARD CEDAR AND JAMES H. SCHWARTZ Department of Microbiology, New York University School ofMedicine, New York, New York 10016 Received for publication 30 July 1968

L-Asparaginase II was synthesized at constant rates by Escherichia coli under anaerobic conditions. The was produced optimally by grown between pH 7 and 8 at 37 C. Although some enzyme was formed aerobically, be- tween 100 and 1,000 times more asparaginase II was produced during anaerobic growth in media enriched with high concentrations of a variety of amino . Bacteria grown under these conditions should provide a rich starting material for the large-scale production of the enzyme. No single amino specifically induced the synthesis of the asparaginase, nor did L-, even when it was used as the only source of . The enzyme was produced at lower rates in the presence of sugars; was the most inhibitory.

Deamidation of L-asparagine by extracts of the conditions which control the production of Escherichia coli was first reported in 1957 by asparaginase II. Tsuji (28). E. coli was later shown to produce two distinct asparaginases (L-asparagine amido- MATERIALS AND METHODS hydrolase, EC 3.5.1 .1) which differ in a number For most of the experiments, we used E. coli K-12 of properties, perhaps most significantly in wild type. We also used strain 22-64, which lacks their markedly different affinities for asparagine (10), and strain 309-1, which lacks (5, 24, 27). The enzyme with the greater affinity, a-ketoglutarate dehydrogenase (B. D. Davis et al., to be located in the Federation Proc., p. 211, 1959); both are mutants of asparaginase II, appears the W strain and were obtained from B. D. Davis. The periplasmic space between the bacterial plasma strain of E. coli B was obtained from J. Teller of membrane and the envelope (6). This as- Worthington Biochemical Corp., Freehold, N.J. paraginase has been purified to apparent ho- Mutants of K-12 which are unable to convert aspara- mogeneity (H. Whelan and J. C. Wriston, Jr., gine to were isolated after mutagenesis Federation Proc., p. 586, 1968). with ethylmethanesulfonic acid (18). After treatment Since the demonstration (2, 3) that an L- with the , cells were diluted and grown over- asparaginase is the component in night on membrane filters (Millipore Co., New Bed- serum which inhibits the growth of certain ford, Mass.) overlying solid growth media. These has been filters were transferred to a detection medium, solidi- tumors (16, 17), there considerable fied with 2% agar (Difco), containing 10 mM aspara- interest in asparaginases from various sources. gine and 20% sucrose. After incubation for 6 hr at Asparaginase II of E. coli even more effectively 37 C, the membrane filters bearing the colonies were inhibits the growth of transplantable mouse and removed, and the underlying detection medium was rat tumors in vivo (5, 20, 26); it is also active carefully blotted for 1 min with Whatman no. 1 filter against lymphoma in the dog (14, 22) and against paper in order to absorb amino acids. After drying, human lymphoblastic leukemia (15, 21). This these papers were dipped in 0.5% ninhydrin in ace- asparaginase, which interferes with the incorpora- tone. The conversion of asparagine to aspartic acid tion of amino acids into tumor in cell was indicated by a blue spot given by aspartic acid corresponding to the position of a colony. Blue spots cultures (4), also inhibits the synthesis of protein were distinguishable from the brownish background under the direction of bacteriophage f2 RNA given by unconverted asparagine. Absence of a blue in cell-free extracts of E. coli (25). Asparaginase spot indicated the position of a colony with impaired I from E. coli is inactive against tumor growth conversion. Eleven mutants were isolated from 1,500 and does not interfere with protein synthesis colonies. in microbial extracts (26). Asparaginase I has Bacteria were grown aerobically on a rotary shaker the lower affinity for asparagine. in 250-ml Erlenmeyer flasks containing a volume of Whereas the production of asparaginase I is 20 ml of culture medium. Anaerobic growth was in jars (Torsion Balance Co., Clifton, N.J.) under a mix- unaffected by the conditions of growth, the ture of 90% nitrogen and 10% dioxide gas at amount of asparaginase II in the bacterial cell a pressure of 3 psi. Bacteria were always adapted to varies greatly. In this paper, we report some of experimental conditions of growth for at least five 2043 2044 CEDAR AND SCHWARTZ J. BACTERIOL. generations. Cells were grown at 37 C, since growth at either higher or lower temperatures yielded lower - A amounts of asparaginase II. 5 4.0 For aerobic growth, the mineral-salts medium A of 0 Davis and Mingioli (7) was used without citrate. For o 3.0 anaerobic growth, the same medium was supple- mented with 2.4 mg of L- per liter (11). Addi- z 2.0 tions to the minimal medium are indicated in the w legends to tables and figures. Tryptone, broth, 0 hydrolysate, and were Difco U- 1.0 products. a-w

Growth was measured turbidimetrically at a wave- 0 / length of 570 nm with a Coleman Junior spectro- *p pd * - photometer. One optical density unit corresponded to 0.93 mg of protein per ml, as determined, after lysis 0.24 B of the cells, by the method of Lowry et al. (19) with uo 0.12 . _. z lysozyme as standard. For determination of w asparaginase II, 15-ml samples were pipetted into ice; CO 0.06 the cells were collected by centrifugation at o 0.03 14,000 X g, washed twice, and finally suspended in a-R 0.8 ml of 0.1 M NaCl buffered at pH 7.8 with 50 mM 0 tris(hydroxymethyl)aminomethane-chloride. Cells 20 40 60 80 100 120 140 160 180 were lysed by treatment with ethylenediaminetetra- TIME (MIN) acetic acid (EDTA) and lysozyme. The suspension was made 1 mm in EDTA and kept at 0 C for 8 min. FIG. 1. Time course of the appearance of aspara- After the addition of magnesium acetate to a concen- ginase II in standing cultures. Bacteria were grown tration of 1 mm, 1 mg of egg white lysozyme (Worth- aerobically with vigorous agitation in 0.05 M sodium ington Biochemical Corp.) was added to give a final phosphate buffer at pH 7.5, supplemented with 1% volume of 1 ml. The samples were incubated for 15 tryptone, 0.1% yeast extract, and 0.1% glucose. At the min at 37 C. Samples (0.01 ml) of these lysates were time indicated by the arrow, the shaker was stopped. assayed for asparaginase II either directly or after Samples were removedfor the assay ofasparaginase II dilution with buffered saline, in a final volume of 0.06 (A) andfor turbidity (B). ml, by following the conversion of '4C-asparagine to aspartic acid with rapid on -ex- in vitro. Incubation of asparaginase at pH 8 change paper (Ecteola, Whatman ET81) (6). The with 10 mm p-hydroxymercuribenzoate or with specific enzymatic activity is given as micromoles of any of the alkylating agents, iodoacetate, iodo- aspartic acid produced per hour per optical density acetamide, or N-ethylmaleimide, also at 10 mM, low unit of the culture. At the concentration of aspara- did not inhibit the enzyme. Asparaginase II in I does gine used this assay (0.1 mM), asparaginase was exposure to air. When not contribute significantly to the products formed not inactivated by (6, 26). the bacteria were aerated after a of anaero- bic growth, the formation of the enzyme stopped. RESULTS Even so, enzyme previously formed in the ab- Formation of asparaginase II in standing cul- sence of air remained at a constant concentration tures. Schwartz et al. (26) observed that asparagi- in the aerated culture. nase II was formed in standing cultures during The production of asparaginase in standing the transition from aerobic to anaerobic growth. cultures depended upon protein synthesis. Cells The time course of the appearance of the enzyme were first grown aerobically. The addition of under these conditions is shown in Fig. 1. Little chloramphenicol at the time when the aeration enzyme was produced by cells during aerobic was discontinued prevented the appearance of growth. As soon as the aeration was discontinued, the enzyme. Dependence of enzyme formation the specific activity increased exponentially on protein synthesis indicated that its appearance until a plateau was reached by 40 min. The resulted from rather than from form of these kinetics suggested that a factor, the conversion of a preexisting but inactive perhaps , inhibitory to the production protein precursor. of the asparaginase was disappearing during Roberts et al. (23) recently reported that, when the period following the cessation of aeration. E. coli is grown for 12 to 16 hr with shaking, These considerations led us to study the formation there is a greater yield of asparaginase in air of enzyme under strict anaerobiosis. than in an of nitrogen. It was con- It was important to establish beforehand that cluded that anaerobiosis depressed the formation the enzyme is stable under aerobic conditions. of asparaginase II. The supply of oxygen is likely Sulfhydryl reagents did not affect the enzyme to be limiting to cultures at high cell densities VOL. 96, 1968 L-ASPARAGINASE II BY ESCHERICHIA COLI 2045

even when shaken in air. Thus, as these workers TABLE 2. Effect ofamino acids on the formation of have also shown (23), under their conditions of asparaginase II by E. coli during anaerobic growtha cultivation, the highest concentration of as- paraginase appeared in cells as the bacteria Addition activitySpecific approached the end of growth. Formation of asparaginase IH during anaerobic None...... 0.4 growth. The experiments with standing cultures Casein hydrolysate . 3.4 suggested that asparaginase II might be produced Asparagine...... 0.5 Casein hydrolysate and asparagine ...... 3.5 continuously and at high rates by bacteria adapted I...... 0.5 to anaerobic conditions of growth. Bacteria II...... 0.3 produced asparaginase II at a constant rate for III...... 0.4 many generations during growth in anaerobic IV...... 0.4 jars. Under these conditions, formation of the I + .0.8 enzyme was stimulated greatly by the enrich- III + IV ...... 0. 6 ment of the mineral-salts-glucose medium with 1 + II + .1.4 tryptone, yeast extract, or casein hydrolysate I+II + IV...... 1.5 (Table 1). I+ II + III + IV ...... 3.2 Similar results were obtained with all of the a Bacteria were grown in anaerobic jars in mini- strains of E. coli tested. Strains B and W under mal medium containing 1% glucose with addi- various conditions of growth had about three tional supplementation as indicated. The amino times as much asparaginase as strain K12. acid groups contained: (1) aspartic acid, iso- Nevertheless, formation of the enzyme in all , , , and ; (II) , , and ; (III) , strains was stimulated to the same extent by , , and ; (IV) , anaerobiosis. , , , and leucine. The Since casein hydrolysate was as effective as final concentration of each added was tryptone or yeast extract, we attempted to de- 0.01%. When added, casein hydrolysate was at a termine whether any single amino acid or group concentration of 0.2%, and asparagine at a con- of amino acids would stimulate the production centration of 0.1%. of asparaginase. Supplementation of the minimal medium with asparagine did not stimulate the acids, however, did not significantly increase production ofthe enzyme (Table 2). Furthermore, enzyme production. When two or more groups the addition of asparagine or together of amino acids were combined, more enzyme was with the casein hydrolysate did not enhance produced. The results for a few of the combina- enzyme synthesis. Commercial casein hydrolysate tions are also shown in Table 2. contains substances other than amino acids. Moreover, supplementation with either a Nevertheless, amino acids alone were sufficient mixture of (biotin, , pantothenic to bring about the increased synthesis of the acid, , and riboflavine, each at a con- asparaginase. As shown in Table 2, supplementa- centration of 1 ,ug/ml) or with purine and tion of the minimal medium with a mixture of 17 pyrimidine bases (adenine, uracil, guanine, and amino acids resulted in the production of the cytosine, each at 1 mg/ml) did not increase the enzyme at almost the same rate as that seen with rate of enzyme synthesis. Casamino Acids also casein hydrolysate. Supplementation of the mini- contain a substantial amount of ; we observed mal medium with any one of the groups of amino no effect of sodium chloride, however, on the synthesis of the enzyme in concentrations up to TABLE 1. Effect of the growth medium on the 5%. Another component of commercial casein formation of asparaginase II by E. coli during anaerobic growtha hydrolysate is iron. The addition of ferric chlo- ride did not influence the production of the en- Addition to Specific minimal medium zyme in concentrations from 0.25 to 1 ,g/ml activity either in the presence or in the absence of the None. 0.4 amino acid mixture. Tryptone (1%). 3.6 As with a number of other catabolic , Yeast extract (0.1%) ...... 3.2 the formation of asparaginase II was inhibited Tryptone (1%) and yeast extract (0.1%)..... 3.4 by the addition of sugars, particularly glucose. Casein hydrolysate (0.2%) ...... 3.4 When xylose, maltose, or galactose was used a Bacteria were grown in anaerobic jars in in place of glucose, enzyme production was minimal medium containing 1% glucose with inhibited to a lesser extent. Galactose allowed additional supplementation as indicated. the highest rate of enzyme synthesis of all the 2046 CEDAR AND SCHWARTZ J. BACamRmO. sugars tested. No matter what carbon source TABLE 4. Production of asparaginase ll when was used, however, enzyme synthesis was en- asparagine served as the sole source hanced by increasing concentrations of amino of nitrogena acids (Table 3). Under all conditions, the addi- tion of glucose inhibited the production of the Specific activity Growth medium enzyme. This inhibition is presumably an example of catabolite repression. The greatest amounts Aerobic Anaerobic of enzyme were obtained in the absence of any A-N + asparagine...... 0.01 0.7 sugar when high concentrations of amino acids A...... 0.02 0.6 were added, although the growth rate under A + casein hydrolysate.. 0.05 5.2 these conditions was low. It can be estimated that, when asparaginase II was produced maxi- a Bacteria were grown under both aerobic and mally, it constituted about 0.1% of the bacterial anaerobic conditions in minimal medium A with cell protein, as the specific activity of the purified the omission of ammonium sulfate (A-N) but con- enzyme is approximately 20,000 ,umoles of am- taining 0.1% asparagine. For comparison, cells monia produced per hr per mg of protein (J. C. were also grown in complete medium A, and in Wriston, Jr., personal communication). medium A supplemented with 0.2% casein hy- anaerobic growth, drolysate. Galactose was added to all media at a During both aerobic and concentration of 1%. E. coli was capable of using asparagine as its sole source of nitrogen. Asparagine could not be used as the sole source of carbon, under tact E. coli. Formation of some of the deaminases either aerobic or anaerobic conditions of growth. studied was stimulated at values above pH 8, The rate of entry of the amino acid into E. coil whereas decarboxylases were produced during appeared to be relatively slow, because the growth growth under acidic conditions. Asparaginase II rate of an auxotroph lacking asparagine syn- was synthesized at optimal rates by bacteria thetase (Cedar and Schwartz, unpublished data) grown between pH 7 and 8. was dependent on the concentration of asparagine Formation of asparaginase H by mutants of below 200 ,ug/ml. For most amino acids, the E. coli. Changes in the concentrations of en- range of concentrations which limits the growth zymes catalyzing the reactions of the rate of E. coli is two or three orders of magnitude cycle are known to occur in bacteria during lower than this. Little asparaginase II was formed anaerobic growth (12, 15). For example, a- either aerobically or anaerobically when aspara- ketoglutarate dehydrogenase is not produced gine was the sole source of nitrogen (Table 4). (1). Mutant 309-1, which lacks this enzyme, In 1943, Gale (8) discussed the effect of and mutant 22-64, lacking citrate synthase, growth at various pH values on the formation were tested for the production of asparaginase of a number of activities in suspensions of in- II. Growth of these mutants aerobically was similar to anaerobic growth of the wild type inasmuch as the was no longer TABLE 3. Effect of the carbon source on the intact. Nevertheless, asparaginase HI was not formation ofasparaginase IH by E. coli during produced by either mutant during aerobic anaerobic growtha growth; production of the enzyme during growth in anaerobic jars was similar to that of the W Casein Specific activity hydrolysate strain wild type. added () Glucose Galactose No added We have not yet been successful in isolating mutants lacking asparaginase -I. Mutants that None 0.4 (0.34) 0.88 (0.34) (0) were identified by a defect in their ability to 0.2 3.2 (0.34) 5.2 (0.34) (0) convert asparagine to aspartic acid when grown 1 6.4 (0.38) 11.2 (0.38) 12 (0.22) as colonies on solid media were found to grow 3 9.2 (0.43) 14 (0.43) 15.6 (0.30) at about one-fifth the rate of the wild-type bac- teria in anaerobic jars. A small amount of aspara- aBacteria were grown in anaerobic jars in mini- ginase II, however, was formed in standing mal medium with casein hydrolysate at the concen- cultures and during the slow anaerobic growth. trations indicated. Sugars, when added, were Thus, these mutants did not selectively lack present at a concentration of 1%. Specific growth rates (the reciprocal of the doubling time in hours asparaginase II, but rather were impaired in multiplied by the natural logarithm of 2) under their ability to grow under anaerobic conditions. the various conditions are indicated in paren- The nature of the defect in these mutants was theses. not investigated further. VOL. 96, 1968 L-ASPARAGINASE II BY ESCHERICHIA COLI 2047 DiSCUSSION a few amino acids are added. Alternatively it seems to us more reasonable to consider that a Anaerobiosis affects the formation of a num- common metabolite acts as a repressor of ber of enzymes other than asparaginase II, asparaginase synthesis, and that the utilization and many of these are involved in the degradation of amino acids during anaerobiosis results in a of amino acids. Stephenson and Gale showed decrease in the concentration of this repressing that the of glutamate (27) and of metabolite. We can speculate that a single mech- serine (9) was enhanced in bacterial suspensions anism controls the formation during anaerobic after growth under anaerobic conditions. Glucose growth of a number of enzymes involved in was also found to inhibit the production of these amino acid degradation. Amino acids are en- activities. Better characterized was the catabolic countered as mixtures in nature, and there might L-threonine deaminase described by Wood and be no advantage in regulating the synthesis of Gunsalus (30) and later studied by Umbarger each enzyme individually. and Brown (29); Umbarger and Brown dis- The use of asparaginase II in the treatment of tinguished this enzyme from the biosynthetic human neoplastic disease makes it important deaminase which appears during both aerobic that the enzyme be produced in large quantities. and anaerobic growth and which functions in The content of the asparaginase varies widely the of . We know of no in different strains of E. coli (5); however, in all studies on the effect of anaerobiosis on gluta- strains the enzyme is localized in the periplasmic minase production. region between the plasma membrane and cell The appearance of specific deaminases during envelope, and its production is stimulated more anaerobic growth may offset the loss of oxidative than 100-fold when the bacteria are grown anaero- deamination reactions which are used aerobically. bically under conditions where amino acids are Halpern and Umbarger (13) observed that the utilized as carbon source. Bacteria grown under formation of aspartase, the enzyme catalyzing these conditions should provide a rich starting the deamination of aspartic acid to fumarate material for the large-scale production of the and , is dependent upon the addition enzyme. of amino acids and is enhanced under anaerobic ACKNOWLEDGMENTS conditions (9; Marcus and Halpern, personal We thank W. K. Maas for helpful discussions. We communication). The production of the enzyme also thank W. K. Maas and J. D. Broome for reading is also inhibited by glucose (9). Hirsch et al. (15) the manuscript critically. showed that a fumarate reductase distinct from Howard Cedar is a predoctoral trainee of the succinate dehydrogenase is formed during anaero- Medical Scientist Training Grant 5 T05 GM 01668-04. bic growth when glycerol and fumarate are This investigation was supported by a Public Health provided as carbon sources. These two reactions Service research career grant GM 28, 125 from the may constitute a terminal deaminating pathway National Institute of General Medical Sciences and by in the of amino acids for which National Science Foundation research grant GB 2771. no specific deaminases exist, since it is likely LITERATURE CITED that much of their amino nitrogen must pass 1. Amarasingham, C. R., and B. D. Davis. 1965. through aspartate after with Regulation of a-ketoglutarate dehydrogenase oxalacetic acid. Asparaginase would serve to formation in Escherichia coli. J. Biol. Chem. feed asparagine from the environment directly 240:3664-3668. into this pathway. A final decision on the function 2. Broome, J. D. 1961. Evidence that the L-aspara- of asparaginase II, however, awaits the isolation ginase activity ofguinea pig serum is responsible for its antilymphoma effects. Nature 191:1114- of mutants which lack the enzyme. 1115. Neither its substrate, asparagine, nor its prod- 3. Broome, J. D. 1963. Evidence that the L-aspara- ucts, aspartic acid and ammonia, appear to act ginase of guinea pig serum is responsible for its specifically to induce the formation of as- antilymphoma effects. I. Properties of the paraginase II. Amino acids generally stimulated L-asparaginase of guinea pig serum in relation the production of the enzyme, the rate depending to those of the antilymphoma substance. J. both on the variety and on the total amounts Exptl. Med. 118 :99-120. of amino acids added. These results might reflect 4. Broome, J. D. 1968. Studies on the mechanism of a system of control involving either a specific tumor inhibition by L-asparaginase. Effects of the enzyme on asparagine levels in the , inducer, which is derived in some way from the normal tissues, and 6C3HED lymphomas of of amino acids during anaerobic mice: differences in asparagine formation and growth, or multiple inducers, no one of which is utilization in asparaginase sensitive and re- available in sufficient concentration to result sistent lymphoma cells. J. Exptl. Med. 127: in the formation of the asparaginase when only 1053-1080. 2048 CEDAR ANE) SCHWARTZ J. BACTERIOL.

5. Campbell, H. A., L. T. Mashburn, E. A. Boyse, guinea pig serum on lymphoma cells in vitro, and L. J. Old. 1967. Two L-asparaginases from discussion. J. Exptl. Med. 98:583-606. Escherichia coli B. Their separation, purifica- 18. Loveless, A., and S. Howarth. 1959. of tion, and antitumor activity. bacteria at high levels of survival by ethyl 6:721-730. sulphonate. Nature 184:1780-1782. 6. Cedar, H., and J. H. Schwartz. 1967. Localization 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and of the two L-asparaginases in anaerobically R. J. Randall. 1951. Protein measurement with grown Escherichia coli. J. Biol. Chem. 242: the Folin phenol reagent. J. Biol. Chem. 193: 3753-3754. 265-275. 7. Davis, B. D., and E. S. Mingioli. 1950. Mutants 20. Mashburn, L. T., and J. C. Wriston. 1964. Tumor of Escherichia coli requiring methionine or inhibition effect of L-asparaginase from Escher- B12. J. Bacteriol. 60:17-28. ichia coli. Arch. Biochem. Biophys. 105:450- 8. Gale, E. F. 1943. Factors influencing the enzymic 452. activities of bacteria. Bacteriol. Rev. 7:139-173. 21. Oettgen, H. F., L. J. Old, E. A. Boyse, H. A. 9. Gale, E. F., and M. Stephenson. 1938. Factors Campbell, F. S. Philips, B. D. Clarkson, L. influencing bacterial deamination. II. Factors Tallal, R. D. Leeper, M. K. Schwartz, J. H. influencing the activity of DL-serine deaminase Kim. 1967. Inhibition of leukemias in man by in Bacterium coli. Biochem. J. 32:392-404. L-asparaginase. Cancer Res. 27:2619-2631. 10. Gilvarg, C., and B. D. Davis. 1956. The role of the 22. Old, L. J., E. A. Boyse, H. A. Campbell, R. S. tricarboxylic acid cycle in acetate oxidation in Brodey, J. Fidler, J. D. Teller. 1967. Treatment Escherichia coli. J. Biol. Chem. 222:307-319. of lymphosarcoma in the dog with L-asparagin- 11. Gorini, L. 1961. Effect of L-cystine on initiation ase. Cancer 20:1066-1070. of anaerobic growth of Escherichia coli and 23. Roberts, J., G. Burson, and J. M. Hill. 1968. New Aerobacter aerogenes. J. Bacteriol. 82:305-312. procedures for purification of L-asparaginase 12. Gray, C. T., J. W. T. Wimpenny, D. E. Hughes, with high yield from Escherichia coli. J. Bacte- and M. R. Mossman. 1966. Regulation of me- riol. 95:2117-2123. tabolism in facultative bacteria. I. Structural 24. Roberts, J., M. D. Prager, and N. Bachynsky. and functional changes in EscIherichia coli as- 1966. The antitumor activity of Escherichia coli sociated with shifts between the aerobic and L-asparaginase. Cancer Res. 26:2213-2217. anaerobic states. Biochim. Biophys. Acta 117: 25. Schwartz, J. H. 1965. An effect of streptomycin 22-32. on the biosynthesis of the coat protein of 13. Halpern, Y. S., and H. E. Umbarger. 1960. Con- coliphage f2 by extracts of E. coli. Proc. Natl. version of ammonia to amino groups in Escher- Acad. Sci. U.S. 53:1133-1140. ichia coli. J. Bacteriol. 80:285-288. 26. Schwartz, J. H., J. Y. Reeves, and J. D. Broome. 14. Hill, J. M., J. Roberts, E. Loeb, A. Khan, A. 1966. Two L-asparaginases from E. coli and MacLellan, and R. W. Hill. 1967. L-Asparagin- their action against tumors. Proc. NatI. Acad. ase therapy for leukemia and other malignant Sci. U.S. 56:1516-1519. neoplasms. J. Am. Med. Assoc. 202:882-888. 27. Stephenson, M., and E. F. Gale. 1937. Factors 15. Hirsch, C. A., M. Rasminsky, B. D. Davis, and influencing bacterial deamination. I. The de- E. C. C. Lin. 1963. A fumarate reductase in amination of glycine, DL-alanine and L-glu- Escherichia coli distinct from succinate dehy- tamic acid by Bacterium coli. Biochem. J. 31: drogenase. J. Biol. Chem. 238:3770-3774. 1316-1322. 16. Kidd, J. G. 1953. Regression of transplanted 28. Tsuji, Y. 1957. Studies on the . IV. Sup- lymphomas induced in vivo by means of normal plemental studies on the amidase action of the guinea pig serum. I. Course of transplanted bacteria. Japan. Arch. Internal Med. 4:222-224. cancers of various kinds in mice and rats given 29. Umbarger, H. E., and B. Brown. 1957. Threonine guinea pig serum, horse serum, or rabbit serum. deamination in Escherichia coli. II. Evidence J. Exptl. Med. 98:565-582. for two L-threonine deaminases. J. Bacteriol. 17. Kidd, J. G. 1953. Regression of transplanted 73:105-112. lymphomas induced in vivo by means of normal 30. Wood, W. A., and I. C. Gunsalus. 1949. Serine guinea pig serum. II. Studies on the nature of and threonine deaminases of Escherichia coli: the active serum constituent: histological mech- activators for a cell-free enzyme. J. Biol. Chem. anism of the regression, tests for effects of 181 :171-182.