JOURNAL OF BACTERIOLOGY, Dec. 1968, p. 2043-2048Copyright @ 1968 American Society for Microbiology

Vol. 96, No. 6Printed in U.S.A.

Production of L-Asparaginase II by Escherichia coliHOWARD 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 underanaerobic conditions. The enzyme was produced optimally by bacteria grownbetween 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 anaerobicgrowth in media enriched with high concentrations of a variety of amino acids.Bacteria grown under these conditions should provide a rich starting material forthe large-scale production of the enzyme. No single amino acid specifically inducedthe synthesis of the asparaginase, nor did L-asparagine, even when it was used asthe only source of nitrogen. The enzyme was produced at lower rates in the presenceof sugars; glucose was the most inhibitory.

Deamidation of L-asparagine by extracts ofEscherichia coli was first reported in 1957 byTsuji (28). E. coli was later shown to producetwo distinct asparaginases (L-asparagine amido-hydrolase, EC 3.5.1 .1) which differ in a numberof properties, perhaps most significantly intheir markedly different affinities for asparagine(5, 24, 27). The enzyme with the greater affinity,asparaginase II, appears to be located in theperiplasmic space between the bacterial plasmamembrane and the cell envelope (6). This as-paraginase has been purified to apparent ho-mogeneity (H. Whelan and J. C. Wriston, Jr.,Federation Proc., p. 586, 1968).

Since the demonstration (2, 3) that an L-asparaginase is the component in guinea pigserum which inhibits the growth of certaintumors (16, 17), there has been considerableinterest in asparaginases from various sources.Asparaginase II of E. coli even more effectivelyinhibits the growth of transplantable mouse andrat tumors in vivo (5, 20, 26); it is also activeagainst lymphoma in the dog (14, 22) and againsthuman lymphoblastic leukemia (15, 21). Thisasparaginase, which interferes with the incorpora-tion of amino acids into tumor protein in cellcultures (4), also inhibits the synthesis of proteinunder the direction of bacteriophage f2 RNAin cell-free extracts of E. coli (25). AsparaginaseI from E. coli is inactive against tumor growthand does not interfere with protein synthesisin microbial extracts (26). Asparaginase I hasthe lower affinity for asparagine.Whereas the production of asparaginase I is

unaffected by the conditions of growth, theamount of asparaginase II in the bacterial cellvaries greatly. In this paper, we report some of

the conditions which control the production ofasparaginase II.

MATERIALS AND METHODS

For most of the experiments, we used E. coli K-12wild type. We also used strain 22-64, which lackscitrate synthase (10), and strain 309-1, which lacksa-ketoglutarate dehydrogenase (B. D. Davis et al.,Federation Proc., p. 211, 1959); both are mutants oftheW strain and were obtained from B. D. Davis. Thestrain of E. coli B was obtained from J. Teller ofWorthington Biochemical Corp., Freehold, N.J.Mutants of K-12 which are unable to convert aspara-gine to aspartic acid were isolated after mutagenesiswith ethylmethanesulfonic acid (18). After treatmentwith the mutagen, cells were diluted and grown over-night on membrane filters (Millipore Co., New Bed-ford, Mass.) overlying solid growth media. Thesefilters were transferred to a detection medium, solidi-fied with 2% agar (Difco), containing 10 mM aspara-gine and 20% sucrose. After incubation for 6 hr at37 C, the membrane filters bearing the colonies wereremoved, and the underlying detection medium wascarefully blotted for 1 min with Whatman no. 1 filterpaper in order to absorb amino acids. After drying,these papers were dipped in 0.5% ninhydrin in ace-tone. The conversion of asparagine to aspartic acidwas indicated by a blue spot given by aspartic acidcorresponding to the position of a colony. Blue spotswere distinguishable from the brownish backgroundgiven by unconverted asparagine. Absence of a bluespot indicated the position of a colony with impairedconversion. Eleven mutants were isolated from 1,500colonies.

Bacteria were grown aerobically on a rotary shakerin 250-ml Erlenmeyer flasks containing a volume of20 ml of culture medium. Anaerobic growth was injars (Torsion Balance Co., Clifton, N.J.) under a mix-ture of 90% nitrogen and 10% carbon dioxide gas ata pressure of 3 psi. Bacteria were always adapted toexperimental conditions of growth for at least five

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generations. Cells were grown at 37 C, since growth ateither higher or lower temperatures yielded loweramounts of asparaginase II.

For aerobic growth, the mineral-salts medium A ofDavis and Mingioli (7) was used without citrate. Foranaerobic growth, the same medium was supple-mented with 2.4 mg of L-cystine per liter (11). Addi-tions to the minimal medium are indicated in thelegends to tables and figures. Tryptone, nutrient broth,casein hydrolysate, and yeast extract were Difcoproducts.Growth was measured turbidimetrically at a wave-

length of 570 nm with a Coleman Junior spectro-photometer. One optical density unit corresponded to0.93 mg of protein per ml, as determined, after lysisof the cells, by the method of Lowry et al. (19) withegg white lysozyme as standard. For determination ofasparaginase II, 15-ml samples were pipetted into ice;the cells were collected by centrifugation at14,000 X g, washed twice, and finally suspended in0.8 ml of 0.1 M NaCl buffered at pH 7.8 with 50 mMtris(hydroxymethyl)aminomethane-chloride. Cellswere lysed by treatment with ethylenediaminetetra-acetic acid (EDTA) and lysozyme. The suspensionwas made 1 mm in EDTA and kept at 0 C for 8 min.After the addition of magnesium acetate to a concen-tration of 1 mm, 1 mg of egg white lysozyme (Worth-ington Biochemical Corp.) was added to give a finalvolume of 1 ml. The samples were incubated for 15min at 37 C. Samples (0.01 ml) of these lysates wereassayed for asparaginase II either directly or afterdilution with buffered saline, in a final volume of 0.06ml, by following the conversion of '4C-asparagine toaspartic acid with rapid chromatography on ion-ex-change paper (Ecteola, Whatman ET81) (6). Thespecific enzymatic activity is given as micromoles ofaspartic acid produced per hour per optical densityunit of the culture. At the low concentration of aspara-gine used in this assay (0.1 mM), asparaginase I doesnot contribute significantly to the products formed(6, 26).

RESULTS

Formation of asparaginase II in standing cul-tures. Schwartz et al. (26) observed that asparagi-nase II was formed in standing cultures duringthe transition from aerobic to anaerobic growth.The time course of the appearance of the enzymeunder these conditions is shown in Fig. 1. Littleenzyme was produced by cells during aerobicgrowth. As soon as the aeration was discontinued,the specific activity increased exponentiallyuntil a plateau was reached by 40 min. Theform of these kinetics suggested that a factor,perhaps oxygen, inhibitory to the productionof the asparaginase was disappearing duringthe period following the cessation of aeration.These considerations led us to study the formationof enzyme under strict anaerobiosis.

It was important to establish beforehand thatthe enzyme is stable under aerobic conditions.Sulfhydryl reagents did not affect the enzyme

5 4.0

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0.24

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20 40 60 80 100 120 140 160 180

TIME (MIN)

FIG. 1. Time course of the appearance of aspara-ginase II in standing cultures. Bacteria were grown

aerobically with vigorous agitation in 0.05 M sodiumphosphate buffer at pH 7.5, supplemented with 1%

tryptone, 0.1% yeast extract, and 0.1% glucose. At thetime indicated by the arrow, the shaker was stopped.Samples were removed for the assay ofasparaginase II(A) andfor turbidity (B).

in vitro. Incubation of asparaginase at pH 8with 10 mm p-hydroxymercuribenzoate or withany of the alkylating agents, iodoacetate, iodo-acetamide, or N-ethylmaleimide, also at 10 mM,did not inhibit the enzyme. Asparaginase II

was not inactivated by exposure to air. Whenthe bacteria were aerated after a period of anaero-bic growth, the formation of the enzyme stopped.Even so, enzyme previously formed in the ab-sence of air remained at a constant concentrationin the aerated culture.The production of asparaginase in standing

cultures depended upon protein synthesis. Cellswere first grown aerobically. The addition ofchloramphenicol at the time when the aerationwas discontinued prevented the appearance ofthe enzyme. Dependence of enzyme formationon protein synthesis indicated that its appearanceresulted from de novo synthesis rather than fromthe conversion of a preexisting but inactiveprotein precursor.

Roberts et al. (23) recently reported that, whenE. coli is grown for 12 to 16 hr with shaking,there is a greater yield of asparaginase in airthan in an atmosphere of nitrogen. It was con-cluded that anaerobiosis depressed the formationof asparaginase II. The supply of oxygen is likelyto be limiting to cultures at high cell densities

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L-ASPARAGINASE II BY ESCHERICHIA COLI

even when shaken in air. Thus, as these workershave also shown (23), under their conditions ofcultivation, the highest concentration of as-paraginase appeared in cells as the bacteriaapproached the end of growth.

Formation of asparaginase IH during anaerobicgrowth. The experiments with standing culturessuggested that asparaginase II might be producedcontinuously and at high rates by bacteria adaptedto anaerobic conditions of growth. Bacteriaproduced asparaginase II at a constant rate formany generations during growth in anaerobicjars. Under these conditions, formation of theenzyme was stimulated greatly by the enrich-ment of the mineral-salts-glucose medium withtryptone, yeast extract, or casein hydrolysate(Table 1).

Similar results were obtained with all of thestrains of E. coli tested. Strains B and W undervarious conditions of growth had about threetimes as much asparaginase as strain K12.Nevertheless, formation of the enzyme in allstrains was stimulated to the same extent byanaerobiosis.

Since casein hydrolysate was as effective astryptone or yeast extract, we attempted to de-termine whether any single amino acid or groupof amino acids would stimulate the productionof asparaginase. Supplementation of the minimalmedium with asparagine did not stimulate theproduction of the enzyme (Table 2). Furthermore,the addition of asparagine or glutamine togetherwith the casein hydrolysate did not enhanceenzyme synthesis. Commercial casein hydrolysatecontains substances other than amino acids.Nevertheless, amino acids alone were sufficientto bring about the increased synthesis of theasparaginase. As shown in Table 2, supplementa-tion of the minimal medium with a mixture of 17amino acids resulted in the production of theenzyme at almost the same rate as that seen withcasein hydrolysate. Supplementation of the mini-mal medium with any one of the groups of amino

TABLE 1. Effect of the growth medium on theformation of asparaginase II by E. coli during

anaerobic growtha

Addition to Specificminimal medium activity

None. 0.4

Tryptone(1%). 3.6

Yeast extract (0.1%) .......... .............. 3.2Tryptone (1%) and yeast extract (0.1%)..... 3.4

Casein hydrolysate (0.2%) ........... 3.4

a Bacteria were grown in anaerobic jars inminimal medium containing 1% glucose withadditional supplementation as indicated.

TABLE 2. Effect ofamino acids on the formation ofasparaginase II by E. coli during anaerobic growtha

Addition Specificactivity

None........................... 0.4Casein hydrolysate. 3.4Asparagine........................... 0.5Casein hydrolysate and asparagine.......... 3.5I............................ 0.5II...........................0.3III............................ 0.4IV...........................0.4I +.0.8III+ IV ........................... 0. 61 + II + .1.4I+II+ IV........................... 1.5I+ II+ III+ IV......................... 3.2

a Bacteria were grown in anaerobic jars in mini-mal medium containing 1% glucose with addi-tional supplementation as indicated. The aminoacid groups contained: (1) aspartic acid, iso-leucine, lysine, methionine, and threonine; (II)glutamic acid, arginine, and proline; (III) alanine,cysteine, glycine, and serine; (IV) phenylalanine,tyrosine, tryptophan, histidine, and leucine. Thefinal concentration of each amino acid added was0.01%. When added, casein hydrolysate was at aconcentration of 0.2%, and asparagine at a con-centration of 0.1%.

acids, however, did not significantly increaseenzyme production. When two or more groupsof amino acids were combined, more enzyme wasproduced. The results for a few of the combina-tions are also shown in Table 2.

Moreover, supplementation with either amixture of vitamins (biotin, niacin, pantothenicacid, thiamine, and riboflavine, each at a con-centration of 1 ,ug/ml) or with purine andpyrimidine bases (adenine, uracil, guanine, andcytosine, each at 1 mg/ml) did not increase therate of enzyme synthesis. Casamino Acids alsocontain a substantial amount of salt; we observedno effect of sodium chloride, however, on thesynthesis of the enzyme in concentrations up to5%. Another component of commercial caseinhydrolysate is iron. The addition of ferric chlo-ride did not influence the production of the en-zyme in concentrations from 0.25 to 1 ,g/mleither in the presence or in the absence of theamino acid mixture.As with a number of other catabolic enzymes,

the formation of asparaginase II was inhibitedby the addition of sugars, particularly glucose.When xylose, maltose, or galactose was usedin place of glucose, enzyme production wasinhibited to a lesser extent. Galactose allowedthe highest rate of enzyme synthesis of all the

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CEDAR AND SCHWARTZ

sugars tested. No matter what carbon sourcewas used, however, enzyme synthesis was en-

hanced by increasing concentrations of aminoacids (Table 3). Under all conditions, the addi-tion of glucose inhibited the production of theenzyme. This inhibition is presumably an exampleof catabolite repression. The greatest amountsof enzyme were obtained in the absence of anysugar when high concentrations of amino acidswere added, although the growth rate underthese conditions was low. It can be estimatedthat, when asparaginase II was produced maxi-mally, it constituted about 0.1% of the bacterialcell protein, as the specific activity of the purifiedenzyme is approximately 20,000 ,umoles of am-monia produced per hr per mg of protein (J. C.Wriston, Jr., personal communication).During both aerobic and anaerobic growth,

E. coli was capable of using asparagine as itssole source of nitrogen. Asparagine could notbe used as the sole source of carbon, undereither aerobic or anaerobic conditions of growth.The rate of entry of the amino acid into E. coilappeared to be relatively slow, because the growthrate of an auxotroph lacking asparagine syn-thetase (Cedar and Schwartz, unpublished data)was dependent on the concentration of asparaginebelow 200 ,ug/ml. For most amino acids, therange of concentrations which limits the growthrate of E. coli is two or three orders of magnitudelower than this. Little asparaginase II was formedeither aerobically or anaerobically when aspara-gine was the sole source of nitrogen (Table 4).

In 1943, Gale (8) discussed the effect ofgrowth at various pH values on the formationof a number of activities in suspensions of in-

TABLE 3. Effect of the carbon source on theformation ofasparaginase IH by E. coli during

anaerobic growtha

Casein Specific activityhydrolysateadded () Glucose Galactose No added

None 0.4 (0.34) 0.88 (0.34) (0)0.2 3.2 (0.34) 5.2 (0.34) (0)1 6.4 (0.38) 11.2 (0.38) 12 (0.22)3 9.2 (0.43) 14 (0.43) 15.6 (0.30)

aBacteria were grown in anaerobic jars in mini-mal medium with casein hydrolysate at the concen-trations indicated. Sugars, when added, werepresent at a concentration of 1%. Specific growthrates (the reciprocal of the doubling time in hoursmultiplied by the natural logarithm of 2) underthe various conditions are indicated in paren-theses.

TABLE 4. Production of asparaginase ll whenasparagine served as the sole source

of nitrogena

Specific activityGrowth medium

Aerobic Anaerobic

A-N + asparagine.......... 0.01 0.7A.....................0.02 0.6A + casein hydrolysate.. 0.05 5.2

a Bacteria were grown under both aerobic andanaerobic conditions in minimal medium A withthe omission of ammonium sulfate (A-N) but con-taining 0.1% asparagine. For comparison, cellswere also grown in complete medium A, and inmedium A supplemented with 0.2% casein hy-drolysate. Galactose was added to all media at aconcentration of 1%.

tact E. coli. Formation of some of the deaminasesstudied was stimulated at values above pH 8,whereas decarboxylases were produced duringgrowth under acidic conditions. Asparaginase IIwas synthesized at optimal rates by bacteriagrown between pH 7 and 8.

Formation of asparaginase H by mutants ofE. coli. Changes in the concentrations of en-zymes catalyzing the reactions of the citric acidcycle are known to occur in bacteria duringanaerobic growth (12, 15). For example, a-ketoglutarate dehydrogenase is not produced(1). Mutant 309-1, which lacks this enzyme,and mutant 22-64, lacking citrate synthase,were tested for the production of asparaginaseII. Growth of these mutants aerobically wassimilar to anaerobic growth of the wild typeinasmuch as the citric acid cycle was no longerintact. Nevertheless, asparaginase HI was notproduced by either mutant during aerobicgrowth; production of the enzyme during growthin anaerobic jars was similar to that of the Wstrain wild type.We have not yet been successful in isolating

mutants lacking asparaginase -I. Mutants thatwere identified by a defect in their ability toconvert asparagine to aspartic acid when grownas colonies on solid media were found to growat about one-fifth the rate of the wild-type bac-teria in anaerobic jars. A small amount of aspara-ginase II, however, was formed in standingcultures and during the slow anaerobic growth.Thus, these mutants did not selectively lackasparaginase II, but rather were impaired intheir ability to grow under anaerobic conditions.The nature of the defect in these mutants wasnot investigated further.

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L-ASPARAGINASE II BY ESCHERICHIA COLI

DiSCUSSION

Anaerobiosis affects the formation of a num-ber of enzymes other than asparaginase II,

and many of these are involved in the degradationof amino acids. Stephenson and Gale showedthat the deamination of glutamate (27) and ofserine (9) was enhanced in bacterial suspensionsafter growth under anaerobic conditions. Glucosewas also found to inhibit the production of theseactivities. Better characterized was the catabolicL-threonine deaminase described by Wood andGunsalus (30) and later studied by Umbargerand Brown (29); Umbarger and Brown dis-tinguished this enzyme from the biosyntheticdeaminase which appears during both aerobicand anaerobic growth and which functions inthe biosynthesis of isoleucine. We know of no

studies on the effect of anaerobiosis on gluta-minase production.The appearance of specific deaminases during

anaerobic growth may offset the loss of oxidativedeamination reactions which are used aerobically.Halpern and Umbarger (13) observed that theformation of aspartase, the enzyme catalyzingthe deamination of aspartic acid to fumarateand ammonia, is dependent upon the additionof amino acids and is enhanced under anaerobicconditions (9; Marcus and Halpern, personalcommunication). The production of the enzymeis also inhibited by glucose (9). Hirsch et al. (15)showed that a fumarate reductase distinct fromsuccinate dehydrogenase is formed during anaero-bic growth when glycerol and fumarate are

provided as carbon sources. These two reactionsmay constitute a terminal deaminating pathwayin the fermentation of amino acids for whichno specific deaminases exist, since it is likelythat much of their amino nitrogen must passthrough aspartate after transamination withoxalacetic acid. Asparaginase would serve tofeed asparagine from the environment directlyinto this pathway. A final decision on the functionof asparaginase II, however, awaits the isolationof mutants which lack the enzyme.

Neither its substrate, asparagine, nor its prod-ucts, aspartic acid and ammonia, appear to actspecifically to induce the formation of as-

paraginase II. Amino acids generally stimulatedthe production of the enzyme, the rate dependingboth on the variety and on the total amountsof amino acids added. These results might reflecta system of control involving either a specificinducer, which is derived in some way from themetabolism of amino acids during anaerobicgrowth, or multiple inducers, no one of which isavailable in sufficient concentration to resultin the formation of the asparaginase when only

a few amino acids are added. Alternatively itseems to us more reasonable to consider that acommon metabolite acts as a repressor ofasparaginase synthesis, and that the utilizationof amino acids during anaerobiosis results in adecrease in the concentration of this repressingmetabolite. We can speculate that a single mech-anism controls the formation during anaerobicgrowth of a number of enzymes involved inamino acid degradation. Amino acids are en-countered as mixtures in nature, and there mightbe no advantage in regulating the synthesis ofeach enzyme individually.The use of asparaginase II in the treatment of

human neoplastic disease makes it importantthat the enzyme be produced in large quantities.The content of the asparaginase varies widelyin different strains of E. coli (5); however, in allstrains the enzyme is localized in the periplasmicregion between the plasma membrane and cellenvelope, and its production is stimulated morethan 100-fold when the bacteria are grown anaero-bically under conditions where amino acids areutilized as carbon source. Bacteria grown underthese conditions should provide a rich startingmaterial for the large-scale production of theenzyme.

ACKNOWLEDGMENTSWe thank W. K. Maas for helpful discussions. We

also thank W. K. Maas and J. D. Broome for readingthe manuscript critically.Howard Cedar is a predoctoral trainee of the

Medical Scientist Training Grant 5 T05 GM 01668-04.This investigation was supported by a Public HealthService research career grant GM 28, 125 from theNational Institute of General Medical Sciences and byNational Science Foundation research grant GB 2771.

LITERATURE CITED1. Amarasingham, C. R., and B. D. Davis. 1965.

Regulation of a-ketoglutarate dehydrogenaseformation in Escherichia coli. J. Biol. Chem.240:3664-3668.

2. Broome, J. D. 1961. Evidence that the L-aspara-ginase activity of guinea pig serum is responsiblefor its antilymphoma effects. Nature 191:1114-1115.

3. Broome, J. D. 1963. Evidence that the L-aspara-ginase of guinea pig serum is responsible for itsantilymphoma effects. I. Properties of theL-asparaginase of guinea pig serum in relationto those of the antilymphoma substance. J.Exptl. Med. 118 :99-120.

4. Broome, J. D. 1968. Studies on the mechanism oftumor inhibition by L-asparaginase. Effects ofthe enzyme on asparagine levels in the blood,normal tissues, and 6C3HED lymphomas ofmice: differences in asparagine formation andutilization in asparaginase sensitive and re-sistent lymphoma cells. J. Exptl. Med. 127:1053-1080.

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10. Gilvarg, C., and B. D. Davis. 1956. The role of thetricarboxylic acid cycle in acetate oxidation inEscherichia coli. J. Biol. Chem. 222:307-319.

11. Gorini, L. 1961. Effect of L-cystine on initiationof anaerobic growth of Escherichia coli andAerobacter aerogenes. J. Bacteriol. 82:305-312.

12. Gray, C. T., J. W. T. Wimpenny, D. E. Hughes,and M. R. Mossman. 1966. Regulation of me-tabolism in facultative bacteria. I. Structuraland functional changes in EscIherichia coli as-sociated with shifts between the aerobic andanaerobic states. Biochim. Biophys. Acta 117:22-32.

13. Halpern, Y. S., and H. E. Umbarger. 1960. Con-version of ammonia to amino groups in Escher-ichia coli. J. Bacteriol. 80:285-288.

14. Hill, J. M., J. Roberts, E. Loeb, A. Khan, A.MacLellan, and R. W. Hill. 1967. L-Asparagin-ase therapy for leukemia and other malignantneoplasms. J. Am. Med. Assoc. 202:882-888.

15. Hirsch, C. A., M. Rasminsky, B. D. Davis, andE. C. C. Lin. 1963. A fumarate reductase inEscherichia coli distinct from succinate dehy-drogenase. J. Biol. Chem. 238:3770-3774.

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17. Kidd, J. G. 1953. Regression of transplantedlymphomas induced in vivo by means of normalguinea pig serum. II. Studies on the nature ofthe active serum constituent: histological mech-anism of the regression, tests for effects of

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19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, andR. J. Randall. 1951. Protein measurement withthe Folin phenol reagent. J. Biol. Chem. 193:265-275.

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21. Oettgen, H. F., L. J. Old, E. A. Boyse, H. A.Campbell, F. S. Philips, B. D. Clarkson, L.Tallal, R. D. Leeper, M. K. Schwartz, J. H.Kim. 1967. Inhibition of leukemias in man byL-asparaginase. Cancer Res. 27:2619-2631.

22. Old, L. J., E. A. Boyse, H. A. Campbell, R. S.Brodey, J. Fidler, J. D. Teller. 1967. Treatmentof lymphosarcoma in the dog with L-asparagin-ase. Cancer 20:1066-1070.

23. Roberts, J., G. Burson, and J. M. Hill. 1968. Newprocedures for purification of L-asparaginasewith high yield from Escherichia coli. J. Bacte-riol. 95:2117-2123.

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27. Stephenson, M., and E. F. Gale. 1937. Factorsinfluencing bacterial deamination. I. The de-amination of glycine, DL-alanine and L-glu-tamic acid by Bacterium coli. Biochem. J. 31:1316-1322.

28. Tsuji, Y. 1957. Studies on the amidase. IV. Sup-plemental studies on the amidase action of thebacteria. Japan. Arch. Internal Med. 4:222-224.

29. Umbarger, H. E., and B. Brown. 1957. Threoninedeamination in Escherichia coli. II. Evidencefor two L-threonine deaminases. J. Bacteriol.73:105-112.

30. Wood, W. A., and I. C. Gunsalus. 1949. Serineand threonine deaminases of Escherichia coli:activators for a cell-free enzyme. J. Biol. Chem.181 :171-182.

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