ARGININE METABOLISM IN PLEUROPNEUMONIA-LIKE ORGANISMSISOLATED FROM MAMMALIAN CELL CULTURE

ROBERT T. SCHIMKE AND MICHAEL F. BARILENational Institute of Arthritis and Metabolic Diseases, and Division of Biologics Standards,

National Institutes of Health, U.S. Public Health Service, Bethesda, Maryland

Received for publication 6 March 1963

ABSTRACTSCHIMKE, ROBERT T. (National Institutes of

Health, Bethesda, Md.) AND MICHAEL F. BARILE.Arginine metabolism in pleuropneumonia-likeorganisms isolated from mammalian cell culture.J. Bacteriol. 86:195-206. 1963.-Arginine degra-dation is a significant metabolic process forpleuropneumonia-like organisms (PPLO; Myco-plasma) isolated from cell culture. The conversionof arginine to ornithine in PPLO-contaminatedcell culture was rapid, and occurred by the ar-ginine dihydrolase pathway involving argininedeiminase, ornithine transcarbamylase, andcarbamyl phosphokinase. In the absence of PPLOcontamination, arginine conversion to ornithinewas minimal and took place by an arginase ac-tivity present in the cell culture, but not in thePPLO. All five PPLO strains isolated from cellculture accomplished the conversion of arginineto ornithine, and contained the requisite enzymeof the arginine dihydrolase system, whereasPPLO-free cell cultures did not. Supplementationof PPLO culture broth with arginine increasedthe extent of PPLO growth. When the argininecontent of the culture limited growth, argininewas completely converted to ornithine. When

During the course of studies of enzymes ofarginine metabolism in cell culture (Schimke,1962a), we were unable to confirm the rapidconversion of arginine to ornithine by cell cultureas found by Piez and Eagle (1958) and Mansonand Thomas (1960). An explanation for thisdifference has been suggested by the studies ofRouse and Bonifas (1962) and Kenny and Pollock(1962), which indicate that cell cultures con-taminated with pleuropneumonia-like organisms(PPLO; Mycoplasma) rapidly deplete the mediumof arginine. Since it has been established thatcontinuous cell cultures are frequently contami-nated with PPLO or L forms of bacteria or both(Robinson, Wichelhausen, and Roizman, 1956;Collier, 1957; Pollock, Kenny, and Syverton,1960; Barile, Malizia, and Riggs, 1962), the needto re-examine arginine degradation in cell cul-tures has become evident. This report deals witharginine degradation in a serially propagated cellculture (HeLa-S3), with and without PPLO con-tamination, and some of the properties of argininedegradation in broth cultures of PPLO isolatedfrom contaminated cell culture.Two known pathways for arginine degradation

resulting in ornithine as a lproduct are:

arginine arginae ornithine + urea

(a.) arginine rginine deiminaseI citrtlline + NH3

(b.) citrulline + P ornithine transcarbamylase ornithine + carbamyl phosphate

(c.) carbamyl phosphate + ADPcarbamyl phosphokinase

Mg++ATP + NH3 + CO2

growth was limited in the presence of excess

arginine, citrulline was the major breakdownproduct. It is suggested that the conversion ofarginine to ornithing constitutes a significant,and possibly major, source of adenosine triphos-phate for this class of organisms.

Reaction 1 is the familiar pathway in ureotelicanimals (Cohen and Brown, 1960) of argininedegradation mediated by the enzyme arginase.Reaction 2, which constitutes the arginine di-hydrolase system first described by Hills (1940),has been described in a number of microorgan-isms, including Streptococcus (Hills, 1940), Lacto-bacillus (Walker, 1953), Clostridium (Schmidt,

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Logan, and Tytell, 1952), Pseudomonas (Horn,1933), and yeast (Roche and Lacombe, 1952).It has also been found to occur in two humanstrains of PPLO (Smith, 1955, 1957).

Evidence will be presented to indicate thatarginine breakdown to ornithine in noncontami-nated HeLa-S3 is minimal and proceeds by re-action 1. In PPLO-infected HeLa-S3, argininedegradation is extensive and proceeds by reaction2. All five PPLO isolates obtained from contami-nated tissue culture were capable of degradingarginine to ornithine, and contained argininedeiminase, ornithine transcarbamylase, and car-bamyl phosphokinase activities. Noncontami-nated tissue culture extracts, on the other hand,contained arginase activity, but none of the en-zyme activities of reaction 2.

MATERIALS AND METHODS

Cell culture. HeLa-S3 in suspension culture waskindly supplied by L. Levintow, National In-stitute of Allergy and Infectious Diseases. Thecells were maintained at concentrations of 2 to 5X 105 cells/ml in Eagle's (1959) spinner mediumsupplemented with 5% dialyzed horse serum.The cells were transferred to monolayer by in-oculation of 2 to 4 X 104 cells into Eagle's (1959)basal medium supplemented with 5% dialyzedhorse serum. This cell line in suspension or mono-layer was consistently found by cultural methods(Barile, Yaguchi, and Eveland, 1958) to be freefrom PPLO.PPLO. The strains used were isolated from

continuous cell cultures (Barile et al., 1962).They were tissue culture PPLO strain HEp-2(human epidermoid carcinoma of skin-Fjelde),S3 (human carcinoma of cervix-Eagle), MS(monkey kidney stable-Tytell), CL (humanliver-Chang), ERKS (embryonic rabbit kidneystable-Westwood). PPLO were maintainedroutinely by infecting PPLO-free primary rabbit-kidney cells (Youngner, 1954) grown at 37 C inmedium No. 199 (Morgan, Morton, and Parker,1950) containing 5% rabbit serum. The kidneycells were fed every 3 to 4 days. The supernatantfluids containing from 105 to 107 PPLO per mlwere used as the working PPLO suspension.PPLO were grown in a medium of Brain HeartInfusion (Difco) broth with 15% horse serumand 1% yeast extract added (Barile et al., 1958).The broth cultures were incubated aerobicallyat 36 C for 1 to 4 days. A dilution-plate count

method was used for the estimation of PPLOpopulation. The PPLO suspension was dilutedin 0.07 M sodium phosphate (pH 7.4) containing0.85% NaCl and 0.2% gelatin; 0.1-ml sampleswere placed on duplicate human blood-y-east agarplates (Barile et al., 1958). The plates were in-cubated aerobically at 36 C for 7 days. Agarmedia were examined for PPLO growth with astereoscopic dissecting microscope. The PPLOcolonies were counted, and the population wasdetermined.Enzyme assay. The PPLO of broth cultures

were sedimented by centrifugation at 15,000 Xg for 15 min. The sedimented cells were washedwith 0.85% NaCl, resuspended in 1 to 4 ml of0.1 M potassium phosphate (pH 6.5), and exposedto ultrasonic radiation for 15 min in a Raytheon10-kc sonic oscillator. The cellular debris wasremoved by centrifugation, and the resulting ex-tract was dialyzed 3 to 4 hr against 0.1 M potas-sium phosphate (pH 6.5) or, when phosphate wasto be excluded, against 0.1 M imidazole Cl (pH6.5). All enzyme assays were performed in du-plicate. Cell-culture cells (HeLa-S3) were scrapedfree from the glass with a Teflon policeman,centrifuged for 10 min at 500 X g, washed withserum-free Eagle's medium, and disrupted asdescribed for the PPLO suspensions. The result-ing extract was used for enzyme assays.

Arginase was assayed as described previously(Schimke, 1962b). Urease was assayed in a sys-tem containing (in 1.0 ml): 10 ,moles of C14 urea(New England Nuclear Corp., Boston, Mass.),specific activity 0.02 mc/mmole; 50 umoles ofpotassium phosphate (pH 6.5); and 1 to 2 mgof extract protein. The incubations were per-formed for 60 min at 37 C. The C1402 liberatedwas collected and measured as described pre-viously (Schimke, 1962a). Blanks with boiledextract were also incubated for 60 min.

Arginine deiminase was assayed by measuringthe rate of citrulline formation. The assay me-dium contained (in 0.5 ml): 50 ,.umoles of potas-sium phosphate (pH 6.5); 50,moles of L-arginine;and 0.5 to 1.0 mg of extract protein. Samples werewithdrawn at 5- to 10-min intervals and assayedfor citrulline by the method of Archibald (1944),as modified by Ratner (1955). Controls consistingof extract without L-arginine and L-argininewithout extract were included with each assay.

Ornithine transcarbamylase and carbamylphosphokinase were assayed by the methods of

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Jones (1962) for measuring citrulline formation.Carbamyl phosphokinase was assayed by cou-pling with ornithine transcarbamylase isolatedfrom rat liver (Burnett and Cohen, 1957). Car-bamyl phosphate, lithium salt, was obtainedfrom Calbiochem.

Arginine, citrulline, ornithine, and urea. PPLOcultures were prepared for estimation of arginine,ornithine, and urea as follows. PPLO were re-moved by centrifugation at 15,000 X g for 10min. Protein in the supernatant fluid was pre-cipitated with trichloroacetic acid at a finalconcentration of 10%. The trichloroacetic acidwas removed by extraction with ether. Argininewas determined by the method of Sakaguchi(1925) as modified by Van Pilsum et al. (1956).Citrulline was determined by the method ofArchibald (1944) as modified by Ratner (1955).Ornithine was measured by enzymatic conversionto citrulline with ornithine transcarbamylase andadded carbamyl phosphate, as outlined by Bur-nett and Cohen (1957). In some experiments, itwas desirable to separate ornithine from citrullinefor accurate quantitative estimates. This wasaccomplished by passing the solution through acolumn (0.3 x 1.0 cm) of Amberlite CG-50 (NH4+form) which retains only the ornithine. Theornithine could then be readily eluted with 2 MNH40H. The ammonia was removed by vacuumdistillation. Urea, as derived from C14-arginine,was measured as described in the urease assay.

Radiochemicals. Guanido-C14-L-arginine was ob-tained from the ChemTrac Corp., Cambridge,Mass.; uniformly labeled C14-L-arginine fromNuclear Chicago Corp., Chicago, Ill.; and ureido-C14-L-citrulline from New England Nuclear Corp.,Boston, Mass. All radiochemicals were subjectedto column chromatography before use to insureabsolute purity. Arginine was chromatographedon an Amberlite CG-50 (NH4+ form) column(1 x 15 cm) with a linear 0 to 2.4 N NH40H gra-dient (see Fig. 1 and 2). Citrulline was chro-matographed on a Dowex-50 X 2 (H+ form)column (0.5 x 10 cm) with a linear 0 to 3 N HCIgradient (see Fig. 3). Samples were counted in aNuclear Chicago gas-flow counter. Similar tech-niques were used for determinations of radio-active products of arginine breakdown in cellculture (Fig. 1, 2, and 3). The measurement ofC1"02 derived from ureido-C14-citrulline (see re-action 2 involving ornithine transcarbamylaseand carbamyl phosphokinase) was accomp)lished

in a manner similar to that used for the ureaseassay.

Protein was measured by the method of Lowryet al. (1951), using crystalline bovine albumin asstandard.

RESULTS

C04-L-arginine breakdown in HeLa-S3 tissueculture with and without PPLO contamination.Figures 1 and 2 show the breakdown products ofuniformly labeled C'4-L-arginine in the mediumof noninfected HeLa-S3 and HeLa-S3 infectedwith a mixture of five cell-culture PPLO strains.Three peaks of radioactivity were found onchromatography of samples of medium on Amber-lite CG-50 columns. The first peak represents theradioactive products of arginine breakdown notadsorbed by the resin. The second and thirdpeaks are ornithine and arginine, respectively,and are eluted with the linear 0 to 2.4 N NH40Hgradient. The pattern of arginine products issimilar in the PPLO-free HeLa-S3 and in mediumwithout any cells (Fig. 1). In contrast, the PPLO-contaminated cells (Fig. 2) show extensive ar-ginine breakdown. The decrease in arginine wasnot accompanied by an equivalent increase inornithine, particularly in the flask incubated for12 hr (Fig. 2). In this case, a substantial amountof radioactivity was not retained on the column.To characterize further the more acidic ar-

ginine breakdown product(s) not retained onthe Amberlite CG-50 column, the nonabsorbedmaterial from the 12-hr sample was chromato-graphed on Dowex-50 X 2 (H+ form) with alinear 0 to 3 N HCl gradient (total volume 400ml). The elution pattern shown in Fig. 3 showsa number of discrete peaks, with the majorityof radioactivity presented in a single peak. Thefractions from this peak were pooled, concen-trated, and further chromatographed on paperin four solvent systems (I. Smith, 1960): phenol-ethanol-water-ammonia (75:20:9:1); phenol-water (80:20); methanol-pyridine-water (20:5:1); and butanol-water-pyridine (1:1:1). Ineach case, a single ninhydrin spot was l)resentwhich also contained the radioactivity, and whichco-chromatographed with authentic citrulline.In addition, the test material produced a chromo-gen identical to that obtained with citrulline whentested by the colorimetric method of Archibald(1944). When tested by the arsenolysis reactionwith purified ornithine transcarbamylase from

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10 20FRACTION NUMBER

FIG. 1. C14-arginine breakdown by controls con-

taining medium only or uninfected HeLa-S3. HeLa-S3 grown in suspension culture was transferred tomonolayer and maintained after transferfor 2 weeks.Uniformly labeled C14-arginine (specific activity, 0.77mc/mmole) was then introduced with a change of me-

dium at a concentration of 0.6 mM (total volume, 25ml). The controls were incubated at 37 C for 24 hrwith the added C'4-arginine. One-half of each me-

dium from which protein was removed was chromato-graphed on an Amberlite CG-50 (NH4+ form)column (0.5 X 15 cm). The column was prepared bywashing the resin with 4 N NH40H and then withwater until the pH was 8.5. The samples of mediumwere washed through the column with 30 ml of water.A linear NH40H gradient from 0 to 2.4 N (totalvolume, 150 ml) was then begun; 4- to 5-ml fractionswere collected, and the radioactivity of each fractionwas determined.

liver (conditions similar to those in Table 4), thetest material was converted to a compound thatco-chromatographed with ornithine, and whichwas retained on an Amberlite CG-50 column.

8

0

C-).::I 4

uLLx

30C-)

0-

30 0

J. BACTERIOL.

10 20 30FRACTION NUMBER

FIG. 2. C'4-arginine breakdown by PPLO-in-fected HeLa-S3 cells. HeLa-S3 grown in suspensionculture was transferred to monolayer and main-tained after transfer for 2 weeks. Some of the flaskswere then infected with 3.5 X 103 cells of a mixtureof five strains of PPLO isolated from other cell-cul-ture lines and grown afurther 14 days. Uniformly la-beled C'4-arginine (specific activity, 0.77 mc/mmole)was then introduced with a change of medium at a

concentration of 0.6 mM (total volume, 25 ml). Priorto the introduction of the C14-L-arginine, the de-canted medium contained 5.6 X 106 PPLO/ml. ThePPLO-infected cells were incubated at 37 C for 24 hrwith the added C'4-arginine. One flask was incubatedfor only 12 hr. One-half of each nmediutm from whichprotein was removed was chromatographed on an

Amberlite CG-50 (NH4+form) column (0.5 X 15 cm).The column was prepared by washing the resin with4 N NH40H and then with water until the pH was

8.5. The samples of medium were washed throughthe column with 30 ml of water. A linear NH40Hgradient from 0 to 2.4 N (total volume, 150 ml) was

then begun; 4- to 5-ml fractions were collected andthe radioactivity of each fraction was determined.

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Thus, it is shown that the major product of ar-

ginine breakdown not retained on the AmberliteCG-50 column of Fig. 1 and 2 is citrulline.The presence of urea in fractions 12 through 15

from the Dowex-50 X 2 column (Fig. 3) was lo-cated by incubating samples of each fraction withurease and measuring C1402 formation. It is evi-dent that urea is not a major product of argininebreakdown in PPLO-infected tissue culture. Theother radioactive peaks have not been furtheridentified, and probably result from assimilationof C'402 liberated during citrulline breakdown(reaction 2).The calculated stoichiometry of arginine break-

down in these experiments is shown in Table 1.In the absence of any cells, arginine depletion oc-

curred to an extent of 1.9% of the original ar-

ginine present. This was accompanied by the ap-

pearance of stoichiometric amounts of urea andornithine. The formation of ornithine and urea

was not observed in Eagle's medium devoid ofserum. This conversion in the absence of any cellsis tentatively attributed to arginase activity pres-

ent in the added serum. The addition of PPLO-free cells increased the formation of urea and or-

nithine only slightly. The depletion of arginine inthe noninfected cells was largely accounted for by

6N HCI

0LINEAR GRADIENT 0-3N HCI

0

20-

CITRULLINE-

no 15

0

X< 10

UREA

5/0

4 12 20 28 36 44 52 60 68 76FRACTION NUMBER

FIG. 3. Chromatography on Amberlite CG-S0 ofnonadsorbed counts of medium from PPLO-infectedHeLa-S3 cells grown in uniformly labeled L-C14-arginine. The nonadsorbed counts from PPLO-in-fected medium (12-hr incubation of Fig. 1) chromato-graphed on Amberlite CG-50 were placed on a Dowex50 X 2 (H+ form) column (100 to 200 mesh; 0.5 X10 cm) and eluted with a linear HCl gradient from0 to 3 N (total volume, 400 ml); 5-ml fractions were

collected and samples were counted for radioactivityin a Nuclear Chicago gas-flow counter.

TABLE 1. Stoichiometry of arginine metabolism byPPLO-infected HeLa-S3

Condition

No cellsHeLa-S3HeLa-S3 +PPLO

HeLa-S, +PPLO

Time

hr

242412

24

Per cent of potential yield from totalC'4-L-arginne initially present

Argin-ine de-pleted

1.911.177.9

98.3

Urea Orni-furea thine

fudfound

1.52.61.6

1.7

1.62.9

42.1

83.1

Citrul-linefound

0

0

21.6

13

Argin-ine

presentin a cellprotein*

-t8.5

5.8

* Cell protein was prepared for counting by themethod of Siekevitz (1951), but Cl2 arginine was

used in the trichloroacetic acid washes.

t Not determined.

TABLE 2. Specific activities of urease and arginasein PPLO-infe ted HeLa-S3*

CultureSpecific activity

Arginase Urease

Uninfected HeLa-S3 ...... 3.2 0PPLO-HeLa-S3 ........... 1.1 0

* Enzyme assays were performed in cells grownin parallel with those used for the experimentsindicated in Fig. 1. Specific activity expressed asumnoles of product per mg of protein per hr.

incorporation into cellular protein as a result ofthe continued growth of the culture. In contrastto the PPLO-free culture, arginine depletion inthe contaminated cultures was accounted forlargely by the appearance of ornithine and smallamounts of citrulline. It is to be noted that thedepletion of arginine is not accompanied by in-creased formation of urea.The extensive breakdown of arginine to or-

nithine by PPLO-infected tissue culture is not theresult of an arginase activity. For the data ofTable 1 to be the result of arginase, an activeurease would be required to degrade urea to am-monia and carbon dioxide, thereby accounting forthe lack of equivalence of urea and ornithineformed. Table 2 indicates that urease was notpresent in extracts of either PPLO-free or PPLO-contaminated HeLa-S3. It was also found thatthe uninfected cell culture contained more ar-

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TABLE 3. Stoichiometry of arginine deiniinaseof PPLO extracts

Time Arginine* Citrulline NH3t

min JAmoles/ml

15 - 18.2 + 17.6 + 16.830 - 35.7 + 34.4 + 33.960 - 47.2 + 46.2 + 50.2

* Initial arginine concentration was 50usmoles/ml.

t Ammonia was determined by the method ofBessman and Bessman (1955).

TABLE 4. Requirements for the formation of C1402from C'4-ureido-citrulline by PPLO extracts

Condition C1402 released

pAmoles

Complete system*.10.6- ADP, Pl, Mg.0.15- ADP, Mg, PI; + 10 ,umoles ofK arsenate.13.2

-ADP.0.40-P1.0.43- MgC2.1.42Boiled extract.0.005

* Complete system contained (in 1 ml): 50umoles of imidazole-Cl (pH 6.5); 25j,moles of C14-ureido-L-citrulline, specific activity 1.5 mc/mmole; 10,UM potassium phosphate (pH 6.5); 10Amoles of ADP; 10,moles of MgCl2; and 0.7 mgof extract protein. Incubated for 60 min.

TABLE 5. Stoichiometry of conversion of citrullineto ornithine by PPLO extracts

Component Amount

,&moles

Citrulline*.-8.6Ornithine... +8.6NH3t.+7.9C02t.+8.8

* Reaction medium initially contained 25,umoles of ureido-C14-L-citrulline.

t Ammonia estimated by the method of Bess-man and Bessman (1955).

t Estimated as C1402 .

ginase activity than that infected with PPLO.The contamination of HeLa-S3 with each of thefive PPLO strains separately was also found todeplete the medium of arginine, as measured bycolorimetric methods.

Studies of arginine breakdown to ornithine byother cell-culture lines, including KB (obtained asa spinner culture from Microbiological Associates,Bethesda, Md.), and a strain of L cells supplied byR. A. Fleishman, National Institute of Allergyand Infectious Diseases, have indicated that theformation of ornithine by these cell lines occursto an extent of not more than 2% of the argininepresent in the medium (0.6 mM) as determined bycolorimetric estimates.

Studies with isolated PPLO. The following stud-ies were undertaken to demonstrate that arginineis actively degraded by way of reaction 2 in ex-tracts of PPLO, i.e., by the combined action ofarginine deiminase, ornithine transcarbamylase,and carbamyl phosphokinase.

Arginine conversion to citrulline (arginine deim-inase). Dialyzed extracts of a mixed culture of thefive strains grown in broth were capable of de-grading arginine to citrulline and ammonia instoichiometric amounts (Table 3) in a system con-taining 0.05 M potassium phosphate (pH 6.5),0.05 M L-arginine, and 0.7 mg of protein, in a vol-ume of 1 ml. With the same dialyzed PPLO ex-tracts in a system consisting of 50 ,umoles of po-tassium phosphate (pH 6.5) and 25 ,umoles ofC'4-argininosuccinic acid (Schimke, 1962a), citrul-line was not detected by color reaction or paperchromatography. Thus, it is shown that the con-version of arginine to citrulline was not due to areversal of the bacterial and animal biosyntheticpathway (see review of Cohen and Brown, 1960)involving argininosuccinate synthetase and ar-ginosuccinase.

Conversion of citrulline to ornithine (ornithinetranscarbamylase and carbamyl phosphokinase).Table 4 indicates that, in extracts of PPLO di-alyzed against 0.05 M imidazole Cl (pH 6.5),adenosine diphosphate (ADP), inorganic phos-phate, and Mg++ were required for the conversionof citrulline to ornithine as determined by C1402formation, and that arsenate could substitute forthese requirements. Table 5 indicates that, in asystem similar to that used in Table 4, for everymole of citrulline converted to ornithine, 1 moleof carbon dioxide and 1 mole of ammonia wereformed.

Since the previous studies were performed withmixed cultures of all five PPLO strains, studies ofenzymes of arginine degradation were undertakenfor each strain. Table 6 indicates that extracts ofeach PPLO isolate contain the necessary enzymes

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for conversion of arginine to ornithine via citrul-line, but essentially no arginase activity. Noattempt was made to quantitate the specific ac-

tivities with the strains CL, MK, and S3 becauseof the small amount of material available, leadingto quantitatively inaccurate values. However, thespecific enzyme activities were of the same orderof magnitude as those found for ERKS andHEp-2.

Characteristics of arginine breakdown by intactPPLO in isolated culture. Table 7 indicates thatthe breakdown of arginine by PPLO does not re-

quire the presence of tissue culture cells, but oc-

curs readily whenthe organisms are growninPPLObroth medium. The five PPLO strains were grownfor 4 days, at the end of which time samples of theculture were used for estimates of PPLO popula-tion and arginine, citrulline, and ornithine deter-

TABLE 6. Activities of arginase, arginine deiminase,ornithine transcarbamylase, and carbamyl phos-phokinase in extracts of PPLO originally iso-lated from contaminated cell cultures*

Arginine Ornithine CarbamylPPLO strains Arginase demnase transcar- phospho-diiaebamylas-e kinase

ERKS ....... .005 3.0 2.1 0.50HEp-2. .... .005 2.6 3.1 0.27CL ........ 0.005 +t + +MS.0. O .005 + + +S3........ 0.005 + + +

* Enzyme activities are expressed as ,umoles ofproduct formed per mg per min.

t Enzyme activities are only noted as beingpresent. The small quantity of each sample madeaccurate quantitative estimates of special ac-

tivity impossible.

TABLE 7. Arginine breakdown by PPLOin broth cultures

Amount (,umoles/ml)Strain time of harvest

Arginine Citrulline Ornithine

Broth only. 12.4* 0 0ERKS. 5.6 X 108 -12.21 +0.12 +11.80HEp-2..... 6.7 X 107 -9.87 +0.37 +9.00MK 106 -2.79 +0.02 +2.54

CL 103, <104 -1.64 +0.01 +1.46

S3...... 103, <104 -0.62 +0.01 +0.41

* Original arginine content of broth medium.

CD

cD 25': 20- 15CD

S 10

5

00 60 120 180 0 60 120 180

MINUTES OF INCUBATION

FIG. 4. Arginine degradation in intact PPLOstrains ERKS and HEp-2. Broth cultures (150 ml)of ERKS and HEp-2 were centrifuged in a ServallSS-1 centrifuge for 15 min at 10,000 X g. The sedi-mented cells were resuspended in 10 ml of 0.86%NaCl and 0.07 M potassium phosphate (pH 7.0),and centrifuged for 10 min at 10,000 X g. The sedi-mented cells were then resuspended in 4 ml of 0.85%NaCI and 0.05 M potassium phosphate (pH 7.0)containing 0.2% gelatin. The protein content of theresuspended PPLO was 8.2 mg/ml and 6.4 mg/mlfor ERKS and HEp-2, respectively, after correctingfor the 0.2% gelatin. C14-L-arginine (specific activ-ity, 0.02 mc/mM) was added to a final arginine con-

centration of 0.050 M. At zero time, and 30-minintervals thereafter, 0.5 ml of each incubation mix-ture was removed, and the cells were removed by cen-

trifugation. The protein was precipitated from themedia with trichloroacetic acid, final concentration10%. The trichloroacetic acid was removed by etherextraction. A portion of each fraction was chromato-graphed on Whatman no. 1 paper in both phenol-ethanol-water-12 N NH40H (75:20:5:1) and phenol-water (80:20). The products of arginine breakdownwere measured with a Vanguard chromatogramscanner (model 880).

minations. In broth cultures of growing PPLOstrains isolated from cell culture, arginine was

converted essentially completely to ornithine.In contrast to the findings with PPLO growing

in broth, Smith (1955) reported that resting, hu-man PPLO strain 07 degraded arginine to citrul-line, but not ornithine, although extracts of thesecells were capable of converting citrulline to or-

nithine (Smith, 1957). Experiments of a type simi-lar to Smith's (1955), using washed PPLO resus-

pended in phosphate buffer containing 0.05 M

L-arginine, are shown in Fig. 4 for strains ERKSand HEp-2. It is evident that in both strains thereis a rapid degradation of arginine, and that bothornithine and citrulline constitute significant deg-radation products. In strain ERKS, ornithine ac-

ERKS HEp-2

_< - ARGININE-r----Ok o- ORNITHINE

h f o/.--ACITRULLINE

_ ...h..

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TABLE 8. Viable population count cfHEp-2 maintained at 87 C in phosph

after centhifugation and washi

PPLO/ml (plateStrain

0 hr

ERKS ........... 2.2 X 1010HEp-2.2.4 X 109

100 e

60to

Jw

40K

IARGININE ADDED20K

8 14 8 22 26 30

HOURS OF INCUBATION

FIG. 5. Effect of supplementation of c

with arginine, glucose, and L-gluttaminstrain PPLO. All supplements were a

rich the medium by 0.050 mi at the onset o

nient, except as indicated by the arrow.

counts for 75 %, of the consumed arginiin HEp-2, ornithine accounts for 41degraded arginine. Citrulline accountsmaining arginine breakdown.The degradation of C14-citrulline

either ERKS or HEp-2 under the co

Fig. 4 is negligible. This finding is in c

of the finding of Smith (1955) that citinot enter these cells. It indicates thatdown of citrulline to ornithine is not t]lysis of PPLO cells during preparatioiinto the suspending medium active extble of degrading citrulline, but ratheconversion has occurred within the ce]The results (Fig. 4) indicating

citrulline formation in PPLO agree wi(1955), and are in contrast to the findincultures (Table 7). However, experircentrifuged PPLO are not comparablein which PPLO growth occurs. As shov8, the centrifugation and washing of P]edly reduces viability during the subse

ERKS and incubation period in phosphate buffer. In otherate buiffer studies, PPLO maintained in the phosphate buffering without centrifugation and washing did not showcount) at any decrease in population over a 24-hr period.

3 hr Effects of arginine on growth of PPLO strainERKS in broth. It has been found that supple-

2.2 X 108 mentation of the infusion broth with 0.05 M addi-4.0 X 105 tional arginine increases the yield of PPLO,

whether measured by turbidimetric techniques,population titer, or protein content of the sedi-

BROTH mented PPLO. Figure 5 shows a growth curve of{ ARGININE ERKS (chosen for its rapid growth rate) obtained

by standard turbidimetric technique (Klett-Summerson photoelectric colorimeter, 540-m,uwavelength). In the unsupplemented broth, whichcontained 12 to 13 mm of arginine, growth ceasedabruptly at a time when the arginine of the me-

T BROTH dium was totally depleted (see Fig. 6). If arginine+GLUCOSE+ GLUTAMINE was added after the cessation of growth, growth

again commenced, and continued at a rate and toan extent similar to that occurring when argininewas added at the onset of the experiment. Ar-ginine supplementation does not increase the rateof growth, but merely the extent. The addition of

qulture broth other p)otential energy sources, glucose and gluta-Ne in ERKS mine (by reversal of the glutamine synthetasetdded to en- reaction) at 0.05 M concentrations, did not resultf the experi- in a more rapid rate or greater extent of growth

than that which occurred without supplementa-tion. In other experiments, the addition of ar-

ne, whereas ginine peptides to the broth in the form of a par-0% of the tial hydrolysate of salmine (Gale, 1945) did notfor the re- stimulate growth to any greater extent than the

limit of the added arginine, in either the presenceadded to or absence of added glucose. This finding is in

nditions of contrast to the results of Gale (1945), who foundDnfirmation that arginine peptides were far superior to ar-rulline does ginine in stimulating Streptococcus growth.,the break- Table 9 shows the relationship among Klett op-,he result of tical density readings, population titers, and pro-n, releasing tein content of cells, and indicates that the use oftracts capa- turbidimetric analysis of ERKS population isr that the valid.[ls. Figure 6 indicates the products of argininesignificant breakdown by ERKS growing in broth under con-ith Smith's ditions where: (i) arginine limits growth at twoigs in broth concentration levels, and (ii) growth ceases in thenents with presence of excess arginine. Samples of broth werewith those removed and analyzed for arginine, ornithine, andvn in Table citrulline, by both colorimetric and radioactivePLO mark- techniques similar to those used for Fig. 4. It isquent 3-hr evident that during all phases of active growth,

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or when growth is limited by arginine, ornithineis the sole product of arginine breakdown. How-ever, when the PPLO cells are no longer growing,but when excess arginine remains, arginine break-down continues, now being accompanied by theformation of citrulline rather than ornithine.Thus, the arginine breakdown product obtainedwill depend on the growth phase of the PPLO cul-ture and the presence of excess arginine in themedium. In other experiments where glucose orglutamine were present in the medium withoutarginine supplementation (Fig. 5), arginine wasalso converted entirely to ornithine.

DISCUSSION

The absence of arginine degradation to or-nithine in HeLa, KB, and L cells indicated inthese studies is in contrast to the reports of Piezand Eagle (1958), who used a HeLa strain, andManson and Thomas (1960), who used L cells.The arginase content of the cell line would appearto have little influence on conversion of arginineto ornithine in the culture medium, since Sanfordet al. (1961) failed to find urea formation in a cellline with far higher arginase levels than the celllines used in this study. These findings of littlearginine breakdown by arginase of cell culturescan be accounted for largely by the high concen-trations of arginine required for significant activ-ity of the enzyme (Km of arginase for arginine =1 to 2 X 10-2 M L-arginine), concentrations neverapproached with the arginine concentrations em-ployed in standard culture media. We thereforeconclude that extensive degradation of arginineto ornithine is not an inherent property of tis-sue culture, but rather is an artifact due to PPLOcontamination. It is of note that Manson andThomas (1960) found that arginine breakdownby L cells was accompanied by the formation of1 mole of ornithine and 2 moles of ammonia, astoichiometry consistent with the pathway ofarginine breakdown by PPLO.The source of the extensive contamination of

cell cultures with PPLO is unknown, although thesuggestion of Rothblat and Morton (1959) thatPPLO contamination results from transformationof bacterial contaminants to stable L forms seemslikely (Barile et al., 1962). Our studies would indi-cate that streptococci, or bacterial species capableof arginine degradation, are potential sources ofsuch PPLO contamination. Neimark and Pickett(1960) previously remarked on the similarities of

TABLE 9. Effect of supplementation of broth witharginine on growth of ERKS strain PPLO

Conditions Optical Cells /ml Protein(Klett) (plate countl (total mg)

Expt 1*Broth 40 8.2 X 108 0.32Broth - argininet 119 2.0 X 109 0.87

Expt 21Broth 37 5.5 X 101 0.39Broth + argininet 111 2.0 X 109 0.96

* Jnitial inoculum: 2 X 107 cells/7 ml.t SupplemeDted by 0.05 M L-arginine.I Initial inoculum: 2 X 10' cells/7 ml.

60 t - -

GROWTH CURVEH 160ur 62 2 MA4RGIN,

120

< 222,MAV2/AVANVN480 22oM mVGi//NE

a.t_40 _ 2,Z2 mMf dRGIAIIIE/

ORNITHINE FORMATiOrT40 -

30

10 5 20 25 30HOURS OF ItICUBATION

". ARGININE DEPLETION

CITRULLINE FORMATION40 -

30 -

z

0-i- 20tr //I

t> 10 - I/

10 15 20 25 30HOURS OF INCUBATION

FIG. 6. Products of arginine degiadation by grow-ing PPLO strain ERKS. ERKS strain was inocu-lated into 8 ml of broth (2 X 107 cells/8 ml) contain-ing C'4-L-arginine under the following conditions:12.2 mM L-arginine, specific activity, 0.39 mc/mmole(0); 22.2 mM L-arginine (0.15 mc/mmole; A); and62.2 mM L-arginine (0.15 mc/mmole; *). Growth wasfollowed by turbidimetric measurements. Sampleswere removed at various times and assayed for argi-nine, citrulline, and ornithine by colorimetricmethods, first separating citrulline from the orni-thine and arginine by means of Amber lite CG-50columns. Samples were also chromatographed inphenol-ethanol-water-ammonia (75:20:5:1) andestimated with a Vanguard chromatogram scanner.

carbohydrate metabolism in PPLO and strepto-cocci. This similarity is further demonstrated bythe studies with arginine breakdown presented inthis paper.

In addition to the artifact of arginine degrada-tion by PPLO, the additional artifact of argininebreakdown by the added horse serum has been en-

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countered (Fig. 1 and 2, Table 1). It is evidentthat added serum may be a potential source of en-zyme activity, and must be considered in studiesof tissue culture metabolism.Evidence has been presented that the conver-

sion of arginine to ornithine in PPLO-contam-inated cell culture, as well as in PPLO brothcultures, occurs by a three-enzyme pathway in-volving arginine deiminase, ornithine transcar-bamylase, and carbamyl phosphokinase (reaction2 of the introduction). This pathway has beenfound in a number of organisms as cited previ-ously, and has been studied in a number of lab-oratories in several species of bacteria (see recentreview by Cohen and Brown, 1960). The reactionproducts and stoichiometry as presented in Ta-bles 3 to 5 are similar to those found for variousbacteria and by Smith (1955, 1957) for humanPPLO strain 07.

All five of the PPLO strains isolated from con-taminated tissue culture degrade arginine to or-nithine (Tables 7) and contain the requisite en-zymes (Table 6). The widespread occurrence ofarginine depletion of culture media in the presenceof PPLO is further indicated by the studies ofPowelson (1961), which consistently demon-strated arginine depletion with several cell linesby use of PPLO isolated from sheep and aviansources.The physiological significance of the active

degradation of arginine to ornithine by PPLO isunknown. The studies of Bauchop and Elsden(1960) suggest that, in Streptococcus faecalis, ar-ginine breakdown by this same pathway is asso-ciated with the formation of adenosine triphos-phate (ATP) used in cellular multiplication. Thatsuch a function for arginine breakdown in PPLOexists would appear plausible in view of the ap-parent limited oxidative capacity of PPLO (Lynn,1960; Neimark and Pickett, 1960; review ofRazin, 1962). P. F. Smith (1960) concluded thatthe possibility of arginine breakdown constitutinga significant energy source seemed unlikely in hu-man PPLO strains because of the lack of conver-sion of arginine to ornithine in resting cells, andthe sluggish activity of the conversion of citrul-line to ornithine in cell-free extracts. Our stud-ies indicate that arginine is in fact convertedto ornithine (Table 7, Fig. 4 and 6). During activePPLO growth, arginine is degraded to ornithinewithout the accumulation of citrulline. Citrullineaccumulates only under resting conditions, i.e.,

in washed cells in phosphate buffer (Fig. 4) andin broth after growth has ceased under conditionswhere arginine is not limiting growth (Fig. 6). Wepropose that such results can be explained on thepremise that arginine breakdown does, indeed,represent a source of high-energy phosphate. Thecontinual breakdown of ATP that occurs in bio-synthetic processes of growth provides a con-tinual supply of ADP which pulls the reversiblecarbamyl phosphokinase reaction (Jones andLipmann, 1960) in the direction of carbamyl phos-phate breakdown. The continual removal of car-bamyl phosphate in turn pulls the ornithinetranscarbamylase reaction in the direction ofcitrulline breakdown. When growth ceases, andthe ATP is no longer utilized, the breakdown ofcitrulline stops. The breakdown of arginine tocitrulline, on the other hand, continues with theresultant formation of citrulline, which can nowbe demonstrated in the medium. The inability ofwashed and sedimented cells to complete argininebreakdown to ornithine (Fig. 4; Smith, 1955) maywell result from lack of growing conditions or fromdamage to the organisms as suggested by loss ofviability (Table 8) and, hence, decreased or lostability to utilize ATP.

Although it is suggested that arginine conver-sion to ornithine constitutes a significant source ofhigh-energy phosphate (ATP) for PPLO, and in-deed cannot take place without formation ofATP, the extent to which this conversion consti-tutes the only or major source of high-energyphosphate is unknown. The continual breakdownof arginine and limitation of growth by this break-down in PPLO is similar to the degradation ofproline by a prolineless mutant of Escherichia coli(Stone and Hoberman, 1953) and of tyrosine byS. faecalis (Kihara, Klatt, and Snell, 1952). Inthese previous cases, as well as in the breakdownof arginine by Streptococcus when grown in a glu-cose-containing broth (Gale, 1945), peptidescontaining the required amino acid were more ef-fective in promoting growth than the free aminoacid, a finding ascribed to a protective effect of thepeptide on degradation of the amino acid. InPPLO, on the other hand, peptides containingarginine did not result in growth greater than oc-curs from the added arginine. If arginine did in-deed furnish the major source of ATP, one wouldpredict that the addition of peptides containingarginine would not stimulate growth to anygreater extent than the free amino acid itself.

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ARGININE METABOLISM IN PPLO

That such is the case would suggest that argininebreakdown is a major source of ATP for at leastthe one Mycoplasma organism studied extensivelyin this paper.

Irrespective of speculation concerning the sig-nificance of arginine breakdown in PPLO, it isclear that the addition of arginine to broth cul-tures results in a two- to threefold increase incellular yield of PPLO (Fig. 5, Table 9). Similarincreased yields of PPLO in contaminated cellcultures have been found after increasing the ar-ginine content of the medium. This finding indi-cates the prime importance of arginine metabo-lism of PPLO, and the desirability of using aPPLO growth medium supplemented with ar-ginine.We are presently investigating whether the ar-

ginine dihydrolase pathway exists in all PPLOorganisms. To date, we have found completeagreement between the presence of arginine de-iminase activity and that of PPLO contaminationby standard culture techniques in 29 cell lines (8negative, 18 positive). These studies are being ex-tended to determine the potential to detect PPLOcontamination of cell culture by a simple enzymeassay for arginine deiminase activity.

ACKNOWLEDGMENT

The authors would like to acknowledge the ex-cellent technical assistance of G. Pla and D. B.Riggs in carrying out these studies.

LITERATURE CITED

ARCHIBALD, R. M. 1944. Determination of citrul-line and allantoin and demonstration of citrul-line in blood plasma. J. Biol. Chem. 156:121-142.

BARILE, M. F., W. F. MALIZIA, AND D. B. RIGGS.1962. Incidence and detection of pleuropneu-monia-like organisms in cell cultures by fluo-rescent antibody and cultural procedures. J.Bacteriol. 84:130-136.

BARILE, M. F., R. YAGUCHI, AND W. C. EVELAND.1958. A simplified medium for the cultivationof pleuropneumonia-like organisms and Lforms of bacteria. Am. J. Clin. Pathol. 30:171-176.

BAUCHOP, T., AND S. R. ELSDEN. 1960. The growthof micro-organisms in relation to their energysupply. J. Gen. Microbiol. 23:457-469.

BESSMAN, S. P., AND A. N. BESSMAN. 1955. Thecerebral and peripheral uptake of ammoniain liver disease with a hypothesis for the mech-

anism of hepatic coma. J. Clin. Invest. 34:622-628.

BURNETT, G. H., AND P. P. COHEN. 1957. Study ofcarbamyl phosphate-ornithine transcarbamyl-ase. J. Biol. Chem. 229:337-344.

COHEN, P. P., AND G. W. BROWN, JR. 1960. Am-monia metabolism and urea biosynthesis, p.161-244. In M. Florkin and H. S. Mason [ed.],Comparative biochemistry, vol. 2. AcademicPress, Inc., New York.

COLLIER, L. H. 1957. Contamination of stock linesof human carcinoma cells by pleuropneu-monia-like organisms. Nature 180:757-758.

EAGLE, H. 1959. Amino acid metabolism in mam-malian cell cultures. Science 130:432-437.

GALE, E. F. 1945. The arginine, ornithine andcarbon dioxide requirements of streptococci(Lancefield group D) and their relation toarginine dihydrolase activity. Brit. J. Exptl.Pathol. 25:225-233.

HILLS, G. M. 1940. Ammonia production by patho-genic bacteria. Biochem. J. 34:1057-1069.

HORN, F. 1933. Uber den Abbau des Arginins zuCitrullin durch Bacillis pyocyaneus. Z.Physiol. Chem. 216:244-247.

JONES, M. E. 1962. Carbamyl phosphate synthesisand utilization, p. 903-925. In S. P. Colowickand N. 0. Kaplan [ed.], Methods in enzymol-ogy, vol. 5. Academic Press, Inc., New York.

JONES, M. E., AND F. LIPMANN. 1960. Chemicaland enzymatic synthesis of carbamyl phos-phate. Proc. Natl. Acad. Sci. U.S. 46:1194-1205.

KENNY, G. E., AND M. E. POLLACK. 1962. Effect ofamino acid depletion by PPLO on growth ofcells in culture. Federation Proc. 21:161.

KIHARA, H., 0. A. KLATT, AND E. E. SNELL. 1952.Peptides and bacterial growth. III. Utiliza-tion of tyrosine and tyrosine peptides byStreptococcus faecalis. J. Biol. Chem. 197:801-807.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

LYNN, R. J. 1960. Oxidative metabolism of pleuro-pneumonia-like organisms. Ann. N.Y. Acad.Sci. 79:538-542.

MANSON, L. A., AND W. J. THOMAS. 1960. Metabo-lism of arginine by mouse fibroblast, StrainL. Proc. Soc. Exptl. Biol. Med. 104:209-211.

MORGAN, J. F., H. J. MORTON, AND R. C. PARKER.1950. Nutrition of animal cells in tissue cul-ture. I. Initial studies on a synthetic medium.Proc. Soc. Exptl. Biol. Med. 73:1-8.

NEIMARK, H. C., AND M. J. PICKETT. 1960. Prod-ucts of glucose metabolism by pleuropneu-

VOL. 86, 1963 205

on July 14, 2019 by guesthttp://jb.asm

.org/D

ownloaded from

SCHIMKE AND BARILE

monia-like organisms. Ann. N.Y. Acad. Sci.79:531-537.

PIEZ, K. A., AND H. EAGLE. 1958. The free aminoacid pool of cultured human cells. J. Biol.Chem. 231:533-545.

POLLOCK, M. E., G. E. KENNY, AND J. T. SYVER-TON. 1960. Isolation and elimination of pleuro-pneumonia-like organisms from mammaliancell cultures. Proc. Soc. Exptl. Biol. Med.105:10-15.

POWELSON, D. H. 1961. Metabolism of animal cellsinfected with Mycoplasma. J. Bacteriol. 82:288-297.

RATNER, S. 1955. Enzymatic synthesis of arginine(condensing and splitting enzymes), p. 356-367. In S. P. Colowick and N. 0. Kaplan [ed.],Methods in enzymology, vol. 2. AcademicPress, Inc., New York.

RAZIN, S. 1962. Nutrition and metabolism ofpleuropneumonia-like organisms, p. 91-104.In E. Klieneberger-Nobel [ed.], PPLO: Myco-plasmataceae. Academic Press, Inc., NewYork.

ROBINSON, L. B., R. H. WICHELHAUSEN, AND B.ROIZMAN. 1956. Contamination of human cellcultures by pleuropneumonia-like organisms.Science 124:1147-1148.

ROCHE, J., AND G. LACOMBE. 1952. Sur l'arginine-desiminase et sur la formation enzymatique decitrulline par les levures. Biochim. Biophys.Acta 9:687-696.

ROTHBLAT, G. H., AND H. E. MORTON. 1959. De-tection and possible source of contaminatingpleuropneumonia-like organisms (PPLO) incultures of tissue cells. Proc. Soc. Exptl. Biol.Med. 100:87-90.

ROUSE, H. C., AND V. H. BONIFAS. 1962. Depletionby pleuropneumonia-like organisms of argi-nine required for adenovirus plaque forma-tion. Bacteriol. Proc., p. 147.

SAKAGUCHI, S. 1925. Uber die Bindungsweise undquantitative Bestimmung des Argininis imProteinmolekule. J. Biochem. (Tokyo) 5:133-142.

SANFORD, K. K., B. B. WESTFALL, E. H. Y. CHU,E. L. KUFF, A. B. COVALESKY, L. T. DUPREE,G. L. HOBBS, AND W. R. EARLE. 1961. Altera-tions in morphology, arginase, and #-glu-curonidase within a clone of mouse tumor cellsin vitro. J. Natl. Cancer Inst. 26:1193-1219.

SCHIMKE, R. T. 1962a. Repression of enzymes ofarginine biosynthesis in mammalian tissueculture. Biochim. Biophys. Acta 62:599-601.

SCHIMKE, R. T. 1962b. Adaptive characteristics ofarea cycle enzymes in the rat. J. Biol. Chem.237 :459-468.

SCHMIDT, G. C. M., A. LOGAN, AND A. A. TYTELL.1952. The degradation of arginine by Clostrid-ium perfringens. J. Biol. Chem. 198:771-783.

SIEKEVITZ, P. 1952. Uptake of radioactive alaninein vitro into the proteins of rat liver fractions.J. Biol. Chem. 195:549-562.

SMITH, I. 1960. Chromatographic and electropho-retic techniques, vol. 1, p. 83-142. IntersciencePublishers, Inc., New York.

SMITH, P. F. 1955. Amino acid metabolism bypleuropneumonialike organisms. I. Generalcatabolism. J. Bacteriol. 70:552-556.

SMITH, P. F. 1957. Conversion of citrulline toornithine by pleuropneumonialike organisms.J. Bacteriol. 74:801-806.

SMITH, P. F. 1960. Amino acid metabolism ofPPLO. Ann. N.Y. Acad. Sci. 79:543-550.

STONE, D., AND H. HOBERMAN. 1953. Utilizationof proline peptides by a prolineless mutant ofEscherichia coli. J. Biol. Chem. 202:203-212.

VAN PILSUM, J. F., R. P. MARTIN, E. KITO, ANDJ. HESS. 1956. Determination of creatine,creatinine, arginine, guanidinoacetic acid,guanidine and methylguanidine in biologicalfluids. J. Biol. Chem. 222:225-235.

WALKER, J. B. 1953. An enzymatic reaction be-tween canavanine and fum-arate. J. Biol.Chem. 204:139-146.

YOUNGNER, J. S. 1954. Monolayer tissue cultures.I. Preparation and standardization of sus-pensions of tryspin-dispersed monkey kidneycells. Proc. Soc. Exptl. Biol. Med. 85:202-205.

206 J. BACTERIOL.

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.org/D

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