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6Loeb, T., and N. D. Zinder, these PROCEEDINGS, 47, 282 (1961).7Notani, G., and N. D. Zinder, unpublished observations.8 Ingram, V. M., Nature, 178, 792 (1956); Katz, A. M., W. J. Dreyer, and C. B. Anfinsen,

J. Biol. Chem., 234, 2897 (1959).9 Hirs, C. H. W., S. Moore, and W. H. Stein, J. Biol. Chem., 219, 623 (1956). Two ml of formic

acid were mixed with 0.1 ml of 30 per cent hydrogen peroxide (superoxal) and allowed to standfor 2 hr at 00. One-tenth ml of this mixture was added to the dried protein and was incubatedfor 1 hr at 00. Oxidation was stopped by the addition of 1 ml of water and the protein was lyo-philized.

AN INDUCIBLE TRYPTOPHAN SYNTHETASE IN TRYPTOPHANAUXOTROPHS OF ESCHERICHIA COLI

BY WM. AUSTIN NEWTON AND ESMOND E. SNELL

DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF CALIFORNIA, BERKELEY

Communicated June 19, 1962

Previous attempts in this laboratory to study the mechanism of the breakdownof L-tryptophan (reaction 1) by tryptophanasel (TPase) have been hampered be-cause all preparations of this enzyme also catalyzed reaction (2), which is catalyzedby tryptophan synthetase (TSase). The latter enzyme from Escherichia coli

TPase1. L-Tryptophan + H20 - indole + pyruvate + NH3.

TSase2. Indole + L-serine ) L-tryptophan + H20.

3. Indoleglycerol phosphate + Lserine L-tryptophan + glyceraldehyde 3-phosphate.

4. Indoleglycerol phosphate + H20 2± indole + glyceraldehyde 2-phosphate.

also catalyzes reactions (3) and (4)2 3 and can be separated into two protein com-ponents, A and B,3 both of which are required for catalysis of each of the three reac-tions, (2), (3), and (4), at maximum rate.To avoid the difficulties presented by this contaminating TSase, a tryptophan

auxotroph of E. coli B, mutant B/1t7, was studied. This mutant has been shownby immunological, genetic, and enzymatic criteria to lack genes A and B,4 5 amongothers, in the tryp region of the chromosome. These genes control synthesis of theA and B components of the repressible TSase,5 which are lacking in this mutant.We confirmed the facts that this mutant grew on tryptophan-supplemented, butnot on indole-supplemented, media and that extracts from cells grown on low levelsof tryptophan did not catalyze reaction (2). However, when the organism wasgrown with high concentrations of tryptophan to induce maximum levels of TPase,we unexpectedly found that high levels of TSase also were present.

This result was surprising not only because of the previous characterization ofthe organism as a deletion mutant lacking TSase,5 but also because of the apparentinduction by high concentrations of tryptophan of a synthetic activity previouslyshown to be repressed by tryptophan in E. coli6 and in Aerobacter aerogenes.7 Itwas also not immediately clear why an organism competent to produce an enzyme

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catalyzing the synthesis of tryptophan from indole grew in minimal medium sup-plemented with tryptophan but not in one supplemented with indole.

This paper describes attempts to clarify these observations.Materials and Methods.-1. Bacterial cultures: The following strains of E. coli8 were used:

Strain B (wild type); B/1t7, a deletion mutant of strain B that lacks all enzymes of the pathwayfor tryptophan synthesis from some point before anthranilic acid; B/1t7-A, a spontaneous mu-tant of strain B/1t7 isolated in this laboratory that grows in minimal medium supplemented witheither indole or tryptophan and forms high levels of both TPase and TSase without inductionby tryptophan; td4, a point mutant of E. coli K-12 that requires tryptophan; and ML-304g, atryptophan-requiring mutant of E. coli ML.

2. Growth conditions and preparation of extracts: The minimal medium contained the inorganicsalts mixture of Rickenberg et al.,9 with either glucose (0.16%) or glycerol (0.16%) as carbonsource, and was supplemented as indicated in individual experiments. The broth medium con-tained 1% yeast extract (Difco), 1% bactotryptone (Difco), 0.5% dibasic potassium phosphateand 0.1% glucose.

Cultures were grown from a 1% inoculum in 100 ml of medium contained in 250-ml Erlenmeyerflasks. These were shaken on a rotary shaker at 300. Cells were harvested by centrifugation atthe end of the exponential phase of growth (12 to 18 hr). Cell densities were calculated from acalibration curve relating dry weight of cells to optical density at 650 m/A.For preparation of cell extracts, freshly harvested cells were washed once with 0.9% NaCl

solution, resuspended in 0.02 M phosphate buffer, pH 7.0, at a concentration of 40 mg (dry weight)per ml, then treated for 20 min in the Raytheon 10 KC sonic oscillator. Cell debris wasremoved by centrifuging at 14,000 X g for 20 min. Small samples were disrupted in 5.0-ml"Lusteroid" centrifuge tubes, which were placed in the chamber of the oscillator in 40 to 50 mlof water.

3. Enzyme assays: TPase: The following procedure for assay of TPase, based partly on pre-vious modifications of the Ehrlich test used by Browder' and by Pardee and Prestidge,'0 hasproved more satisfactory in our hands for the assay of cell-free extracts than other procedures.To a 10-ml Erlenmeyer flask are added 0.02 ml of pyridoxal phosphate solution (0.20 mg per ml),0.01 ml of 0.005 M reduced glutathione, 0.02 ml of 1.0 M phosphate buffer, pH 8.3, enzyme solu-tion, and sufficient distilled water to give a final volume of 0.30 ml. This solution is layered with1.0 ml of toluene and incubated for 5 min at 37°. The reaction is started by addition of 0.1 mlof L-tryptophan solution (5.0 mg per ml). The flask is stoppered and the incubation carried outwith gentle shaking on a Brunswick rotary shaker for 10 min. The reaction is stopped and thecolor developed by the .ddition of 3.5 ml of a freshly mixed acid-Ehrlich reagent (5 volumes of5% p-dimethylaminobenzaldehyde in 95% ethanol plus 12 volumes of 5% sulFuric acid in n-butanol). After 20 min, the color intensity is read at 570 my in 0.5-inch tubes. The assay islinear to about 0.050,Mmole of indole. One unit of TPase is that amount of enzyme which forms0.10 Mmole of indole in 10 min. The specific activity is expressed as the number of units per mgof protein.

TSase: This assay is similar to that of Yanofsky1' with the principal changes being a fourfoldincrease in the iserine concentration and a reduction of the volume of the incubation mixture to0.2 ml. The smaller volume allows the color reaction for indole to be performed on the entirecontents of the assay tube while maintaining the absorbance in a readable range. To a conical12-ml centrifuge tube are added 0.08 ml of 0.40 M L-serine, 0.01 ml of 0.005 M reduced gluta-thione, 0.01 ml of 1.0 M phosphate buffer, pH 8.3, 0.005 ml of pyridoxal phosphate solution (1.0mg per ml), and enzyme. The mixture is diluted to 0.18 ml with distilled water and incubated at370C for 20 min. The reaction is initiated by the addition of 0.02 ml of 0.005M indole (or indole-glycerol phosphate (InGP)) to the assay tube and appropriate smaller amounts of indole to othertubes containing no enzyme for the preparation of a standard curve. After 40 min of incubationwith rapid shaking, the reaction is stopped and the color developed by the addition of 5.0 ml of thefreshly mixed acid-Ehrlich reagent.12 After 20 min at room temperature, the absorbance is readin 0.5-inch tubes at 540 m1A. One unit is that amount of enzyme which causes the disappearanceof 0.10 Mmole of indole in 40 min under these conditions. The specific activity is expressed asunits per mg of protein.

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This same enzymatic assay mixture is used when tryptophan is determined microbiologically.The reaction is stopped by heating at 100'C for 5 min. The solution is diluted to 1 ml and cen-trifuged, and aliquots of the supernatant solution are used for the determination.

4. Miscellaneous: Permeability of the B/1t7 mutant and wild type E. coli cells to indole wasdetermined by the Conway diffusion technique.'3 Inulin was the control compound. Proteinwas determined by the method of Lowry et al.'4 Indoleglycerol phosphate was prepared enzy-matically;"5 other chemicals were from commercial sources.

Results.-Inducibility of TSase activity by tryptophan: When mutant B/1t7 isgrown in the presence of only 10 ,gg of tryptophan per ml, the TSase activity is ator below the lower limit of detectability (Table 1). Extracts of cells grown in thesame medium with high levels of tryptophan or tryptone, or in broth, show an ex-tremely high level'6 of both TSase and TPase.The close parallelism between the two activities, as reflected in the constancy of

the activity ratios, suggested as one possibility that the indole and pyruvate formedby the TPase reaction might induce TSase formation. However, only extremelylow levels of TSase were produced when indole and pyruvate were supplied togetherwith low levels of tryptophan (Table 1). 5-Methyltryptophan, which cannot sub-stitute for tryptophan in protein synthesis,'7 induces high levels of TPase and TSaseunder similar conditions (Table 2). The potency of tryptophan as an inducer thusdoes not result from a high level of this amino acid being required for enzyme syn-thesis (cf. Vogel et al.'8). The possible involvement of enzyme inhibitors or acti-vators was excluded by mixing experiments in which extracts from induced and un-induced B/1t7 cells were assayed separately and in combination without significantdepartures from additivity in the results obtained.The parallel influence of different inducers and different carbon sources on produc-

tion of TSase and TPase is evident in Table 2. The repressive effect of glucose,noted previously on induction of TPasel9 and several other enzymes,20 is also ob-

TABLE 1EFFECT OF COMPOSITION OF THE MEDIUM ON THE TSASE AND TPASE ACTIVITIES OF EXTRACTS OF

E. coli, STRAIN B/1t7,-Specific Activity of Cell Extract---, Ratio

Medium and supplement TSase TPase TSase/TPaseMinimal medium:*+ L-Tryptophan (10 jg per ml) 0 -+ L-Tryptophan (1 mg per ml) 18.0 10.5 1.7

fIndole (100 Mg per ml)+ <Pyruvate (100 ,ug per ml) 0.08

L-Tryptophan (10 ,ug per ml)+ Tryptone(I%) 4.0 3.3 1.2Broth 38.0 21.5 1.6

* Glucose and 0.1% acid-hydrolyzed casein were also present.

TABLE 2COMPARATIVE INDUCTION OF TPASE AND TSASE BY TRYPTOPHAN AND 5-METHYLTRYPTOPHAN IN

THE PRESENCE OF GLUCOSE OR GLYCEROL AS CARBON SOURCE-Supplement to Minimal Medium* Specific Activity

Carbon source Other, jg per ml TSase TPase TSase/TPaseGlucose L-tryptophan, 100 5.6 5.0 1.1Glycerol L-tryptophan, 100 17.0 13.4 1.3Glucose L-Tryptophan, 10 + 5-methyl-DL- 12.7 9.1 1.4

tryptophan, 100Glycerol L-tryptophan, 10 + 5-methyl-DL- 15.0 12.0 1.2

tryptophan, 100* The medium also contained 0.1% of acid-hydrolyzed casein.

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served with TSase. These results and those from Table 1 indicate that tryptophanand 5-methyltryptophan probably induce' TSase and TPase by acting specificallyon some common regulatory mechanism.The possibility was considered that the induced TSase formed by strain B/lt7

corresponds structurally to the repressible TSase present in the parent wild typeE. coli B. This situation might arise if translocation of the A and B genes of thetryp region of the chromosomes occurred to a region where their function in themutant was controlled by the TPase regulatory mechanism. This possibility iseliminated by data of Table 3, which show that the inducible TSase is not peculiarto strain B/1t7 but is also produced by two tryptophan auxotrophs derived fromtwo other E. coli strains.2' These mutants, ML-304g and td4, are phenotypicallysimilar to the B/1t7 strain in that they will not grow on minimal medium that issupplemented only with indole. Although the level of induced synthetase activityin strain ML-304g is six times that in strain td4, the ratio of TSase to TPase is com-parable in the two extracts and is about the same as that found in extracts of in-duced B/1t7 cells. Strain td4 has been mapped as a point mutant in the B gene ofthe tryp region of the chromosome4 and consequently forms an altered B proteintogether with normal A protein corresponding to the repressible TSase. Since italso forms the tryptophan-induced TSase, this enzyme very likely represents an-other distinct protein and not the product of translocated A and B genes.

TABLE 3INDUCTION OF TSASE AND TPASE BY TRYPTOPHAN IN OTHER TRYPTOPHAN

AUXOTROPHS OF E. coliL-Tryptophan added to Specific Activity of

minimal medium* Cell ExtractsStrain (pg/ml) TSase TPase TSase/TPase

ML-304g 10 0 0200 8.25 6.25 1.4

td4 10 0 0200 1.62 0.98 1.6

B/1t7-A 10 45.2 32 1.4* The minimal medium contained glycerol and 0.1 per cent of acid-hydrolyzed casein.

If the above interpretations are correct, inducible TSase should be formed whenE. coli B, the parent strain of mutant B/1t7, is grown with high concentrations oftryptophan. Unpublished results indicate this is the case.

Catalytic activity of the induced TSase: No indole disappearance resulted in eventhe most active extracts when serine was omitted from the TSase assay. Chro-matography of complete assay mixtures on paper with two different solvent systems(pyridine:acetic acid:water, 50:35:15 and n-butanol:acetic acid:water, 60:15:25)showed the presence of a ninhydrin-positive compound which ran well ahead ofserine and cochromatographed with authentic tryptophan. Finally, microbiologi-cal assay of these reactions mixtures with Leuconostoc mesenteroides P60, whichspecifically requires L-tryptophan and cannot utilize indole in its place, confirmedthe fact that tryptophan was formed (Fig. 1A). The rate of tryptophan produc-tion was unaffected by addition of 0.010 umole of tryptophan to the assay mixture,thus demonstrating that under these conditions the high levels of TPase activitypresent in these extracts do not interfere significantly with the determination oftheir TSase activity. The relation of indole disappearance to the concentration ofcell extract under these assay conditions is shown in Figure 1B.

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.050

G~~~~~~~~~W .040_ .080 °

~.030- 2 .060I

0. Aia, .020o .040a.g

a.0104 .020 0

II ~~00 10 20 30 10 20 30

TIME (MIN) ILg PROTEIN

FIG. 1.-A: Rate of L-tryptophan formation in the TSase assay with (Curve 1) andwithout (Curve 2) the addition of 0.010 Mmole of L-tryptophan to the assay tubes. r-tryp-tophan was determined microbiologically.

B: Relation of tryptophan synthesis (measured by indole disappearance) to the amountof crude cell extract from broth-grown B/1t7 cells. A similar extract was used for the de-termination of part A.

While the induced TSase actively synthesizes tryptophan from indole and serine,it differs markedly from the previously studied repressible TSase in that it does notcatalyze reaction (3) (InGP + serine tryptophan). The latter reaction wascatalyzed by mixtures of the A and B components of the repressible TSase underthe same conditions (Table 4). Furthermore, additions of either the A or the B

TABLE 4THE COMPARATIVE SYNTHESIS OF TRYPTOPHAN FROM INDOLE OR INDOLEGLYCEROL PHOSPHATE BYTHE INDUCIBLE TSASE AND BY A AND B COMPONENTS OF THE REPRESSIBLE TSASE FROM E. coli

Additions to Reaction Mixture (jA) . -L-Tryptophan Formed*---B/1t7-

A proteint B proteint extract§ From Indole From InGP2 10 48.0 28.0

20 - 0.4 2.410 5 47.5 41.0

25 - 29.0 05 36.5 0

25 020 5 42.5

25 5 57.Ott -20 - 25 0

25 25 2.2* Expressed mpmoles per assay tube per 40 min. L-Tryptophan formed was determined by microbiologica

assay with L. mesenteroides. Difco assay medium was used.t 365 units per ml (cf. ref. 3).t 200 units per ml (cf. ref. 3).§ Broth-grown cells were used for preparation of the extract.ft Calculated value (65.5) is beyond the linear portion of the assay curve.

protein in excess to extracts of induced B/1t7 cells failed to increase significantlysynthesis of tryptophan from indoleglycerol phosphate or indole above the "blank"levels. Extracts of the constitutive B/1t7-A cells also catalyze reaction (2) butnot reaction (3). Similar experiments showed that the inducible TSase also did notcatalyze reaction (4). Altered A or B proteins, as well as the normal proteins, willgreatly increase the activity of the normal B or A protein, respectively, under con-ditions similar to those described above.5 Thus, the inducible TSase is a distinct,previously unobserved enzyme and appears unrelated to either the A or the B pro-tein of the repressible TSase.

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1436 BIOCHEMISTRY: NEWTON AND SNELL PROC. N. A. S.

Constitutive mutant: A spontaneous mutant of strain B/lt7, designated asB/1t7-A, was isolated which, unlike the parent mutant, grows on minimal mediasupplemented with indole. The normal pathway for tryptophan synthesis is stillabsent in this organism since it does not grow when indole is replaced by anthranilicacid. Cells of this mutant grown in minimal media supplemented only with lowconcentrations of tryptophan contain high levels of both TPase and TSase (Table3). The mutation has, therefore, altered a control mechanism in such a way thatboth TPase and the inducible TSase are formed constitutively. While both activ-ities are higher than those observed in extracts of induced B/1t7 cells, the ratio ofactivities is nearly the same as that characteristic of the parent organism. Theseresults further support the conclusion that, whatever the structural relationshipbetween TPase and the inducible TSase, they share a common regulatory mecha-nism.

Growth response of E. coli B/1t7 to indole: Three possible explanations wereconsidered for the inability of the B/1t7 cells to grow on indole-supplemented media:(1) the mutants are not permeable to indole, (2) the TSase formed is defective inthe sense that it cannot catalyze tryptophan synthesis in vivo at a rate sufficientfor growth, or (3) the level of tryptophan required for induction of the enzyme can-not be furnished endogeneously as tryptophan synthesized from indole.

Appropriate experiments showed that B/1t7 cells possess an active uptake mech-anism for indole, thus excluding (1).

00 500 To determine whether the inducedJTS ase ---o- TSase was functional for growth, cells

F 80 ase (-0{--0) 400 were grown in a medium in which highTRYPTOPHAN r levels of the two enzymes are formed.w 60 1-

/ ¢ - 300 O They were then harvested, washed, andsn transferred to minimal media contain-

F 40 r\, /s/ INDOLE ing the supplements indicated in Figure40-1-020 200 2. The cells now grew in the presence

0 20 _ >100 _ of tryptophan or indole but not in the{NONE presence of anthranilate, thus showing

00- 20-- W 0 that the enzyme was functional and40 60 80 00 excluding (2). Unlike growth withTIME (HIRS.)

tryptophan, however, that with indoleFIG. 2.-Correlation of growth on various ttophan, howevert witindemedia with the TSase and TPase activities stopped after 4 to 5 cell generations.

of E. coli B/1t7. Assays for TPase and TSase conductedSolid curves (right ordinate): Growth in Aafras and thase cutedminimal-glucose medium supplemented with on cell extracts from these cultures at

10 ,ug per ml of tryptophan, indole, or anthr- the indicated times (Fig. 2) showedanilate. A washed inoculum from a broth-grown culture in the log phase was used to that the level of these enzymes per cellsupply an initial cell density of 1.5 (O ), had fallen in parallel during growth to3.0 (-A-), or 15(-0-) jg per ml. Growthresponse to anthranilate was the same as very low levels when growth ceased;that shown for no supplement. the total enzyme content of the cultureDashed curves (left ordinate): Relative thealentth eculturespecific activities of extracts of cells harvested decreased less than 50% during this

from the indole-supplemented medium at time. It thus appears that growth onvarious times following inoculation with 15jug per ml of the broth-grown culture. Specific the indole-supplemented medium iSactivities of the inoculum at zero time was permitted by the initially high level of36 (TSase) and 21.5 (TPase) units per mg of TSase supplied by the broth-grownprotein, respectively.

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VOL. 48, 1962 BIOCHEMISTRY: NEWTON AND SNELL 1437

inoculum and that dilution of this pre-existing enzyme as a consequence of _/0_cell division leads to growth stoppage. /The experiment supports (3) as the 2explanation for the failure of B/1t7 to

L 0 /grow on indole. This explanation is 4_

zalso consistent with the occurrence of /the constitutive mutant, B/lt7-A, J 200which grows with indole as the only c,supplement to minimal medium. 34While cells of B/1t7 obtained from 00 -

media low in tryptophan will not grow 0 20 40 60 80 o0 120on an indole-supplemented minimal TIME (HRS.)mediuadditionof the normallyFIG. 3.-Growth of E. coli B/1t7 on

medium, addition of the normally bac- minimal-glucose medium supplemented withteriostatic 5-methyltryptophan22 to tryptophan, indole or 5-methyltryptophan.amupigarInoculum: 15 gg per ml of cells grown in

such a medium permits growth after minimal medium plus low tryptophan.an initial lag period (Fig. 3). An at- Curve 1, L-tryptophan, 10 A.g per ml; Curve 2,aninitiallagpriodFig.3indole, 10 ug per ml + 5-methyl-DI-trypto-tractive explanation for the growth- phan, 100 ,ug per ml; Curve 3, indole, 10promoting properties of the 5-methyl- ,sg per ml; Curve 4, 5-methyl-DL-tryptophan,tryptophan, which cannot itself sup- 100 Mg per ml.port growth, lies in its action as anunnatural inducer of the TSase (cf. Table 2) which then permits synthesis of tryp-tophan from indole and serine. However, other explanations of this striking effectare possible and have not been excluded.23Discussion.-Although this constitutes a new instance of the induction of a bio-

synthetic enzyme by its reaction product, the situation is not comparable to that inwhich enzymes of the arginine biosynthetic pathway are induced by arginine.18, 24The latter enzymes are apparently those responsible for the biosynthesis of argininein wild type E. coli and their level is increased only several fold in the B strain by thepresence of arginine. In contrast, the inducible TSase studied here apparently isnot normally involved in the biosynthesis of tryptophan in wild type E. coli.That a single mechanism controls the formation of the inducible TSase and TPase

is indicated by the constancy of the ratio of the two activities under the severaldifferent cultural conditions employed and also by the isolation of mutant B/1t7-Awhich constitutively forms both activities (cf. ref. 25). The simplest possible expla-nation of the results reported here would be that a single protein possesses both TPaseand TSase activity. The postulated mechanisms of action of these two enzymesare closely similar.26 Although Burns and DeMoss report that a highly purifiedTPase preparation does not catalyze the TSase reaction,27 this does not exclude thepossibility that a single protein catalyzes both reactions. This possibility, theequally possible occurrence of two distinct proteins controlled by a common in-ductive mechanism, and other possible explanations of the results presented here areunder current investigation.Summary.-Tryptophan-requiring mutants of three different strains of E. coli

contain a previously unobserved TSase that is induced in parallel with TPase byhigh concentrations of tryptophan. The inducible nature of the enzyme appearsresponsible for the inability of indole to substitute for tryptophan in growth of the

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mutants. The inducible TSase catalyzes synthesis of tryptophan from indole plusserine but not from indoleglycerol phosphate plus serine, and it does not hydrolyzeindoleglycerol phosphate. This difference in catalystic activity, together withprevious enzymatic and genetic studies of mutant strains that produce the newTSase, demonstrate that this enzyme is different from either the A or the B com-ponent of the repressible TSase of E. coli. Both the inducible TSase and TPaseare formed in constant ratio under a variety of cultural conditions. Possible ex-planations of the results include (a) a single mechanism that controls the syn-thesis of two distinct proteins responsible for the two activities and (b) a single pro-tein that possesses both catalytic activities.

We are indebted to Charles Yanofsky and Gunther Stent for valuable discussions during thecourse of this work.

* Supported in part by a grant (E-1575) from the National Institutes of Health, U.S. PublicHealth Service.

' Browder, H. P., doctoral dissertation, University of Texas, 1959.2 Crawford, I. P., Biochim. et Biophys. Acta, 45, 405 (1960).3Crawford, I. P., and C. Yanofsky, these PROCEEDINGS, 44, 1161 (1958).4Yanofsky, C., and E. S. Lennox, Virology, 8, 425 (1959).5 Yanofsky, C., and I. P. Crawford, these PROCEEDINGS, 45, 1016 (1959).6 Cohen, G., and F. Jacob, C. R. Acad. Sci. (Paris), 248, 3490 (1959).7Monod, J., and G. Cohen-Bazire, C. R. Acad. Sci. (Paris), 236, 530 (1953).8 We wish to thank the following individuals for cultures: E. coli B/1t7, Charles Yanofsky;

E. coli td4, Martin Rachmeler; E. coli ML-304g, Jacques Monod.9 Rickenberg, H., C. Yanofsky, and D. M. Bonner, J. Bacteriol., 66, 683 (1953).10 Pardee, A. B., and L. S. Prestidge, Biochim. et Biophys. Acta, 49, 77 (1961).11 Yanofsky, C., in Methods in Enzymology, ed. S. P. Colowick and N. 0. Kaplan (New York:

Academic Press, 1955), vol. 2, p. 233.12 In some TSase reaction mixtures, a precipitate (presumably inorganic phosphate) appeared

on addition of this reagent. This was avoided by adding 0.1 ml of water per 5 ml of the reagentmixture. High concentrations of -SH compounds (e.g., glutathione) should be avoided, sincethey interfere in these "direct' TPase and TSase assays, where indole is not separated prior todetermination of its concentration.

13 Mitchell, P., and J. Moyle, in Bacterial Anatomy, Sixth Symposium of the Society for GeneralMicrobiology, ed. Spooner and Stocker (Cambridge University Press, 1956), p. 150.

14Lowry, 0. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265(1951).

16 We are grateful to Charles Yanofsky for generously supplying the purified A and B proteinsand to Martin Rachmeler for the indoleglycerol phosphate.

16 The levels of the enzymes from cells grown on the minimal medium with various supplementswere apparently influenced by contaminants present at the time in the distilled water. This andother possible nutritional factors influencing production of the induced TSase are now understudy.

17 Pardee, A. B., V. G. Shore, and L. S. Prestidge, Biochim. et Biophys. Acta, 21, 406 (1956).18Vogel, H. J., A. M. Albrech, and C. Cocito, Biochem. Biophys. Research Communs., 5, 115

(1961).19 Freundlich, M., and H. C. Lichstein, J. Bacteriol., 80, 633 (1960).20 Magasanik, B., F. C. Neidhardt, and A. P. Levin, in Physiological Adaptation, ed. C. Ladd

(Washington, D.C.: American Physiological Society, 1958), p. 159.21 Dr. Yanofsky (personal communication) has now found this induced TSase in a number of

E. coli tryptophan auxotrophs.22 Moyed, H. S., J. Biol. Chem., 235, 1098 (1960).23 Beerstecher, E., and E. J. Edmonds, Federation Proc., 15, 216 (1956).24 Gorini, L., and W. Gunderson, these PROCEEDINGS, 47, 961 (1961).

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25 Cf. i-o-galactoside mutants in Jacob, F., and J. Monod, J. Mol. Biol., 3, 318 (1961).26 Snell, E. E., Vitamins and Hormones, 16, 77 (1958).27 Burns, R. O., and R. D. DeMoss, personal communication.

CONSTITUENTS OF THE THYMUtJS GLAND AND THEIRRELATION TO GROWTH, FERTILITY, MUSCLE, AND CANCER*

BY ALBERT SZENT-GY6RGYI, ANDREW HEGYELI, AND JANE A. MCLAUGHLIN

INSTITUTE FOR MUSCLE RESEARCH AT THE MARINE BIOLOGICAL LABORATORY, WOODS HOLE,

MASSACHUSETTS

Communicated June 15, 1962

The fact that no low-molecular biologically active substances have unequivocallybeen isolated yet from the thymus gland indicates that either there are none presentor else that their demonstration and isolation is connected with major difficulties.In the absence of characteristic chemical reaction, isolation of such substances de-pends on a test, based on some biological reaction. Growth involves the interactionof a great number of reactions, and so we hoped to be able to demonstrate activematerial, possibly present, by its influence on growth. The thymus is most activein young, growing animals.Normal growth being a slow process, we turned to the faster pathological growth

of cancer. This work led us three years ago1 to the conclusion that "our extractscontained two active substances, the one promoting, the other inhibiting malignantgrowth, and the result depended on their balance." In a crude extract of thegland, both substances are present and compensate one another, and no activityis observed. The inhibitor and promotor action can be demonstrated only afterthe separation of the two. Since the chemical or physical properties of the two sub-stances are very similar, this separation is difficult, which may explain why theseactivities have hitherto not been unequivocally demonstrated. Another propertywhich makes isolation of the two substances difficult is that they show no cleancutwater or fat solubility and tend to adhere to any substance or precipitate producedduring the course of preparations. They have no acute drug action either.

For convenience, we will call the substance promoting malignant growth "pro-mine," the retarding substance "retine."

The Test.-Groups of 5-10 mice were injected subcutaneously with ascites tumor.Two days later, daily injections of various extracts were begun. The growth of thetumor was followed by noting its outlines. Final results were obtained on the tenthday by sacrificing the animals, isolating and weighing the tumors, and comparingtheir weight with that of the tumors in the control group. Various factors had tobe considered.Animal material: To exclude individual variations, inbred Swiss albino mice

were used. The growth of tumors in such animals depends on the age of the ani-mal, growth being faster in young than in old animals. We found, for instance,that in four-week-old mice the average weight of the tumor was 4,823, in four-month-old mice 2,735, and in mice older than one year 542 mg three weeks after

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