Vol. 69 MICRODIALYSIS OF STEROIDS IN BLOOD PLASMA 103The author wishes to expres his gratitude to Professor

C. C. Lucas, of the Banting and Best Department ofMedical Research, for helpful suggestions with respect tothe design of the microdialysis cell, and to Mrs D. Parrottfor her able technical help.

This work was carried out during tenure of a SeniorMedical Research Fellowship awarded by the NationalResearch Council of Canada. It was faoilitated by the abletechnical assistance rendered by Mrs Dorothy Parrot.

REFERENCES

Axelrod, L. R. & Zaffaroni, A. (1954). Arch. Biochem.Biophys. 50, 347.

Burton, R. B., Zaffaroni, A. & Keutmann, E. H. (1951).J. biol. Chem. 188, 763.

Bush, I. E. (1952). Biochem. J. 50, 370.Bush, I. E. (1954). Recent Progr. Hormone Re8earch, 9, 449.Cartland, G. F. & Kuizenga, M. H. (1936). J. biol. Chem.

116, 57.Eik-Nes, K., Nelson, D. H. & Samuels, L. T. (1953).

J. din. Endocrin. Metab. 13, 1280.Haines, W. J. (1952). Recent Progr. Hormone Re8earch, 7,

255.Hanes, C. S. & Isherwood, F. A. (1949). Nature, Lond., 164,

1107.

Harwood, C. T. & Mason, J. W. (1956). J. din. Endocrin.Metab. 16, 790.

Kalant, H. (1958). Biochem. J. 69, 93.Levy, H. & Kushinsky, S. (1954). Recent Progr. Hormone

Re8earch, 9, 357.Lewis, B. (1956). Biochim. biophy8. Acta, 20, 396.Lombardo, M. E., Hudson, P. B., Mann, P. H. & Mittel-man, A. (1954). Fed. Proc. 13, 254.

Morris, C. J. 0. R. & Williams, D. C. (1953). Biochem. J.54, 470.

Nelson, D. H. & Samuels, L. T. (1952). J. clin. Endocrin.Metab. 12, 519.

Nelson, D. H., Samuels, L. T., Willardson, D. G. & Tyler,F. H. (1951). J. din. Endocrin. 11, 1021.

Pechet, M. M. (1953). J. din. Endocrin. Metab. 13, 1542.Richards. J. B. & Sweat, M. L. (1953). Proc. Soc. Exp.

Biol., N. Y., 84, 125.Sakal, E. H. & Merrill, E. J. (1953). Science, 117, 451.Savard, K. (1954). Recent Progr. Hormone Besearch, 9,

185.Schmidt, H. & Staudinger, Hj. (1953). Biochem. Z. 324, 128.Szego, C. M. & Roberts, S. (1946). Proc. Soc. Exp. Biol.,

N.Y., 61, 161.Weichselbaum, T. E. & Margraf, H. W. (1955). J. clin.

Endocrin. Metab. 15, 970.Zaffaroni, A. (1953). Recent Progr. Hormone Re8earch, 8,51.

The Free Amino Acids in Growing and Non-GrowingPopulations of Escherichia coli

BY J. MANDELSTAMNational In8titute for Medical Re8earch, Mill Hill, London, N.W. 7

(Received 21 Augu8t 1957)

It was reported by Taylor (1947) that there wereno free amino acids in Escherichia coli and otherGram-negative bacteria, and this was considered torepresent a fundamental distinction between themand the Gram-positive organisms. Proom &Woiwod (1949), using the more sensitive method ofpaper chromatography, showed that extracts ofcoliform bacteria contained a large selection ofamino acids. From the conditions of their experi-ments it was difficult to be sure that the freeamino acids they found were not the result of con-tamination from the hydrolysed casein of thegrowth medium. More recently free amino acidshave been demonstrated in Gram-negative bacteriagrown in glucose and ammonium salts, where thereis no possibility of contamination by the medium(see Mandelstam, 1955, 1956a; Britten, Roberts &French, 1955; Markovitz & Klein, 1955).Although it seems likely that the free amino

acids are on the pathway of protein synthesis(Britten et al. 1955), there has not yet been anydetailed study of their behaviour in E. coli. In the

present paper the level of the free amino acidsduring growth and during nitrogen or carbonstarvation is described. Experiments were done onseveral coliform strains in an attempt to ensure thatthe conclusions should be generally valid and notdue to the peculiarities of a single strain.

MATERIALS AND METHODS

Organi8m. A coliform organism called Bacterisum cada-verw by Gale & Epps (1944) (NCTC 6578) and the followingstrains of E. coli were used: ML30, K 12, NCTC 1433,ML328c (leucine-requiring), 160-37 (requiring arginine orornithine), NCTC 4139 (proline-requiring). The organismswere grown with shaking at 350 in the following medium:NHEC1, 0.5 g.; (NH)2SO4, 05 g.; KH2PO4, 13-6 g.;MgSO4, 20 mg.; Fe(NH4),(S04),,6H30, 15-6 mg.; glucose,20 g.; water to 1 1. The pH was adjusted to 7-2 with NaOH.For the amino acid-requiring strains the medium was sup-plemented with L-arginine HCI (100OAg./ml.) or DL-leucine(300,ug./ml.) or L-prolne (150pg./ml.). The bacteria weregenerally harvested towards the end of the exponentialphase of growth, when the culture contained about 0-8mg.

J. MANDELSTAMdry wt. of bacteria/ml. The cells were washed twice with005M-phosphate buffer (prepared from KH2PO4 broughtto pH 7 with NaOH) and then incubated with shaking inthe same buffer at 350 under specified conditions at abacterial density of about 1 mg. dry wt. of bacteria/ml.Analyss of amino acids. For analysis of the free amino

acids a sample of the suspension, 30-40 ml., was removedand centrifuged. The supernatant solution was treatedseparately, and amino acids found in it will be referred toas the extracellular amino acids. The bacteria were sus-pended in 10 ml. of water and heated at 1000 for 20 min. toliberate the intracellular amino acids. The suspension wascentrifuged and the clear extract poured off. This extractand the supernatant were put on to columns (3 cm.2 cross-section) of Zeo-Karb 225 (5 ml.) in the H+ form. Thecolumns were washed with about 50 ml. of water and theamino acids were then eluted with an excess (25 ml.) of aq.1-5N-NH, soln. The eluates were evaporated to dryness andthe residue was dissolved in 0-2 ml. of water and transferredwith two washings of 0-1 ml. to Whatman no. 3 paper. Inmost experiments the chromatograms were developed withbutanol-acetic acid-water (63:10:27) for 18-24 hr. Forone-dimensional separation of glycine, serine and threo-nine, m-cresol-water was used. Leucine and isoleucine wereseparated from each other with tert.-amyl alcohol (Work,1949). Two-dimensional chromatograms were run onWhatman no. 1 paper, phenol-ammonia followed bybutanol-acetic acid-water being used.The amount of amino acid in each spot was determined

by a method essentially similar to that of Kay, Harris &Entenman (1956). In our experience the method wasaccurate to within about 10% for amounts of amino acidexceeding 10 ug.; below this level the error increased con-siderably. It was necessary to run standards for everyestimation because the intensity of the ninhydrin colourvaried somewhat from day to day. Results are expressedas ,ug. of amino acid/ 100 mg. dry wt. of bacteria. The totalamino acid content was obtained by summing the indi-vidual fractions.The complete isolation and estimation of an amino acid

was not carried out unless it was necessary for determina-tion of specific radioactivity or for some other specialreason. In general, the one-dimensional chromatogramobtained with butanol-acetic acid was used, and the aminoacids were grouped into several fractions for estimation.Two-dimensional chromatograms and the special solventsreferred to above showed that these fractions had thefollowing composition. The 'leucine' fraction consisted ofleucine and isoleucine; the 'valine' fraction consistedmainly of valine but also contained some methionine; the'glycine' fraction contained glycine, serine and asparticacid (the last being present in very small amounts in com-parison with the other two components); the 'glutamic'fraction contained mostly glutamic acid and threonine;'tyrosine' and 'alanine' fractions consisted almost entirelyof tyrosine and alanine but contained small amounts ofother amino acids which were not identified; the 'arginine'fraction contained basic amino acids and possibly gluta-thione. To avoid confusion, quotation marks will be usedwhen referring to a fraction.

Specific radioactivity of leucine. The specific radioactivityof [1-14C]leucine was determined by separating the leucineas described above. The radioactivity was measured on thechromatogram with the scanning instrument described by

Piper & Arnstein (1956), and the amount of leucine wasthen determined by the method of Kay et al. (1956).Radioactive standards containing 25 and 50 zg. of leucinewere run on each sheet.

EXPERIMENTAL

Free amino acids of Escherichia coliExtracts from freshly harvested E. coli yielded atleast a dozen distinct ninhydrin-reacting spots inone-dimensional chromatograms, and more wereobtained by two-dimensional analysis. The follow-ing amino acids have Deen identified in wild-typestrains (ML 30 and 6578): glycine, alanine, serine,threonine, aspartic acid, glutamic acid, arginine,lysine, methionine, valine, phenylalanine, tyrosine,isoleucine, leucine. In addition, most extractscontained several unidentified amino acids orpeptides.Although the same amino acids were found in all

the strains examined, the relative concentration ofany particular amino acid not only varied from onestrain of bacteria to another, but also dependedupon the stage of growth (see below). Similarvariations were encountered in the total pool,which varied from about 0-25 to over 1% of thebacterial dry weight.The amino acid content was not significantly

decreased by repeated washing in phosphate bufferor by short periods of incubation in the presence ofglucose. Analysis of the washings in such experi-ments showed that only negligible traces of aminoacids were being removed.

Free amino acids during growthWhen E. coli (ML 30) was grown in 500 ml. of

medium in a 51. conical flask with the normalshaking rate of 90 oscillations/min. with a hori-zontal excursion of 8 cm., growth remained ex-ponential until the bacterial density had reached1 mg./ml. With a horizontal excursion of only4 cm. the aeration was insufficient and the growthrate began to fall slowly at about 0 3 mg./ml. Thevariation in the intracellular free amino acid ofsuch a culture was followed by removing samples,each containing 40 mg. of bacteria, and analysingthem as described. Contrary to the findings ofDagley & Johnson (1956), no extracellular aminoacids could be found.As growth proceeded the content of intracellular

free amino acids fell more or less linearly (Fig. 1 a).The relationship between the level of the freeamino acids and growth was roughly linear asshown in Fig. 1 (b), where the specific rate ofgrowth calculated from the data of Fig. 1 (a) hasbeen plotted against the size of the pool, the valuesfor the first sample being taken as 100 in bothcases. An arbitrary measure of the specific growth

104 I958

FREE AMINO ACIDS IN E. COLIrate was obtained by calculating the percentageincrease in unit time at various points on thegrowth curve of Fig. 1 (a).The proportionality between growth rate and the

free amino acid content held also for most of theamino acid fractions that were examined: 'leucine',' valine', 'alanine' and 'glutamic'. Exceptionswere 'tyrosine', whose concentration remained un-changed throughout, and the 'glycine' fractionwhose concentration fell slightly at the lastsample.

Maximum content of intracellular free amino acidsAn attempt was made to measure the maximum

amount of free amino acid that could accumulateintracellularly by allowing bacteria to synthesizeamino acids but not to incorporate them intoprotein. This was done in two ways. The firstmethod consisted of incubating wild-type E. coli at1 mg./ml. in the complete growth medium con-taining chloramphenicol (20 ,ug./ml.) to inhibitprotein synthesis. In the second method, anamino acid-requiring strain was used, and proteinsynthesis was prevented by omitting the essentialamino acids from the medium.Both procedures yielded results that were

similar in all respects and a typical example ofamino acid accumulation is shown in Fig. 2. At theend of 40 min., when the first sample was taken, thetotal intracellular amino acid content had risenfrom 475 to 710,g./100mg. and 400pug./lOOmg.had been secreted into the external medium.Further incubation did not lead to any increase inthe intracellular amino acids, but amino acidscontinued to accumulate in the external medium.The behaviour of the various amino acid fractions

closely resembled that of the total amino acid inthat each had reached its maximum at the end of40 min. and did not change significantly during theensuing 2 hr.

100-

2775D

¢ 50

0

0w4so3 2

(b)

.

I I I Iu 25 50 75 100

Intracellular free amino acid (arbitrary units)

Fig. 1. (a) Free amino acid content of E. coli (ML30)during growth under sub-optimum conditions of aera-tion (see text). *, Bacterial density; 0, total intra-cellular free amino acids. (b) Linear relationshipbetween growth rate and intracellular amino acid con-tent in E. coli (ML30). Specific growth rate, calculatedfrom the data of (a), is plotted against the free amino acidcontent. Values at 1 hr. 25 min. (see a) were arbitrarilytaken as 100.

u

-o

E-8

0to80

._.

c);

0 30 60 90 120 150Time (min.)

Fig. 2. Accumulation of free amino acids under conditionsof blocked protein synthesis. Arginine-requiring bacteriawere incubated with glucose and ammonium salts,arginine being omitted from the medium to preventprotein synthesis. 0, Intracellular amino acids; *,extracellular amino acids.

I I

Vol. 69 105

J. MANDELSTAM

The fixed levels reached by the total intracellularamino acids and by each of the constituentfractions were characteristic of the bacterial strain.In Table 1 the maximum values are shown for fourstrains of bacteria: two wild-type (ML 30 and6578) and two amino acid-requiring strains(160-37 and 4139). There is considerable variationbetween the strains, not only in the total amount ofamino acid (0.3-1.5 % of the bacterial mass) butalso in the relative distribution of the differentfractions.

It is perhaps significant that the strain with avery low free amino acid content (6578) grew muchmore slowly than any of the other strains. It hada generation time of 90 min., whereas the genera-tion time for the other strains varied from 55 to65 min.

Effect of azide and of 2:4-dinitrophenol upon thelevel of intracellular free amino acid8

The effect of azide and 2:4-dinitrophenol wastested to determine whether normal energy meta-

Table 1. The capacity offour 8train8 of Escherichiacoli to accumulate intraceUular free amino acidaAccumulation was caused by allowing amino acid

synthesis from glucose and ammonium salts to proceed for160 min. under conditions of blocked protein synthesis.The amino acids have been divided into various fractions(see Methods).

Amino acid content(.tg. of amino acid/100 mg.

of bacteria)

Strain ... ...

Amino acid fraction'Leucine''Valine''Tyrosine''Alanine''Glutamic''Glycine'' Arginine '

Total

ML 30 6578 160-37 4139

3322020

440571130135

1549

17 13 1613 165 16134 25 4333 163 25081 259 63593 65 10528 140 166

299 830 1376

bolism was required to maintain the level of theintracellular amino acids.Washed bacteria (ML 30) were incubated either

with 1% (w/v) glucose or with 1 % (w/v) glucoseand inhibitor. The final concentration of sodiumazide was 12 mm and that of the dinitrophenol was0-6 mm. The results appear in Table 2.

Incubation with glucose alone caused the intra-cellular amino acids to increase from 425 to574,g./100 mg. in 20 min. This effect will be con-sidered in detail in the next section. In contrastwith this, treatment with azide caused the level tofall from 425 to 355 pg./100 mg. Most of thematerial lostwas derived from the 'valine', 'alanine'and 'glutamic' fractions. The 'glycine' fractionwas not significantly affected, and the 'arginine'fraction increased.The effect of dinitrophenol was more marked and

more general. The total amino acids decreased byabout 50% (425-215 ,g./100 mg.) and all the con-stituent fractions of the pool except 'glycine' wereaffected. Some of the fractions such as 'leucine'and 'valine' were lost altogether.

Free amino acid8 during nitrogen 8tarvationIn the previous section it was noted that when

washed bacteria were incubated with glucose thefree amino acid content increased. This effect wasfollowed by incubating a similar suspension andtaking samples for analysis over a longer period.In addition, the extemal medium was also ex-amined for the presence of amino acids.

In a typical experiment (see Fig. 3a) the intra-cellular amino acids increased steadily from 376 to608,ug./100mg. in the first 80min. and then re-mained constant. In addition 166 ,tg./100 mg. wasfound in the external medium. The appearance ofamino acids in the external medium under theseconditions is essentially similar to that reported byDagley & Johnson (1956). In these experimentsthere was always some loss of material to themedium even though the maximum internal level

Table 2. Effect of azide and of 2:4-dinitrophenol upon the intracellular free amino acids of Escherichia coliWashed celLs (ML30) were incubated for 20 min. with glucose alone (control) or with glucose and inhibitor. Concentra-

tions: 2:4-dinitrophenol, 0-6 mM; azide, 2 mM. Amino acid content(jug. of amino acid/100 mg. of bacteria)

Amino acidfraction

'Leucine''Phenylalanine''Valine''Tyrosine''Alanine''Glutamic''Glycine''Arginine '

Total

Beforetreatment

155

421581999375

425

Control2418424

1561929048574

2:4-Azide Dinitrophenol

90151054879090355

0008183010851215

106 I958

FREE AMINO ACIDS IN E. COLIhad not been attained. Nevertheless, the greaterproportion of extra amino acid that appeared in thecourse of the experiment was retained by the cells.A rather different result was obtained by omit-

ting glucose, i.e. by shaking the bacteria in phos-phate buffer. It will be seen (Fig. 3 b) that, whereasthere was, as before, an increase in the total freeamino acids, all the extra material was lost to theexternal medium and the prolonged carbonstarvation produced a slow decline (586-476 /Ag./100 mg.) in the level ofthe intracellular amino acids.Although nitrogen starvation

produced an increase in the totait was difficult to obtain quantita

-C

ti 600.0tD

E2 400

v-

Q 200:4)._ 0

O-

0

_ I

30 60 90Time (mir

(b)

Time (min.)Fig. 3. (a) Accumulation of amino E

starvation. Washed bacteria (E. c

bated with glucose in the absence0, Intracellular amino acids; 0,

acids. (b) Accumulation of amincand nitrogen starvation. Washed biwere incubated in buffer in the absesource of carbon or nitrogen. 0acids; *, extracellular amino acids

results from one experiment to another even withthe same strain of bacteria. The probable reason forthis will be discussed below.

Origin of the free amino acid8 relea8ed duringnitrogen 8tarvation. While protein was the mostobvious source for the amino acids released duringnitrogen starvation, they might also have beensynthesized from traces of ammonium saltscarried over from the growth medium, or alter-natively through the utilization of nitrogen fromnon-protein metabolites.

almost invariably An attempt was made to determine the origin of1l free amino acids, the released amino acids by using a strain oftively reproducible bacteria (ML 328c) which was unable to synthesize

leucine (Hirsch & Cohen, 1953), and carrying outthe same type of nitrogen starvation procedure. If

__0 this strain liberated leucine in the same way as thewild type (ML 30), breakdown of protein would beindicated. If, on the other hand, leucine did notappear together with the other amino acids it

(a) would indicate that the increase was due to pro-duction of amino acids from a non-protein source.In preliminary experiments itwas found that leucineappeared together with the other amino acids inthe same way as it had done in the wild type.But there still remained the possibility that the

block in the synthesis of leucine was not absolute,and that the bacteria were able to synthesize a

120 150 small amount of leucine, insufficient for growth,but sufficient to account for the increase observedin the experiment. The experiment was thereforecarried out with labelled leucine as follows (see alsoMandelstam, 1956a). The bacteria were grown froma small inoculum (0-04 mg. dry wt. of bacteria) in500 ml. of synthetic medium supplemented with100,ug./ml. of DL-[1-14C]leucine (approx. 0-5,uc/mg.). They were harvested at a density of 0-80-0-85 mg./ml., washed twice in phosphate buffer andsuspended in 410 ml. of the same buffer containing

° 1/I% of glucose. Duplicate samples, each containing100 mg. of bacteria, were taken at the beginning ofthe experiment and after 2 hr. To conserve radio-active leucine, the extracellular and intracellularsamples were pooled and estimated together.The increase in total free amino acid was 255 ,g./

100 mg. of bacteria (Table 3), which is equivalent toabout 0-45% of the bacterial protein. Leucine

Is accounted for 6-5% of the total increase, which120 150 180 would be expected since leucine constitutes about

6% of the protein in E. coli (Polson, 1948).acids during nitrogen Quadruplicate estimations of specific radio-o1t ML30) were incu- activity showed that the leucine before incorpora-of a nitrogen source. tion had an activity of 36-4-38-6 counts/min./,ig.extracellular amino of 1acids during carbon eucie; the values for the leucine released by the

acteria (E. coli ML30) bacteria during the incubation were 35-4 andance of any exogenous 36-7 counts/min./,g. This was within the error ofIntracellular amino the method, and demonstrated that the released

leucine had not been synthesized by the bacteria.

Vol. 69 107

J. MANDELSTAM

Table 3. Production offree amino acid8 byEscherichia coli during nitrogen 8tarvation

Leucine-requiring bacteria were washed and incubatedin phosphate buffer with 1% glucose for 2 hr., and sampleswere taken at the beginning and end of the incubation.Leucine was estimated separately; the other amino acidswere pooled and estimated together. In the last column theamount of amino acid produced is expressed as a per-

centage of the amino acid in the cell protein.

LeucineAll other amino acids

Amino acid content(p&g. of amino acid/100 mg.

of bacteria)

Before After %incubation 2 hr. produced

Trace 16-5 0 47228 483 0445

DISCUSSION

The experiments on the capacity of the bacteria tohold amino acids indicate that there is a level forthe amino acids beyond which no more materialcan be retained in the cell. This finding could beexplained by assuming that there are fairly specificadsorption sites for the amino acids and that thesebecome saturated under conditions where proteinsynthesis is blocked and amino acids accumulate.A theory of specific adsorption sites was put

forward by Cohen & Rickenberg (1955) to accountfor their observations on the concentration of[14C]valine. They had found that the valine was

rapidly taken up by E. coli in the presence of a

source of energy. The valine could be recovered byheating the cells at 1000. Alternatively, it could bedisplaced rapidly by unlabelled valine and also byleucine or isoleucine. The addition of azide or

2:4-dinitrophenol at the beginning of the experi-ment prevented the uptake of labelled valine, andthe addition of azide after uptake of the valine ledto a slow loss of it from the cells.

In a later communication Cohen & Rickenberg(1956) abandoned their earlier theory, althoughthey admitted that it accounted satisfactorily forthese phenomena. They preferred, however, thetheory of a specific catalytic concentrating mech-anism ('permease'), thus bringing their interpre-tation into conformity with that advanced for theconcentration of galactosides in E. coli (Rickenberg,Cohen, Buttin & Monod, 1956).The 'permease' theory is as follows:

PermeaseExternal valine >

Internal valine External valine.'Permease' is considered to be a catalytic con-

centrating mechanism which transports valine intothe cell and whose action is competitively in-hibited by similar molecules such as leucine and

isoleucine. The transport system requires energyand is inhibited by azide and dinitrophenol. Thevaline which has been concentrated is expelledfrom the bacteria by another mechanism, A.According to this model there is thus a rapid circu-lation of valine through the cell so that r14C]valineis rapidly displaced from the bacteria by additionof valine (or leucine or isoleucine). If azide isadded, however, the loss of valine is much slower,and to explain this fact the authors find it necessaryto assume that azide inhibits A as well as permease.They also suggested that the concentration ofamino acids generally is effected by means ofspecific permeases.

This scheme appears to contain a number of un-necessary postulates. First, there seems to be in-sufficient justification for assuming the existence ofa catalytic concentrating mechanism, and if thisassumption is made it becomes necessary topostulate the system A and to postulate furtherthat it is inhibited by azide. Even with theseadditional assumptions the theory fails to providea satisfactory explanation for our present findingsthat all amino acid above a certain fixed level islost from the cells whereas below this level it isfirmly retained. This is particularly noteworthywith lysine which, in the absence of an energysource, appears to diffuse fairly freely in coliformbacteria (Mandelstam, 1956b) so that its concen-tration is the same inside and outside the cells.Nevertheless, a small amount of intracellular lysinedoes not seem to be able to equilibrate in thismanner since it is always found in the cells. Itcannot be removed even by repeated washings, nor(in Bacterium cadaveri8) is it destroyed by lysinedecarboxylase which is always present in this strain(Mandelstam, 1954).

It seems that the present experimental findingscan be more simply and more adequately explainedin terms of Cohen & Rickenberg's (1955) originaltheory that there are specific adsorption sites forthe amino acids and that a good supply of energy isnecessary for rapid equilibration between addedand adsorbed amino acid. This follows from thefact that very little labelled valine is taken up bythe cells without a source of energy, whereas in thepresence of a source of energy there is rapid ex-change between the labelled valine and the un-labelled valine which the bacteria contain whenthey are harvested. However, the maintenance ofthe pool seems to require much less energy, becauseprolonged carbon starvation produces only a slightfall in the intracellular pool (Fig. 3b). That someenergy is needed is indicated by the fact that azideand dinitrophenol increase the rate of loss ofamino acids from the cells.The increase found in free amino acids during

nitrogen starvation is similar to that observed

108 I958

FREE AMINO ACIDS IN E. COLI

when nitrogen-starved yeast cells were incubatedwith alcohol instead of glucose (Halvorson, Fry &Schwemmin, 1955). In E. coli the increase in freeamino acids is obtained equally in the presence ofglucose or of succinate or, indeed, with no exo-genous carbon source at all. The experiments withlabelled leucine show that this amino acid, at least,is not synthesized by the bacteria from non-proteinnitrogen. This does not formally prove that all theamino acids thus released arise from the breakdownof protein, but, in the next paper, independentevidence is presented to show that there is a fairlywell-balanced degradation and resynthesis of pro-tein in nitrogen-starved bacteria. The processcontinues for 3-4 hr. at a rate equivalent to about5% of the total protein/hr. (Mandelstam, 1957).Since the amino acid pool is equivalent to onlyabout 0-5-2 % of the total protein, the amount ofmaterial passing through the pool is large incomparison with what is already there. It is clearthat a relatively small imbalance in the rates ofsynthesis and degradation of protein could lead toappreciable changes in the size of the pool. Thusthe change in free amino acid reported in Table 3could be accounted for if the rates of degradationand resynthesis of protein were 5 and 4-7 %/hr.respectively. This may explain the difficulty ex-perienced in getting quantitatively reproducibleresults in the nitrogen-starvation experiments.The relationship of the intracellular free amino

acids to protein synthesis may now be considered.Britten et al. (1955) showed that 14C-labelledproline was apparently adsorbed by suspensions ofE. colti, and that the radioactivity incorporatedinto protein during growth was directly propor-tional to the specific activity of the proline in theadsorbed state. They stated that even if only asmall fraction of the proline entering the proteinhad by-passed the adsorbed pool it would havebeen detected by their method. The fact that therate of growth is proportional to the total freeamino acid content (Fig. 1 b) suggests that thefindings from the experiments with proline mayapply to the other amino acids as well, and that theadsorbed pool as a whole is on the pathway ofprotein synthesis.

There is no evidence to indicate the manner ofadsorption or the nature of the adsorbing surface,but Britten et al. (1955) suggested hydrogen-bonding on a surface of ribonucleic acid. Now theamino acid pool when 'full' contains 1000-1500 ,ug. of amino acid/100 mg. dry wt. of bacteria,i.e. about 10 ,umoles of amino acid/100 mg. Theamount of ribonucleic acid in the cells is roughly50,moles of ribonucleic acid nucleotide residues/100 mg. (Britten et al. 1955), and thus there isample nucleotide material to account for theadsorption of the amino acid pool. While this

calculation shows that nucleic acid could be theadsorbing surface, there is no evidence to provethat it is, and it could be argued with equal forcethat the amino acids are adsorbed on protein.There are roughly 100 amino acid residues inprotein for every amino acid molecule in the freestate, so that again there seems to be ampleprotein to provide the adsorbing surface.

If it is accepted tentatively that adsorption ata specific site is an essential preliminary step inprotein synthesis, it is of interest to calculate therate of flow of material through the adsorptionsites during growth. With a pool equivalent to1 % of the bacterial protein, and a generation timeof 1 hr., the turnover of amino acids in the poolwould be 100% in 0.77 min., i.e. each adsorptionsite would, on average, take up a fresh amino acidmolecule about once every 45 sec. This result maybe compared with one derived from the penicil-linase-forming system of Bacillu8 cereus (M. R.Pollock, personal communication). The rate ofinduced penicillinase formation is dependent uponthe number of penicillin molecules specificallyfixed by the bacteria (about 200/cell). If it isassumed that each molecule allows one enzyme-forming site to become functional, it can becalculated that the rate of formation of theenzyme is of the order of one molecule/site/min.,i.e. the enzyme-forming site has about the sameturnover-time as the amino acid-adsorbing sites.This agreement may be considered additionalsupport for the view of Britten et al. (1955) that theamino acids are adsorbed at the actual sites ofprotein synthesis.

SUMMARY

1. Coliform bacteria grown in a medium con-taining glucose and inorganic salts contained atleast 15 amino acids in the free state. Together theseconstituted 0-25-1-5 % of the dry weight, de-pending upon the type of bacteria.

2. The total free amino acid content varieddirectly with the specific growth rate of thebacteria.

3. The results support the theory that there isa limited number of adsorption sites for eachamino acid.

4. Retention of the free amino acids within thecell was linked with energy metabolism.

5. WVhen bacteria were starved of nitrogen orcarbon, free amino acids accumulated and this hasbeen shown to be due to protein degradation.

6. The possible role of the free amino acids inprotein synthesis is discussed.

I wish to thank Dr T. S. Work for advice on chromato-graphic procedure and I am greatly indebted to Miss R.Coyle for invaluable technical assistance.

Vol. 69 109

110 J. MANDELSTAM I958REFERENCES

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Turnover of Protein in Growing and Non-GrowingPopulations of Escherichia coli

BY J. MANDELSTAMNational In8titute for Medical Re8earch, Mill Hill, London, N. W. 7

(Received 21 August 1957)

The classical theory of the dynamic state ofproteins was based upon isotopic experimentswith mammals. Recently the concept has beenquestioned on the basis of experiments withbacterial systems. Three independent groups ofworkers reported that the proteins of E8cherichiacoli were stable and that the rate of turnover ofprotein in growing suspensions was negligible(Rotman & Spiegelman, 1954; Hogness, Cohn &Monod, 1955; Koch & Levy, 1955). Hogness et al.(1955) went on to suggest that proteins in themammalian cell also were stable and that theobserved turnover was not true intracellular turn-over but was due to replacement of materialreleased by secretion or cell lysis.However, a number of facts pointed to the

occurrence of degradation and synthesis of proteinin non-growing populations, and suggested that itmight not be valid to extrapolate from growingbacteria to mammalian systems where the cellpopulation is, by comparison, virtually static.Thus Podolsky (1953) had reported a slow rate ofprotein degradation (about 0-25 %/hr.) and Mel-chior, Klioze & Klotz (1951) had found that [35S]_methionine was incorporated into the proteins ofwashed E. coli. Further evidence for the synthesisof protein is to be found in the fact that washed, oreven nitrogen-starved, bacteria can synthesizeinducible enzymes (for examples see Mandelstam,1956; Pollock, 1958).The present paper is a report of experiments

designed to measure the extent of protein turnover

in non-growing populations of E. coli by separatedetermination of the rates of degradation and ofsyn-thesis. Factors affecting both processes have beenstudied and, in addition, the rates of degradation ingrowing and non-growing populations have beencompared. In this paper the term non-growing willbe used to denote suspensions of bacteria in whichthere is no net synthesis of protein.The experiments were carried out with mutant

strains of E. coli with specific amino acid require-ments. For the measurement ofprotein synthesis innon-growing suspensions, leucine- or arginine-requiring strains were used. The bacteria were firststarved of the essential amino acid and then incu-bated with an excess of labelled glycine. The condi-tions were therefore such that protein synthesis,with concomitant incorporation of glycine, couldnot take place until leucine or arginine was releasedby the degradation of existing protein. The validityof this method as a measure of protein synthesiswill be considered in the Discussion.For investigation of protein breakdown a strain

of E. coli was used which required both leucine andthreonine for growth. The proteins were labelledby growing the bacteria in the presence of labelledleucine. The bacteria were then washed and incu-bated with an excess of unlabelled leucine to traplabelled leucine liberated from the proteins. In theexperiments with non-growing suspensions, onlya carbon source was added; when a growing popu-lation was required the medium was supplementedwith ammonium salts and threonine.