JOURNAL OF BACTERIOLOGY, Dec., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No. 6Printed in U.S.A.

Protein Turnover in Escherichia coli as Measuredwith an Equilibration Apparatus

ELLIOT M. LEVINE1Laboratory of Biochemical Pharmacology, National Institute of Arthritis and Metabolic Diseases, National

Institutes of Health, Bethesda, Maryland

Received for publication 14 July 1965

ABSTRACTLEVINE, ELLIOT M. (National Institutes of Health, Bethesda, Md.). Protein turn-

over in Escherichia coli as measured with an equilibration apparatus. J. Bacteriol.90:1578-1588. 1965.-Intercellular protein turnover (the reutilization by one cell ofamino acids derived from the protein of another cell) occurs at a rate of 0.16 to 0.18%per hour in nongrowing cultures of Escherichia coli, as determined in an apparatus thatrapidly equilibrates the culture fluids of two separated bacterial suspensions.

Protein turnover may be defined as a dualprocess: (i) degradation of protein molecules topeptides or amino acids and (ii) their subsequentreincorporation into protein molecules. Thenature of protein turnover in microbes has beenwidely discussed (Rotman and Spiegelman, 1954;Weinbaum and Mallette, 1959; Cohn, 1957;Borek, Ponticorvo, and Rittenberg, 1958;Markovitz and Klein, 1958a, b; Mandelstam,1960). As will be seen, a critical evaluation ofprotein turnover measurements requires that anydescription of protein turnover in a cell popula-tion should distingusih among three possibilities:(i) that each cell degrades and reforms proteinexclusively within itself (hereafter termed intra-cellular turnover), (ii) that amino acid residuesderived from damaged or secreting cells arereutilized by other cells (intercellular turnover),and (iii) that both intracellular and intercellularturnover are operative under physiological condi-tions.The usual procedures employed for measuring

protein turnover in microorganisms (Borek et al.,1958; Halvorson, 1958; Mandelstam, 1958,1960) cannot distinguish intracellular turnoverfrom intercellular turnover. Generally, cells whoseprotein has been prelabeled with a radioactiveamino acid are incubated in the presence of anexogenous "trap" of unlabeled amino acid, andthe appearance of radioactive amino acid in themedium is taken as a measure of protein turn-over. This procedure does not measure intra-cellular turnover exclusively, because labeledamino acid found in the medium may be derived

1 Present address: Department of Cell Biology,Albert Einstein College of Medicine, Bronx, N.Y.

from sources other than the endogenous pool ofviable cells (e.g., from lysed, damaged, or secret-ing cells). Thus, the usual determinations reflectboth intercellular and intracellular turnover,a value which may be termed total turnover.

Determining intercellular turnover, therefore,is important not only because the phenomenonitself is of interest, but because intracellularturnover cannot be determined unless inter-cellular turnover has been accurately measured.Furthermore, because experimental manipula-tion can damage cells (Koch, 1959), a given valuefor total turnover may be greatly increased bycertain experimental procedures. Only an accu-rate determination of intercellular turnover candetect such an artifact. The object of the presentstudy has been to determine the contributionof intercellular turnover to total turnover inEscherichia coli.

Previous studies have attempted to evaluateintercellular turnover in several ways. Cellularlysis, damage, and amino acid secretion wereassessed by measuring cell viability and appear-ance of endogenous cellular constituents oramino acids in the medium. Such estimates maybe misleading. For example, viable counts areconsidered neither precise nor sensitive, and donot reflect cell lysis in a nongrowing culture ifnew cells grow by utilizing dead cells as nutrients(Koch, 1959; Mandelstam, 1960). The method ofassaying for appearance of endogenous enzymesin the medium (Mandelstam, 1958; Halvorson,1958; UrbA, 1959), has also been subject toreservations (Koch, 1959). As shown withBacillus subtilis and E. coli, some endogenousenzymes are not totally released into the mediumafter cell damage, and others are inhibited by

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extracellular bacterial products (Pollock, 1961;Gershanovitch, Avdeeva, and Goldfarb, 1963;Neu and Heppel, 1964a, b). As far as detectionof amino acids in the medium is concerned, this isdifficult if the uptake rate into the cell is com-parable to the exit rate; the consequent steady-state level of amino acid in the medium can bevery small despite a high rate of protein turnover.Finally, the rate of amino acid appearance in amedium initially devoid of amino acid only re-flects the degradative phase of intercellularturnover. Overall intercellular turnover can bedetermined only by measuring the rate of aminoacid reincorporation into cells.

Clearly, for all the above reasons, intercellularprotein turnover in bacteria has not been ade-quately studied, and, thus, the true magnitude ofintracellular turnover still remains in question.However, a successful experimental approach hasbeen devised to study intercellular turnover inmammalian cells (Eagle et al., 1959). By couplingtwo culture vessels together, a monolayer oflabeled mammalian cells adhering to a glasssurface of one vessel was allowed to share thesame culture fluid as a monolayer of unlabeledcells in a second vessel. In the absence of anexogenous amino acid "trap" in such a system,incorporation by the unlabeled cells of thelabeled amino acid released into the mediumproceeds almost as efficiently as reincorporationby the initially labeled cells themselves. Therate of incorporation reflects the rate of inter-cellular protein turnover.To utilize this principle for studies with bac-

terial suspensions, certain modifications werenecessary. This report describes experimentswith E. coli performed in a new apparatus de-signed for rapid equilibration of the culturefluids of two bacterial suspensions separated by aMillipore filter. Cells of an E. coli threoinine-tryptophan auxotroph were prelabeled withthreonine-C'4 and were then suspended in mediumdevoid of threonine and tryptophan; the transferof threonine-C'4 to an unlabeled culture of thesame auxotroph was measured. The results showthat a definite intercellular turnover does occurin nongroving E. coli, but at a slow rate.

MATERIALS AND METHODS

Amino acids. All unlabeled amino acids were"A" grade reagents purchased from Calbiochem.L-Threonine-C'4, uniformly labeled (specific ac-tivity, 165 ,c/,umole i 5%), was purchased fromNew England Nuclear Corp., Boston, Mass.

Other materials. Cab-O-Sil was purchased fromPackard Instrument Co., LaGrange, Ill. E. coliK-12 strain PA209 was kindly furnished by F.Jacob, Pasteur Institute, Paris, France. This

strain requires L-histidine, L-leucine, L-trypto-phan, L-threonine, and thiamine for growth, andcannot ferment lactose, galactose, and severalother sugars (Jacob and Wollman, 1961).

Preparation of cells. E. coli K-12 strain PA 209was grown in a medium containing, in a totalvolume of 1 liter: K2HPO4, 1.5 g; KH2PO4, 0.5 g;MgSO4, 1.0 g; D-glucose, 5.0 g; (NH4)2SO4, 2.5 g;thiamine, 10 mg; L-threonine, 100 mg; L-trypto-phan, 100 mg; L-histidine, 100 mg; L-leucine, 100mg; L-isoleucine, 100 mg; and glycine, 100 mg;final pH was 7.0. Cells grown in the above mediumwith L-threonine-C'4 contained 95% of theirradioactivity in protein-bound threonine. Unlessunlabeled glycine and isoleucine were included inthe growth medium, radioactivity appeared in theglycine and isoleucine residues of the cell protein.Media were filtered through PH (pore size,

0.30,A) Millipore filters for sterilization; this alsoremoved particulate matter which could clog theequilibration apparatus. The culture was grownto a density of approximately 150 Klett units(no. 540 filter, Klett-Summerson photometer,equivalent to an absorbancy of 0.450 at 540 m, in aBeckman DU spectrophotometer, 1-cm light path;the dry weight of cells was 0.46 mg/ml and theweight of bacterial protein was 0.22 mg/ml). Thefollowing procedure was used to wash cells freefrom growth medium. Cells were centrifuged atroom temperature, the supernatant fluid wasdecanted, and cells were suspended in filteredmedium devoid of (NH4)2SO4, L-threonine, L-tryptophan, L-isoleucine, and glycine. Resus-pended cells were incubated with shaking for ap-proximately 10 min in a water bath at 37 C. Cellswere centrifuged, suspended, and incubated withshaking twice more, and were then suspended to adensity of 50 Klett units. This washing procedurediluted threonine carried over from the originalsupernatant fluid to less than 5% of the lowestconcentration (0.12 ,u,mole/ml) added in any ex-periment.

Equilibration apparatus. Several simple ap-paratuses have been described for the equilibra-tion of two bacterial cultures. In these, cultureswere separated by cellophane membranes(Nurmikko, 1955, 1956, 1957) which allowed onlya slow equilibration (a matter of days) or ultrafinesintered-glass filters (Davis, 1950) which cloggedafter several transfers of culture medium. In thenew apparatus, Millipore filters were used toseparate the bacterial suspensions, and alternateapplications of air pressure and vacuum wereemployed to flush the culture fluid between thetwo bacterial suspensions. The culture fluids ofthe two bacterial suspensions were equilibratedquite rapidly: 57% after 20 sec, 92% after 40 sec.The details of a typical experiment, including acomplete description of the apparatus, its assem-bly, and operation, follow.As shown in Fig. 1, culture flasks I and II (modi-

fied 125-ml suction flasks) were joined by a filteringunit consisting of two Millipore filters mountedbetween two fritted-glass filter bases. The filter

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Al R- From saturatorand trop

FILTERI NGUN IT

FIG. 1. Equilibration apparatus. The apparatus itself is shown in the center of the diagram; the upperleft insert is a wiring diagram, and the lower right insert an enlarged view of a filter holder (one-half ofthe filtering unit). In the diagram of the apparatus, "A" and "B" represent electric hosecocks; "A" isshown open, and "B" is closed. Air entered the apparatus through the shaded tubing; vacuum wasapplied through the plain tubing. See text for details. The following materials were used in the constructionand assembly of the apparatus: vacuum gauge, pressure gauge, glass joint clamps no. 28 and no. 18,Pyseal cement, and electric hosecocks, purchased fromr Fisher Scientific Co., Pittsbuirgh, Pa.; MFcement (formulation no. 1), Millipore filters (25 mm, types PH and HA), and Pyrex microanalysisfilter holders purchased from the Millipore Filter Corp., Bedford, Mass.; Siliclad (a siliconizingagent) obtained from Clay-Adams, Inc., New York, N.Y.; Caf6-au-lait rubber stoppers (no. 2) obtainedfrom Emil Greiner Co., New York, N.Y. (used because they are more pliable than conventional stop-pers); an interval timer purchased from. Harco Products, Philadelphia, Pa.; and a single-pole-double-throw relay obtained from Potter and Brumfield, Princeton, Inc.

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bases (shown in the lower insert of Fig. 1) weresimilar to those available from the MilliporeFilter Corp., except that the ordinary stem wasreplaced with thick-walled capillary tubing (2-mmbore). A coarse-grade fritted-glass disc (19 to 20mm in diameter) was sealed into the supportingglass tube with its top surface about 0.5 mm belowthe tubing; the end of the protruding tubing wasground flat, and its inner edge was beveled smoothwith a carborundum stick (Fig. 1, lower insert).The internal volume of the entire filtering unitwas approximately 2 ml. To prepare the filteringunit for use, MF cement was placed on the groundends of the filter bases, and a 25-mm type PHMillipore filter (pore size, 0.30 jA) was mounted oneach holder. The holders then were butted to-gether and secured by two ball-and-socket jointclamps (no. 18 and no. 28). The junction of thetwo filter holders was sealed with Pyseal cement.The filter holder stems, previously inserted intocafe-au-lait rubber stoppers (no. 2), were placedin the culture flasks through openings made forthat purpose.

After the filtering unit was in place, the appara-tus could then be employed to equilibrate themedia of two bacterial suspensions, one in unit Iand the other in unit II, by alternate applicationof pressure and vacuum to each unit. Air pressure(shaded tubing) was supplied to the equilibrationapparatus at 20 psi and vacuum (unshaded tubing)at 15 inches (38.1 cm) of mercury ("house line")through heavy-walled rubber tubing. Those por-tions of the lines which pass through electrichosecocks A and B were of thin-walled small-diameter tubing. Air first passed through a centralsaturator (a hot water bath at 70 C) and a centraltrap (neither shown in Fig. 1) located outside theincubator containing the culture flasks. Inside the37 C incubator were two additional small traps,one for each symmetrical half of the apparatus.These latter traps contained several layers ofwater-soaked absorbant cotton. The air wassaturated with water vapor to reduce evaporationof culture fluid during the vacuum cycle. The twosmall traps were connected to the two cultureflasks in the shaking incubator at 37 C. Air pres-sure was applied to each culture flask through itsside arm from the adjoining trap; vacuum wasapplied through the top opening of the cultureflask.The mechanism for controlling the application

of pressure and vacuum consisted of two electrichosecocks, A and B, regulated by an intervaltimer and an electric relay connection as indicatedin Fig. 1 (upper left insert). There were two sepa-rate circuits, one for hosecock A and the other forB. The interval timer in circuit B closed hosecockB for a certain portion of a given cycling time; theclosing of circuit B also activated a relay whichbroke circuit A, opening hosecock A. Thus, whenhosecock B was closed, hosecock A was opened,and vice versa; the pressure and vacuum lineswere arranged so that pressure for unit I and

vacuum for unit II were controlled by hosecock A;hosecock B controlled pressure for unit II andvacuum for unit I. At the stage of operation de-picted in Fig. 1, air pressure was being applied tounit I and vacuum to unit II; i.e., culture fluidwas flowing from unit I into unit II.A typical experiment was preceded by testing

the apparatus with sterile medium as follows.After the central saturator reached 70 C and unitsI and II were at 37 C, 20 ml of washing medium(see Preparation of cells) were added to each cul-ture flask. The electric current was turned on, andthe interval timer was set so that hosecocks A andB opened and closed every 10 sec. Air pressureand vacuum were applied gradually to the appara-tus by means of valves in increments per cycle of2 psi and 3 inches (7.6 cm), respectively, untiloperating pressure and vacuum were reached. Ifno leaks appeared at the junction of the filterholders and filtration rates were equal in bothdirections, the washing medium was removedfrom the flasks and filtering unit.

Immediately after testing the apparatus, 10 mlof washed cells of strain PA 209 (absorbancy, 50Klett units) were added to each culture flask. Theexperiment was begun as above by gradually in-creasing air pressure and vacuum until the operat-ing levels were reached. The culture fluids of theseparated bacterial suspensions were equilibratedfor 1 hr at a filtration rate of approximately 7 mlper 10 sec. The filtration rate was determined bymeasuring the amount of fluid delivered in 10 secinto one unit when the other unit contained 14 mlof cell suspension.

After each experiment, Millipore filters in thefiltering unit were tested as follows for microscopicleaks. At 10 min before the end of an experiment,0.1 to 0.2 ml of a suspension (ca. 35 Klett units,equivalent to 0.07% of the total cells in unit I) ofa prototrophic strain of E. coli (lactose-positive)was added to the culture flask in unit I. In contrastto the auxotrophic strain PA 209, the prototrophgrew on acid casein hydrolysate and formed char-acteristic galactose-positive colonies oIn eosin-methylene blue-agar plates. At the end of the ex-periment, small portions of the cultures in bothunits were streaked onto three types of agarplates: nutrient broth, eosin-methylene blue, andacid casein hydrolysate. The agar plates were in-cubated for 24 hr at 37 C. If the filters were intact,no prototrophic cells entered unit II, no growthwas apparent on casein hydrolysate, and no galac-tose-positive colonies appeared on the eosin-methylene blue plates. [Preliminary experimentsof this type indicated the necessity of using typePH filters (pore size, 0.30,u); type HA filters (poresize 0.45 p) did not prevent the passage of someprototrophic E. coli cells from one unit to theother.] Several experiments were performed inwhich all the cells in unit I were prototrophs: nocells were transferred to unit II after 1 or 2 hr ofincubation. In many experiments, additional evi-dence that cells were not transferred from unit I

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to unit II was provided. By careful removal of thePyseal coating with a razor blade, the two holderswith their filters were separated without disturb-ing the cement bond between each holder and itsfilter. The exposed filter faces were then pressedonto nutrient agar plates. If the filters were intact,no bacteria were present on the outer surfaces ofthe filters (i.e., the surfaces not in direct contactwith bacteria), and no growth occurred on nutrientagar plates.Sampling. After 1 hr, the equilibration was

stopped, the cell suspensions were removed fromeach unit, and their absorbancies were determined.Then, the cells from each unit were collected onseparate Millipore filters for analysis of theirradioactivity. Several procedures were adoptedwhich assured better than a 90% recovery of cellsin the apparatus. Treatment of the glass parts ofthe apparatus with Siliclad aided in the removal offluid from the culture flasks. The cells remainingin the capillary stems of each unit were recoveredby flushing with washing medium and were alsocollected on two separate Millipore filters. Theonly significant amount of cells still remaining inthe apparatus (about 10% of the cells added) wasfound on the filters within the filtering unit. Thesefilters were removed from their ifiter holders withthe aid of a razor blade. In each experiment, threepairs of filters were analyzed for radioactivity: thetwo filters used to collect the cells in the initialsamples from units I and II, the two filters usedfor collecting cells obtained by flushing units Iand II, and the two filters from the filtering unit.

Analysis of bacterial protein. Cellular proteinwas obtained by extracting cell samples with hot5% trichloroacetic acid. The total radioactivityof this fraction was measured, and in some casesthe sample was hydrolyzed overnight in 6 N HCI at107 C, subjected to paper chromatography, andanalyzed for radioactivity in individual aminoacid fractions.

Radioactive analysis of chromatograms. Standardsolutions of threonine-C14, and acid hydrolysatesof labeled bacteria, were chromatographed in aphenol-borate buffer (93:7) solvent (Wade, Mathe-son, andHanes, 1961). TheRe values of threonine,serine, glycine, and alanine were: 0.35, 0.18, 0.25,and 0.46, respectively. Chromatograms were de-veloped overnight and dried, and radioactive spotswere located by use of a Vanguard model 880 auto-scanner.

Radioactivity determination with a gas-flowcounter. Cells collected by filtration were depositedwithin an area of 2.5 cm2 in the center of Milliporefilters. All radioactive solutions were placed withinan area of 2.5 cm2 in the center of stainless-steelplanchets. Samples were counted in a thin end-window gas-flow counter (efficiency for Cl4, 35%;background, 20 count/min) to a standard error ofless than 5%.The activity of samples collected on filters was

multiplied by 1.18 to correct for reduced back-scatter in these samples (Weatherford and Larson,1959). A correction factor for self-absorption was

applied to samples containing cells: for example,the factor for the cells from 10 ml of a culturehaving a reading of 50 Klett units was 1.21. Aself-absorption correction factor was applied toall samples of culture fluid: for example, the factorfor 2 ml of culture fluid was 3.5.

Radioactivity determination with a scintillationcounter. Samples were analyzed in a liquid scintil-Jation counter by use of a water-miscible dioxanephosphor (Davidson and Feigelson, 1957). Thesample (3 ml) and phosphor (15 ml) were mixedin a scintillation vial filled with Cab-O-Sil (Ottet al., 1959), a gelling agent that prevents salt-phosphor precipitates from settling. The countingefficiency was approximately 45%. Addition ofCab-O-Sil or culture medium did not decrease theefficiency.

RESULTSThreonine-C14 uptake. Amino acid uptake is an

obligatory step in intercellular turnover and isessential to the measurement of that process inan equilibration apparatus. If the amino acid up-take rate is very fast, then C'4-threonine releasedinto the medium of unit I may be reincorporatedby unit I cells before the medium can be transfer-red to unit II. The uptake of i-threonine wasexamined at added concentrations of 0.12 to 280,u,umoles/ml. As shown in later experiments, thethreonine concentration in media from turnoverexperiments was 6 to 8 ,ujmoles/ml.Two types of uptake experiments were per-

formed: threonine-C'4 uptake by a culture wasstudied under normal conditions in an Erlenmeyerflask, and the relative uptake of threonine-C'4 byunlabeled unit I cells as compared with unlabeledunit II cells was studied in the equilibration ap-paratus.

Uptake under normal conditions. The initial rateof threonine-C'4 uptake was a linear function ofthe exogenous threonine concentration over arange of 5 to 250 ,u,umoles/ml of added threonine-C14 (Fig. 2). Approximately 8% of the amino acidin the medium was removed by the cells in thefirst minute of incubation. Such a rapid uptakerate would vitiate results obtained under condi-tions where equilibration of the two cultures wasslow (no flushing of the culture fluid); however,the apparatus described here, which equilibratedthe two culture fluids in 40 sec, theoretically wasable to serve its purpose.

Uptake under equilibration conditions. The ef-fectiveness of the apparatus also was tested di-rectly. Initially unlabeled cultures were placed inboth unit I and unit II, and trace amounts oflabeled threonine were added to unit I while thetwo cultures were equilibrating. After 1 hr, thecells from both sides were harvested and counted.Figure 3 shows that unit I cells accumulated an

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0 bo~0 200THREONINE- C 14 ADDED

(M/. moles/ml)FIG. 2. Uptake of threonine-C14 by strain PA 209

under normal conditions. Cells were prepared asindicated for turnover experiments, but were incu-bated with shaking in Erlenmeyer flasks at 37 C.Threonine-C14 was added at various concentrationsto different batches of cells. Samples were taken atzero-time and at 1 -min intervals for at least the first5 min. Some cultures also were sampled at 10, 15,30, and 60 min. Cells were collected on Milliporefilters, but were not washed, since adhering mediumdid not alter rate calculations. The radioactivity incollected cells was measured in a Geiger counter.For all threonine concentrations tested, a linearuptake rate was obtained for the first 5 min. Theseinitial rates are plotted in the figure as a functionof external threonine concentration. The percentageof added threonine-C14 taken up in the first minute ofincubation was obtained from the slope of the uptakecurve:

rate of threonine-C14 uptake into cells(counts per min per ml of culture/min) X 1 X 100.concentration of threonine-C14 added min(counts per min per ml of culture)An average uptake in the first minute of 8% of theadded threonine occurred within the concentrationrange tested.

average of 54% of the total isotope incorporatedby both units. Such a small deviation from atheoretical 50/50 distribution was not of quanti-tative significance in these studies. The appara-tus, then, transferred culture fluid between unitsI and II so rapidly that both units incorporatedamino acid added to unit I to the same extent.

Turnover as measured in the equilibration appa-ratus. The above experiments demonstrated thefeasibility of using the equilibration apparatus instudies of intercellular turnover. As detailed inMaterials and Methods, intercellular turnoverwas determined by measuring the transfer ofthreonine-C'4 from protein of prelabeled cells toprotein of initially unlabeled cells. After placing

labeled cells in unit I and unlabeled cells in unitII, the culture fluids were equilibrated in the ap-paratus for 1 hr. The cells of each unit were har-vested and analyzed for radioactivity as indicatedpreviously.

Table 1 presents data from three equilibrationexperiments with labeled cells in unit I and anunlabeled "trap" culture in unit II. The amountof turnover per hour was calculated by dividingthe activity of unit II by that of unit I and mul-tiplying by a factor of 2 to allow for equal rein-corporation of released threonine by both units(Eagle et al., 1959). The values for intercellularturnover ranged from 0.16 to 0.18% per hour,whether data from whole cells or extracted cellswere used in the calculations. (Cells in both unitsapparently have the same minor percentage ofcounts in the hot water- and hot trichloroaceticacid-soluble fractions.) This intercellular turnoverrate compares with a total turnover rate for non-growing E. coli of 3 to 4% per hour, as determinedby Mandelstam (1958) and verified for strainPA 209 in this laboratory.

Control experiments. As shown in Table 2, onlyabout 20% of all the isotopic material released byinitially labeled cells in unit I was subsequentlydetected in unit II cells; the remaining 80% wasfound in the medium and on the filters in the

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FIG. 3. Uptake of threonine by strain PA 209under equilibration conditions. Unlabeled cells wereprepared as for turnover experiments (see text) andadded to both units of the equilibration apparatus.While the culture fluids were equilibrating, traceamounts of L-threonine-C'4 were added to unit 1.Cells were harvested after 1 hr and assayed for radio-activity as in turnover experiments. In the figure,the abscissa is the total amount of threonine in-corporated into all cells (units I and II); the ordinateis the percentage of total counts detected in unit Icells. The dashed line represents the theoretical levelof incorporation (50%) into unit I cells, if both unitstrapped added isotope with equal efficiency.

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TABLE 1. Transfer of threonine-C14 from prelabeledunit I cells to unlabeled unit II cells

Expt C

1. Whole cells ......... 24,300 21.1 0.087 0.172. Cell residue after

hot water extrac-tionc ................ 192,000 150d 0. 078 0. 16

3. Cell residue afterhot water and hottrichloroacetic acidextractionc......1,080,000 962, 0.0890.18

a Refers to counts per minute present after 1 hrin the cells in 1 ml of a culture having a Klettreading of 50. Some cultures exhibited slight ab-sorbancy changes (less than 10%); these werecorrected for in calculating counts per minute permilliliter of culture. All results were normalizedto counts per minute which would have been de-tected if samples were plated at infinite thinnessand counted in a Geiger counter. The specific ac-tivities of the L-threonine-C14 used to preparelabeled cells for unit I were: experiment 1, 794,000counts per min per umole; experiment 2, 6,260,000counts per min per gumole; and experiment 3,52,400,000 counts per min per ,umole.

b Per cent per hour transfer was calculated asfollows:

counts per minute/unit II cellsin 1 ml of culture

counts per minute/unit I cellsin 1 ml of culture

Intercellular turnover rate was obtained by multi-plying the per cent per hour transfer by 2, tocompensate for the equal reincorporation of iso-tope by units I and II.

c Cells were extracted for 20 min with water(90 C). In experiment 3, this was followed by a

20-min extraction with 5% trichloroacetic acid(90 C). Samples of extracts were plated andcounted in a Geiger counter. Trichloroacetic acidextracts were washed with ether before plating;none of the isotopic material was found in theether phase. The counts per minute found in theextracts were subtracted from the counts perminute found in unextracted whole cells to givethe values for extracted cells.

d Represented 88% of the total radioactivity inunextracted whole cells.

e Represented 77% of the total radioactivity inunextracted whole cells. After extraction with hotwater, only 2% of the total radioactivity in unex-tracted whole cells was soluble in hot trichloro-acetic acid.

center filtering unit. In the light of these data, aseries of control experiments was performed toeliminate the following possible artifacts as causesof intercellular turnover: (i) labeled cells, lysed ordamaged by equilibration, may have contributedlarge amounts of isotopic material to the mediumwhich subsequently was incorporated by unit IIcells; (ii) the isotopic material in the medium mayhave been derived from healthy labeled cells, butmay have been adsorbed (rather than incorpora-ted) by unit II cells; extraction with trichloroace-tic acid may not have removed adsorbed material;

TABLE 2. Radioactivity released by unit I cells anddetected in the cells, medium, and center

filter of unit II

Radioactivity (counts per min per ml)* Percentageof totalreleased

detected inunit IIExpt UiIIcells

cells Mediumt Center filtert u______(A) (B) (C) A+B+C

x_oo)

2 150 499 141 193 962 2,170 1,270 224 354 739 686 20

* The cells, medium, and center filter were allfrom unit II; the sum of their isotopic content wastaken as the total radioactivity released by 1 ml ofunit I cells (normalized as in footnote a, Table 1).The counts per minute in unit II cells were nor-malized as in Table 1. The counts per minute inthe medium were normalized to the equivalent of1 ml of culture with a Klett reading of 50. Thetotal radioactivity on a center filter was assumedto be derived from 10 ml of culture; the values inthe table are given per milliliter of culture.

t When medium harvested at the end of turn-over experiments was chromatographed in buf-fered phenol solvent, about 10% of the total radio-activity was localized in a peak having the Rp ofthreonine; the remainder was found in a sharppeak at the origin (14%), a peak of fluorescentmaterial (18%; RF, 0.19), anda large, diffusepeakat the solvent front (47%). The threonine peakwas increased to 40% when hydrolyzed medium(6 N HCl, 18 hr, 107 C) was chromatographed.

t Most of the radioactivity in center filters didnot represent adhering labeled cells, but rathermaterial adsorbed from the medium, since it waspresent on a unit II center filter when labeledcells in unit I were equilibrated with only mediumin unit II. The exact nature of this material is notclear, but the following characteristics indicateit is not free threonine-CU4: it could not be elutedfrom the filter by washing with 10 ml of unlabeledL-threonine (1 mg/ml), 1 M NaCl, 5% trichloro-acetic acid (0 or 90 C), nor displaced with serumalbumin or redissolved trichloroacetic acid-precipitable bacterial protein.

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MEASUREMENT OF PROTEIN TURNOVER IN E. COLI

or (iii) at the start of a turnover experiment, themedium in unit I may have contained enoughlabeled material to account for all the isotope in-corporated by unit II cells in the following hourof equilibration.

Evidence that cells incubated under equilibra-tion conditions did not suffer gross damage wasprovided by experiments in which absorbancyreadings of experimental and control cultureswere compared. In a control Erlenmeyer flask inwhich a culture in deficient medium was incu-bated with shaking for 1 hr, the Klett reading de-creased from 50 to 48. The Klett readings of aculture equilibrated in the apparatus under thesame conditions were identical with those of thecontrol flask. More convincing evidence that cellswere not damaged by the experimental procedurecomes from measurements of the labeled intra-cellular pool before and after 1 hr of equilibration.If cell membranes were damaged, the intracellularpool size should decrease, since metabolites nor-mally concentrated within the cell would leak outto the medium. In experiment 2, a hot water ex-tract of 1 ml of unit I cells contained 3,710 counts/min before and 3,400 counts/min after 1 hr ofequilibration; in experiment 4, the values were10,000 counts/min before and 10,900 counts/minafter equllibration. No significant change in thesize of the labeled pool was apparent, and, thus,no membrane damage was indicated by this cri-terion.

Because it affects the initial size of the intra-cellular threonine-C14 pool, the method used inwashing cells before equilibration (see Preparationof cells) should be mentioned here. Labeled cul-tures were not incubated, prior to experiments,in media containing unlabeled threonine to re-move the intracellular pool of labeled threonine.This procedure was omitted to eliminate relabel-ing of the intracellular pool as a complicatingfactor in the transfer of labeled protein-boundthreonine from unit I cells to unit II cells. At thestart of the equilibration, the amount of labeledthreonine in the intracellular pool of unit I cellsshould equal exactly the amount of unlabeledthreonine in the intracellular pool of unit II cells.Whatever additional labeling of unit II cell pro-tein is derived from the intracellular pool oflabeled unit I cells should be counterbalanced bythe initial lag in labeling caused by the pre-exist-ing unlabeled intracellular pool in unit II cells.

Further evidence that little, if any, cell damageoccurred as a result of equilibration was providedby measurements of total and trichloroaceticacid-soluble radioactivity in media from culturesequilibrated in the apparatus and from culturesincubated in Erlenmeyer flasks with shaking(control flasks). If appreciable cell damage occur-red during equilibration, there should have been

more radioactivity in media from the apparatusthan in media from controI1 flasks. Furthermore,any severe physiological insult to the cells mighthave resulted in leakage of macromolecules(Koch, 1959; Pollock, 1961) precipitable by tri-chloroacetic acid. However, similar total amountsof labeled material were found in media from theapparatus and from control flasks (499 and 533counts per min per ml in experiment 2, and 2,170and 1,689 counts per min per ml in experiment 3),and in both cases most of the material was solublein trichloroacetic acid (406 and 518 counts permin per ml in experiment 2, and 1,750 and 1,594counts per min per ml in experiment 3). No at-tempt was made to evaluate the relative amountsof material found on center filters from the appa-ratus as compared with filters used to harvestmedia from labeled control cultures. Finally,when chromatographed, media from both sourcesexhibited similar distributions of radioactive andninhydrin-positive material (see second footnote,Table 2).The second series of control experiments (Table

3) was designed to show that the amount of iso-topic material adsorbed (rather than incorpora-ted) by unit II cells was negligible, and that theradioactivity in the mediurn at zero-time couldnot account for the level of incorporation by unitII cells after 1 hr of equilibration. In these controlexperiments (lines 2 and 4), labeled medium washarvested from the apparatus at the beginning orend of turnover experiments (lines 1 and 3) andadded to unlabeled "acceptor" cells in an Erlen-meyer flask. The resultant cell suspension wasincubatedfor 1 hr at either 37 or 0 C, and the radio-activity detected in "acceptor cells" (lines 2 and4) was compared with that in unit II cells at theend of turnover experiments (B as % A).That the level of adsorbed labeled material is

low is shown by a comparison of the turnover ex-periment on line 1 and its control experiment online 2. In these experiments, the culture fluid atthe end of the turnover experiment contained onlyenough radioactive material to subsequently label"acceptor" cells 13% as much as unit II cells.This low level of adsorption was not due to a lackof adsorbable material in the medium, since ad-sorption could be demonstrated again with me-dium already treated with "acceptor" cells.Furthermore, "acceptor" cells incubated withmedia from control flasks exhibited a similar lowlevel of adsorbed material.That the level of "zero-time" incorporation is

also low is shown by a comparison of the turnoverexperiment on line 1 and its control experimenton line 4. Clearly, the culture fluid from the turn-over experiment at zero-time contained onlyenough radioactive material to label "acceptor"

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TABLE 3. Levels of adsorption and zero-time incorporation in turnover measurements

Radioactivity(counts per min

per ml)'

Determination Turnover Medium source for Control incubation B as % Aincubation controlsb (B)(A) "Ac-

Unit IIIC ceptor"cellsd

I. Level of adsorption1. Turnover Apparatus, _ 962

expt 3 1 hr, 37 C2. Controld Medium from Erlenmeyer 128 13.0

end of turn- flask, 1 hr,over expt 3 0 C

II. Level of zero-time incor-poration

3. Turnover Apparatus, 150 -expt 2 1 hr, 37 C

4. Controld Medium at zero- Erlenmeyer - 14 9.3time of turn- flask, 1 hr,over expt 2 37 C

See first footnotes to Tables 1 and 2.b The medium harvested after 1 hr contained approximately 10% free threonine. The total radio-

activity in the medium was: lines 1 and 2, 2,170 counts per min per ml; line 3, 499 counts per min perml; and line 4, 117 counts per min per ml. Other characteristics of the medium are given in Table 2,second footnote.

c Unit I cells contained 1,080,000 counts per min per ml for line 1 and 192,000 counts per min per mlfor line 3.

d For the adsorption control experiment on line 2, unlabeled "acceptor" cells were prepared as forturnover experiments, and were resuspended to a Klett reading of 50 in medium harvested from unit II(see Medium Source for Controls) at the end of the turnover experiment (line 1). The resulting controlcultures were incubated in an Erlenmeyer flask with shaking at 0 C, since adsorption should not betemperature-dependent. After 1 hr, "acceptor" cells were collected on Millipore filters and counted asusual. The zero-time incorporation control experiment on line 4 was performed as follows. Immediatelybefore the start of the turnover experiment (line 3), medium was harvested from a portion of a labeledculture prepared for unit I (see Medium Source for Controls) and was added to an equal volume of anunlabeled "acceptor" cell suspension (prepared as in turnover experiments but having a Klett readingof 100). The resulting control culture (Klett reading 50) was incubated in an Erlenmeyer flask withshaking at 37 C. After 1 hr, "acceptor" cells were collected on Millipore filters and counted as usual.

cells 9.3% as much as unit II cells were labeledafter 1 hr in the apparatus.One further observation is also of interest. If

the control experiment on line 2 is performed at37 C instead of 0 C, the level of incorporation isstill only 31% of that which occurred in the turn-over experiment on line 1. This indicates thatcontinued equilibration with labeled unit I cellsis required for maximal isotope incorporation byunit II cells.

All the above experiments support a scheme ofintercellular turnover in which cells constantlyrelease amino acids derived from proteins intothe medium and continually reincorporate aminoacids from the medium into proteins.

DISCUSSIONAs determined with the equilibration appara-

tus, a definite intercellular turnover of proteins

occurs in nongrowing E. coli at a rate between0.16 and 0.18% per hour. The reliability of thisvalue depends upon: (i) the ability of "trap"cultures (unit II) to take up released threonine-C14 from the medium as rapidly as initially labeledcultures (unit I); (ii) the absence of abnormal celllysis or damage in the apparatus; (iii) the lack ofnonspecific adsorption of extraneous labeled ma-terial by "trap" cultures (unit II).By inference from the threonine uptake rate

for strain PA 209 (Fig. 2) and by direct test inthe apparatus (Fig. 3), one may conclude thatthreonine-C'4 released by unit I cells was "trap-ped" effectively by unit II cells. As judged byKlett readings, intracellular pool measurements,and culture fluid characteristics (see Control ex-periments), cultures equilibrated in the apparatusshowed no evidence of cell damage over thatwhich may occur in cultures incubated in control

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MEASUREMENT OF PROTEIN TURNOVER IN E. COLI

Ehrlenmeyer flasks. Finally, the radioactivity"trapped" by unit II cells was not nonspecificallyadsorbed material, but rather was incorporatedthreonine-C'4 derived from unit I cells and takenup via the medium (Table 3).

In addition to the above considerations, thepossibility that labeled threonine is being de-graded before it can be incorporated by the"trap" culture should be noted. However, in anexperiment using cells prelabeled with L-leucine-C14 approximately the same turnover rate (0.2%per hour) as that obtained with cells prelabeledwith L-threonine-C'4 was obtained. It seems un-likely that both threonine and leucine are de-graded appreciably and to the same extent.

In nongrowing E. coli, then, intracellular turn-over accounts for almost all of the observed totalprotein turnover of 3 to 4% per hour (Mandel-stam, 1958) because intercellular turnover is only0.16 to 0.18% per hour. Although the magnitudeof intercellular turnover in E. coli now has beendetermined, the exact nature and function of thisprocess still remain to be elucidated. The experi-ments reported here do not provide evidence fordistinguishing between minimal cell lysis or dam-age, or some form of secretion, as the source ofamino acids transferred between cells. A majorunanswered question is whether intercellularturnover is merely the fortuitous result of leakageand uptake of amino acids produced during intra-cellular turnover, or whether intercellular andintracellular turnover are separate processes.Clearly, however, leakage of nutrients from cellsmay "condition" the medium for subsequent cul-ture growth and account for population effectsobserved in bacterial cultures (Jannasch, 1962)in the same way as it does for population effectsfound in mammalian cell cultures (Eagle and Piez,1962).

ACKNOWLEDGMENTSI am greatly indebted to Herbert Tabor for his

advice and encouragement during the course ofthis investigation. I am grateful also to the mem-bers of the Laboratory of Biochemical Pharma-cology for many helpful discussions.

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