l-asparagine uptake in escherichia - journal of bacteriology

JOURNAL OF BACTERIOLOGY, Sept. 1975, p. 937-945Copyright 0 1975 American Society for Microbiology

Vol. 123, No. 3Printed in U.S.A.

L-Asparagine Uptake in Escherichia coliR. C. WILLIS' AND C. A. WOOLFOLK*

Department of Biochemistry and Molecular Biology, University of California at Irvine, Irvine, California 92664

Received for publication 31 March 1975

The uptake of L-asparagine by Escherichia coli K-12 is characterized by twokinetic components with apparent Km values of 3.5 MM and 80 MM. The 3.5 ,MKm system displays a maximum velocity of 1.1 nmol/min per mg of protein, whichis a low value when compared with derepressed levels of other amino acidtransport systems but is relatively specific for L-asparagine. Compoundsproviding effective competition for L-asparagine uptake were 4-carbon analoguesof the L-isomer with alterations at the f,-amide position, i.e., 5-diazo-4-oxo-L-nor-valine (K, = 4.6 MuM), fj-hydroxyamyl-L-aspartic acid (Ki = 10 MM), andL-aspartic acid (K, = 50 MM). Asparagine uptake is energy dependent and isinhibited by a number of metabolic inhibitors. In a derived strain of E. colideficient in cytoplasmic asparaginase activity asparagine can be accumulatedseveral-fold above the apparent biosynthetic pool of the amino acid and 100-foldabove the external medium. The high affinity system is repressed by culture ofcells with L-asparagine supplements in excess of 1 mM and is suggested to benecessary for growth of E. coli asparagine auxotrophs with lower supplementconcentrations.

There have been numerous investigations ofthe transport of various amino acids by Esche-richia coli which have been the subject of recentreviews (12, 14). These studies have revealed anumber of highly specific systems for individualamino acids as well as more general systems forgroups of related amino acids. Several systemsmay occur simultaneously which are capable oftransporting the same amino acid but withquite different apparent affinities and modes ofgenetic regulation.Although the transport of all other natural

amino acids found in protein has been investi-gated in E. coli (12), a transport system forL-asparagine has, as yet, not been described.The results of competition studies characteriz-ing the specificity of transport systems for theclosely related amino acids aspartate (6), gluta-mine (15), and glutamate (3) suggest thatasparagine is not a substrate for these systems.Similar studies of other amino acid transportsystems have not included asparagine. Suchstudies would be complicated by the presence ofseveral asparaginase activities in this organism(1), but the existence of an asparagine auxotrophof E. coli K-12 (2) establishes that asparaginemay enter this cell without destruction.

In a recent paper (16) we have describednutritional experiments with an asparagineauxotroph of E. coli K-12. The results suggested

I Present address: Department of Medicine, University ofCalifornia at San Diego, La Jolla, Calif. 92037.

the presence of at least two systems mediatingthe entry of asparagine: a high-affinity systemwhich appeared to be repressed by culture ofcells with 1 mM asparagine and was required forgrowth of cultures of an asparagine auxotrophat supplement concentrations of 10 to 100 AML-asparagine and a second, low-affinity systemutilized by cells when asparagine was the solenitrogen source of the culture. This paper char-acterizes a high-affinity kinetic component ofasparagine uptake in E. coli K-12 strains whichis relatively specific for L-asparagine and isrepressed by growth of cultures with 1 mML-asparagine. Transport systems have also beendescribed for asparagine in the gram-positivebacteria Lactobacillus plantarum and Strepto-coccus faecalis where the specificity of thesystems differ significantly from that reportedhere (4).

(A portion of this paper was submitted by thesenior author in partial fulfillment of the re-quirements for the Ph.D. degree in BiologicalSciences from the University of California atIrvine.)

MATERIALS AND METHODS

Chemicals. L- [U- '4C Jasparagine (218 gCi/ymol),L- [U- "C Jaspartate (208 ACi/gmol), L- [U- 14C ]gluta-mine (36.3 UCi/1mol), L-[U-'4C ]glutamate (249 ,ICi/Amol), and L-[U-14C]leucine (311 ;Ci/Amol) werepurchased from Amersham-Searle. 5-Diazo-4-oxo-L-norvaline was a gift from R. E. Handschumacher,

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938 WILLIS AND WOOLFOLK

Department of Pharmacology, Yale University. L-f,-Hydroxamyl aspartic acid, bovine serum albumin, E.coli B asparaginase, and natural amino acids werepurchased from Sigma Chemical Co. Oxalacetic acid,f8-ketoglutaric acid, fumaric acid (sodium salt), ma-lonic acid, D-lactic acid (lithium salt), fl-cyano-L-ala-nine, O-phospho-L-serine, and chloramphenicol werepurchased from Calbiochem Co. L-Alanyl-L-aspara-gine, L--y-aminobutyric acid, a-methyl-L-aspartic acid,N-methyl-DL-aspartic acid, and D-asparagine werepurchased from Nutritional Biochemicals. Oxamicacid, oxalic acid dihydrate, and succinamide werepurchased from Aldrich Chemical Co. All other mate-rials were obtained from the usual commerical sourcesand were either reagent grade or the highest chemicalpurity available. Where required, solutions of basicand acidic materials were adjusted to neutrality withhydrochloric acid or potassium hydroxide, respec-tively.

Bacteria. All strains used in this study werederivatives of E. coli K-12. Strain W3110 was ob-tained from D. L. Wulff, Department of Biochemis-try, University of California, Irvine. Other strainsused were ATCC 25287 (asn-, thi-), originally isolatedby E. Reich, Rockefeller University, and 47, a cyto-plasmic asparaginase-deficient mutant (16) of strain25287.

Culture. The maintenance and culture of bacterialstrains has been described previously (16). Changes inthe media composition are noted. Carbon-starvedcells were prepared from cultures originally supple-mented with 0.04% glucose and harvested for 4 h aftergrowth ceased.

Cell suspensions. Cells used for transport assayswere harvested in the mid-exponential phase ofgrowth. The cells were harvested from cultures andcollected from wash solutions by centrifugation for 10min and 10,000 x g at room temperature. The cellswere washed twice by resuspension into 0.5 originalvolume buffer A (10 mM potassium phosphate, pH6.9, 1.0 MM magnesium chloride, 100 Mg. of chloram-phenicol per ml). After the last wash, the cell pelletswere resuspended at 6 to 8 mg of cellular protein/ml inbuffer A. The washed cells were stored at either roomtemperature or 4 C until used for assay. Cells stored at4 C were incubated for 15 min at 37 C before dilutionfor assay. Transport activity remained constant for atleast 4 h under either condition of storage.Transport assays. All operations, except where

noted, were performed at room temperature andmaterials were prepared in buffer A. Transport wasassayed in 60-Ml reaction mixtures containing, inorder of addition: 20 ul of appropriately diluted cellsuspension; 20 Ml of energy source, routinely 75 mMglucose; and after a 10-min incubation period 20 Ml of"C-labeled substrate (20 to 70 MCi/Mmol) and,where indicated, unlabeled competitor. Assays wereterminated by transferring 50 Ml of assay mixture tothe center of a wetted Millipore filter (0.45 Mm,HAWP) under vacuum and the collected sample waswashed by vacuum filtration with 5 ml of buffer A.The backside of the filter containing the washedsample was blotted and the filter was rapidly trans-ferred to a scintillation vial containing 2 ml of Aquasol(New England Nuclear). Radioactivity was deter-

mined by scintillation counting. Standardization ofcounts per minute to microcuries was performed byadding a known amount of radioactivity to a samplecounted previously, recounting the sample, and deter-mining the difference in counts per minute. All data isreported in nanomoles of asparagine taken up perminute per milligram of protein. Protein was deter-mined by the method of Lowry et al. (9).

Routinely, assay mixtures contained 2 to 3 mg ofcellular protein/ml, i.e., 0.10 to 0.15 mg of protein/fil-ter, assay times were 30 s, and the specific activity ofthe isotope was 20 to 70 MCi/Mmol. The cell concentra-tions used in all assays were predetermined such thatless than 10% of the substrate was removed during a30-s incubation. In experiments requiring many sam-ples to be taken with time, the assay mixtures wereincreased proportionally in volume and a 28-mm outerdiameter scintillation vial was used in place of a tube(13 by 100 mm) as the reaction vessel. The vial wasused to allow an increase in the surface area-volumeratio of the reaction mixture and thereby preventoxygen starvation of cells during long-term incuba-tion.

Extraction and identification of accumulatedmaterials. Immediately after the collection and washof a sample by membrane filtration, the filter wastransferred to a 28-mm outer diameter scintillationvial containing 5 ml of 50% ethanol equilibrated to95 C. The vial was recapped and sample was extractedby continued incubation at 95 C for 10 min. The vialwas then vigorously vortexed and the solution wastransferred to a centrifuge tube. The extraction wasrepeated a second time and the solutions were com-bined. The extract solutions were clarified by centrifu-gation at 16,000 x g for 15 min and the volume of theclarified extract solution was adjusted to 10 ml withwater. The solution was divided and evaporated todryness at 60 C with a rotary Evapomix (BuchlerInstruments). One fraction was resuspended in 50 Mlof 50% methanol. The other fraction was incubated for120 min at 37 C with 4 to 5 units of E. coli Basparaginase in a total volume of 100 Ml of 50 mMammonium bicarbonate, pH 7.6. The solution wasevaporated to dryness and resuspended in 50 Ml of 50%methanol. Equal volumes of both fractions werespotted on adjacent lanes of a 250-Mm surface depthSilica Gel G thin-layer chromatography plate (Brink-mann) and chromatographed with a chloroform-methanol-ammonium hydroxide-water (8:8:3:1) sol-vent system. Developed plates were dried and autora-diograms were prepared (16). The location of aspara-gine on the chromatograms was determined by theabsence or loss of radioactivity in samples treatedwith asparaginase. Similarly, the position of aspartatewas detected by the increase in radioactivity insamples treated with asparaginase. The areas ofinterest were removed from the chromatogram byscraping, and the radioactivity of the scraped samplewas determined by scintillation counting in 5 ml oftoluene scintillation solution (16).

RESULTS

Optimum conditions for measurement ofasparagine uptake. The standard assay condi-

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ASPARAGINE UPTAKE IN E. COLI 939

tions for measurement of asparagine uptakewere determined by studies of the pH-bufferoptimum and cation dependence. The initialrate of asparagine uptake displays a pH op-timum over the range of 6 to 7 with 50 mMdibasic potassium phosphate-monobasic potas-sium phosphate buffer. Uptake activity isgreatly diminished below pH 5.0 with 50 mMsodium acetate-acetic acid buffer, and abovepH 7.5 with 50 mM trizma base-hydrochloricacid buffer. At similar pH both sodium acetateand tris(hydroxymethyl)aminomethane bufferswere inhibitory in comparison to uptakes mea-sured in the presence of a respective potassiumphosphate buffer. The optimum potassiumphosphate buffer concentration was in the rangeof 10 to 50 mM. In studies with 10 mMpotassium phosphate buffer, the pH of assaymixtures containing cell densities equivalent to2 mg of cellular protein per milliliter wasmaintained for 20 min at 37 C. The uptake ofasparagine was not dependent on added sodiumion in studies utilizing concentrations of sodium

chloride from 0.01 to 100 mM. At concentra-tions in excess of 100 mM both sodium andpotassium chloride inhibited the apparent ini-tial rate of asparagine uptake. Magnesium chlo-ride at concentrations from 1 to 10 ,M providedthe maximum uptake activity. Calcium chlo-ride had little effect.The uptake of asparagine was dependent on

temperature and the initial rate was linear withtemperature from 10 to 50 C. Incubation of cellsfor longer than 2 min at temperatures greaterthan 50 C destroyed the asparagine uptake ca-pacity of cells.Metabolism of ["4C]asparagine during up-

take assays. Asparagine is rapidly metabolizedby E. coli K-12 cells with cytoplasmic aspa-raginase activity (16). To show that metabolismof the transported asparagine does not interferewith initial rate measurements, uptake wasmeasured in an isogenic pair of strains, 25287and 47; strain 47 is deficient in cytoplasmic as-paraginase activity. The results of these mea-surements (Fig. 1) indicate that the initial rates

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o 15 l

00

0 5 10 15 20

Minutes

FIG. 1. Time course of asparagine uptake. Strain 25287 (closed symbols) and cytoplasmic asparaginase-deficient strain 47 (open symbols) were incubated in assay mixtures initially containing [14C]asparagineat concentrations of 7.5 MM (circles), 27.5 MM (triangles), and 207 IAM (squares). Incubation mixtures weresamples at the times indicated and radioactivity accumulated by the cells was determined as described inMaterials and Methods.

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of asparagine uptake, 30-s determinations, arenearly identical whether metabolism is blockedor normal. The measurement of radioactivityaccumulated during 30-s incubations of cellswith ["4C]asparagine is therefore a relativelyvalid estimation of the initial rate of asparagineuptake.

Distribution of radioactivity accumulated.Figure 2 shows the distribution of radioactivityaccumulated by cells with normal and blockedasparagine metabolism when incubated for 10min in the presence of 10 MM [14C0]asparagine.Greater than 80% of the radioactivity accumu-lated by the asparaginase-deficient strain 47was recovered as asparagine, whereas only 20%of the radioactivity accumulated by strain25287 with normal asparaginase activity wasrecovered as asparagine. When experimentswere similarly performed with 1-min incuba-tions, greater than 90% of radioactivity accu-mulated by 47 cells and approximately 60% ofthe radioactivity accumulated by 25287 wasrecovered as asparagine. In the experimentshown in Fig. 2, strain 47 accumulated 5 nmol ofasparagine/mg of cell protein during the 10-minincubation period. It can be estimated on thebasis of cell volume-protein ratios (10) that thecellular concentration of extractable asparagineis 0.9 mM. This value represents nearly a100-fold accumulation or maintenance of theintracellular asparagine pool above the initialexternal level. The accumulated pool is rapidlylost after dilution of cells into medium contain-ing 1 mM 2,4-dinitrophenol. Either asparagineuptake or the maintenance of the internalasparagine pool would theti appear to involve anactive process (7).Dependence of asparagine uptake on meta-

bolic energy. Cells prepared from either car-bon-starved or normal cultures retain consider-able endogenous energy for the transport ofasparagine (Table 1). As expected, cells fromcarbon-starved cultures have less capacity foruptake than cells from normal cultures. Theinitial rate of transport in both cell preparationscan be stimulated above the endogenous levelsby incubation with the carbon sources glucose,D-lactate, or succinate. No preference for aparticular energy source is observed with theexception of succinate. At concentrations above25 mM, succinate inhibited the transport ofasparagine as well as aspartate, glutamine, andglutamate. This effect may be due to the highconcentrations of cation associated with dicar-boxylic acid.The initial rate of asparagine uptake is inhib-

ited by a variety of metabolic inhibitors rou-

L-

co

4)c4)

-o0ul-a

4)

0(L)

cm From originFIG. 2. The distribution of the radioactivity ac-

cumulated during incubation of strains 25287 and 47with [14C]asparagine. Shown are the densitometrictracings of autoradiograms (16) of the thin-layer-chromatographed cell extracts. Cells were incubatedfor 10 min with 10MgM ["'CJasparagine (100 MCi/Mgmol).Assays were terminated and extracts were preparedand chromatographed as described in Materials andMethods. The [14CJasparagine accumulated by thecells is identified by the loss of radioactivity in areas ofchromatographed untreated sample (solid lines) whencompared to an equivalent fraction of asparaginase B-treated sample (broken lines).

tinely used to establish the dependence oftransport on energy including the succinatedehydrogenase inhibitor, malonate, and lactatedehydrogenase inhibitors, oxamate and oxalate(Table 1). The glucose-stimulated asparagineuptake is not as sensitive to inhibition bymalonate, oxamate, or oxalate as the succinate-

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stimulated uptake with malonate or D-lactate-stimulated uptake with oxamate or oxalate.These results suggest that the energy providedfor the transport of asparagine is not exclusiveto any one particular dehydrogenase (8), butmay be provided by several different metabolic

reactions leading to an energized state of thecytoplasmic membrane.Concentration dependence of asparagine

uptake. The effect of asparagine concentrationon the initial rates of asparagine uptake isshown in Fig. 3. The reciprocal plot can be

TABLE 1. Dependence of asparagine uptake by strain W3110 on metabolic energya

Culture Additionscconditions' UptakedEnergy source Inhibitor

Normal None None 100

0.025 M glucose None 1800.025 M glucose 0.050 M malonate 510.025 M glucose 0.050 M oxalate 250.025 M glucose 0.050M oxamate 180.025 M glucose 0.002 M 2,4-dinitrophenol 50.025 M glucose 0.010M potassium cyanide 510.025 M glucose 0.001 M iodoacetate 640.025 M glucose 0.020 M sodium azide 560.050 M succinate None 600.025 M succinate None 1140.025 M succinate 0.050 M malonate 190.050M D-lactate None 1900.025 M D-lactate None 1860.025 M D-lactate 0.050M oxalate 60.025 M D-lactate 0.050M oxamate 3

Carbon None None 59starvation 0.025 M D-lactate None 144

0.025 M glucose None 135

'Uptake was measured during a 60-s incubation period with 20 OM [ICT asparagine.'The preparation of normal and carbon-starved cells is described under Materials and Methods.cThe inhibitors were added to the cell suspensions 20 min before initiation of the assay and energy source 10

min before initiation of the assay. Basic and acidic compounds were neutralized with hydrochloric acid andpotassium hydroxide, respectively.

d One hundred percent uptake was 0.71 nmol/min per mg.

B

_C.)B E

c -

8 Evm --F-

Cv

Asparagine (pM)

FIG. 3. Concentration dependence of asparagine uptake. Asparagine uptake was measured with strain 47at the initial [14C]asparagine concentration indicated. The incubation mixtures were sampled at 30-s intervalsfor 2 min. Closed symbols represent the initial velocity of asparagine uptake. Open symbols represent C/Vtransformations of the initial rate data. C is in units ofuM and V is in nmol/min per mg.

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represented by two linear segments and sug-gests that uptake of asparagine may be medi-ated by two systems. The apparent Km and Vmaxvalues determined are 3.5 MM and 1.1 nmol/minper mg for the high-affinity component and 80gM and 33 nmol/min per mg for the low-affinitycomponent, respectively.Regulation of the high-affinity component

of asparagine uptake. When the K-12 strainsused in this study were cultured in minimalmedium supplemented with less than 100 ,Masparagine, the low-affinity component of as-paragine uptake was not apparent in the C/Vtransformations of initial velocity (v) measure-ments at substrate concentrations (c) below 20MM. However, the supplementation of cultureswith 1 mM asparagine repressed the high-affin-ity component of asparagine uptake. The repres-sion is observed in Fig. 4 as the appearance ofthe low-affinity component in the transformedinitial velocity data at substrate concentrationsbelow 20 gM.The growth of the asparagine-requiring auxo-

troph, 47, in nutrient broth is limited due toinsufficient asparagine. Supplementation of nu-trient broth with 100 MM L-asparagine over-comes this deficiency and 47 cultures establishdoubling times and achieve cell densitiesequivalent to 3110 cultures. The high-affinitycomponent of asparagine transport is dere-pressed under these conditions. As observedwith the minimal medium cultures supplemen-tation of nutrient broth with 2 mM L-asparaginerepresses the high-affinity component of aspar-agine transport. These results suggest that therepression is specific to asparagine.

Asparagine uptake does not appear to besubject to catabolite repression. Culture of the

!) v 5

0 8 16I uuII I

0 8 16

Asparagine (pM)

FIG. 4. Repression of asparagine uptake. StrainW3110 was cultured under (-) standard conditionsand (0) with a 1 mM L-asparagine supplement.Asparagine uptake was measured by a 30-s incuba-tion with the concentrations of ["CJasparagine in-dicated.

K-12 strains in minimal medium with 1% succi-nate replacing glucose did not significantlychange the kinetic parameter of uptake fromthose indicated for glucose-grown cultures inFig. 3.

Specificity of the high-affinity componentof asparagine uptake. Several compounds,including amino acids, asparagine analogues,and keto acids, were tested for inhibition ofasparagine uptake (Table 2). The system medi-ating asparagine uptake at low concentrations(5 AtM asparagine) appears to be stereospecificfor L-asparagine and recognizes only the L-iSo-mers of analogues of asparagine with substitu-tion at the f3-amide position. Compounds pro-viding greater than 20% inhibition of uptake

TABLE 2. Specificity of the high-affinity componentof asparagine uptake by strain W3110O

Unlabeled compound' Maximumeuptake

Asparagine ............................ 5Aspartate ............................. 60Glutamine ............................ 80Glutamate ............................ 102Leucine ............................. 100Proline ............................. 90Serine ............................. 85Phenylalanine ......................... 92Methionine ........................... 96Lysine ............................. 94D-Asparagine .......................... 80Alanyl asparagine ........... ......... 80fi-Cyanoalanine ....................... 70,8-Aspartyl hydroxamate ....... ........ 10Diazo-oxo-norvaline .......... ......... 5a-Methyl aspartate ........... ......... 97N-methyl-DL-aspartate ........ ........ 98Succinamide .......................... 101Pyrazole-DL-alanine ........ ........... 90Diamino propyl phosphonic acid ........ 94O-phospho serine ...................... 105O-phospho threonine ......... ......... 109L-y-amino butyrate ........... ......... 85Succinate ............................. 99Fumarate.97Malate ............................. 102Oxaloacetate .......................... 100NH,Cl (pH = 6.9) ............. ........ 95

a Uptake was measured with a 60-s incubationperiod and assay mixtures contained 5 AsM L- [14C las-paragine.

'Five hundred AM L-isomer unless indicated other-wise. Acidic compounds were neutralized with potas-sium hydroxide. Basic compounds were neutralizedwith hydrochloric acid.cOne hundred percent uptake was 1.2 nmol/min

per mg.

0- 0.9.0 EQ. --D) .'E

E0.0c 46

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as 0.2

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when present at concentrations 100-fold in ex-

cess of L-["Clasparagine were 5-diazo-4-oxo-L-norvaline, f3-hydroxyamyl-L-aspartic acid, L-as-partic acid, and L-fl-cyanoalanine. Compoundsproviding greater than 10% inhibition of aspara-gine uptake were, in addition to those listedabove, L-glutamine, D-asparagine, L-alanyl-L-as-paragine, L-y-amino butyrate, and L-serine. Theapparent K; values determined as shown in Fig.5 of compounds providing greater than 20% inhi-bition of L-asparagine uptake are summarizedin Table 3.The inhibition of asparagine uptake by diazo-

oxo-norvaline is noteworthy. Effects by theanalogue are observed on both the slope andintercept of double reciprocal plots of experi-ments when the analogue and asparagine are

added simultaneously (Fig. 6). If cells are firstpretreated with the diazo derivative, washed toremove the compound, and subsequently testedfor inhibition by the analogue, the intercepteffect is no longer observed and the inhibitionappears to be exclusively of the competitive

v

2

([14C}Asparagine, MM) -1

FIG. 5. K, determination for [12C aspartate and12C ]asparagine inhibition of ["4C]asparagine uptake.Strain W3110 was incubated 30 s with the indicatedconcentrations of [14C]asparagine and 25 uM [12C ]_aspartate or 25 MM [12C ]asparagine. V is in units ofnmoles of asparagine/min per mg. Symbols represent:(0) no inhibitor; (0) 25 AM L-aspartate; (0) 25 gML-asparagine.

TABLE 3. K, values determined for inhibitors ofasparagine uptake by strain W3110a

Unlabeled competitor K, (AM)

L-asparagine ........................... 3.0L-5-diazo-4-oxo-norvaline ....... ........ 4.6bL-fB-hydroxamyl aspartate ........ ....... 10L-aspartate ............................. 50L-/B-cyanoalanine ....................... 275

a K, values were determined as shown in Fig. 5 of1/V1 versus 1/c plots where V, is velocity in presence ofan inhibitor and c is the initial concentration ofL- [140 Jasparagine.

bThe K, value was determined with cells pretreatedwith 5 JIM L-5-diazo-4-oxo-norvaline as described inthe text.

type (Fig. 7). Comparison of the two experi-ments (Fig. 6 and 7) suggest that the action ofthe inhibitor may in part be explained by anirreversible inhibition of a portion of the trans-port activity. Direct evidence that the velocityis affected in an irreversible manner is providedby the experiment of Fig. 8. The Km for aspara-gine uptake in the absence of the inhibitor doesnot seem to have been appreciably affected bythe preincubation. The effect appears to berelated to asparagine uptake since kinetic pa-rameters of aspartate and glutamine transportare not altered in the pretreated cells.

DISCUSSIONEarlier studies suggested that E. coli may not

actively accumulate asparagine (1, 2). Ourresults indicate that asparagine uptake is medi-ated by two transport systems which are distin-guishable on the basis of specificity and regula-tion. The high-affinity component of asparagineuptake (Km = 3.5 AM) does provide an energy-dependent accumulation of asparagine andmost likely represents the system which main-tains the endogenous asparagine pool requiredfor protein synthesis. This system is repressedby culture of cells in medium containing aspar-agine supplements of 2 mM.

In a previous paper (16) we reported lags inthe establishement of characteristic growthrates when cultures of the asparagine auxotroph25287 growing with 1 mM asparagine supple-ments were subcultured into medium contain-ing 0.01 to 0.1 mM asparagine supplements.The lag periods were not observed if subcultureswere initiated from cultures grown with 0.05 to0.07 mM asparagine supplements. A repressionof the high-affinity asparagine transport systemand, subsequently, the time required for depres-sion of the system with the shift of cells into

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0.8

E

06~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~J

.-'~~~~~~~~~~~~01/010 15 do 25

Asparagine (oIM)

FIG. 6. Inhibition of asparagine uptake by diazo-oxo-norvaline. Strain W3110 was incubated for 30 s inassay mixtures containing diazo-oxo-norvaline and [14C]asparagine at the concentrations indicated. Symbols:(0) no diazo-oxo-norvaline; (0) 2 M1M diazo-oxo-norvaline; (Q) 5 MM diazo-oxo-norualine; and (e) 10 /MMdiazo-oxo-norvaline. V is in units of nanomoles of asparagine per minute per milligram.

40r medium containing the low-asparagine supple-ments provide an explanation for these previ-

3_ /° ~~~~~~OUSresults. The lags in growth rate observedEv / ~~~~~~~withthe asparagine auxotroph represent the2 time required for return of the high-affinity^{ ~~~~~transport system to a level of activity which is

/ ~~~~~~notgrowth rate limiting.E

1( ' ~~~~~~~~The low-affinity system (Km > 80,uM) hasE 05.s;o not been studied in any detail but appears to be-151.05 10 1necessary for E. coli to use asparagine as a

Asp/cogi nitrogen source (16). Preliminary experimentsFIG. 6.Inibitin of sparaineutakeydiaooxonorvaindicatethat both aspartate a ndammonia are

assay mixturescontainig diazo-xo-norvaine and4Capotent inhibitorsof asparagine degradation byC0) no diazo-oxo-norvaline; (0) O2AM d_azo oxonorvawholecells, whereas neither provides significantdiazooxonorvaline V is inunitsofnanomolesofasparaginhibition of asparagine degradation by toluene-

0 mdFIG. 7. Uptake of asparagine in the presence (0)-eCir ~~~~~~~~~andabsence (0) of 5 MM diazo-oxo-norvaline by cells

pretreated with 5 MM diazo-oxo-norvaline for 30 mi2 6 8 FO }F ~~~~at37 C in buffer A followed by centrifugation and

resuspension in buffer A. The cells were then used toAsporogine wt)measure uptake in the standard manner.

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90

8C

u 7

6CL

ID)-, 502

e 40D

. 30

-2cr20

2 10

K~~~~~ mI

4-

2 4 6 8 iu 3uPreincubation (Minutes)

FIG. 8. Effect of preincubation of cells with diazo-oxo-norvaline on asparagine uptake. Cells were pre-

incubated with 5 AM diazo-oxo-norvaline in buffer Aat 37 C for the times indicated and washed to removediazo-oxo-norvaline as indicated for Fig. 7, and theuptake of asparagine by the washed cells was meas-

ured in the standard manner. The activity of the cellsis expressed in percent relative to the uptake of cellssimilarly manipulated without the exposure to diazo-oxo-norvaline.

treated cells or cell extracts. These preliminaryresults suggest that aspartate and ammoniamay be preferred substrates for the transportsystem(s) represented by the low-affinity com-

ponent of asparagine uptake. A similar con-

clusion is suggested from the results of [I'N ]-ammonia sparing studies of Roberts et al. (13).However, other workers have found that aspar-

agine does not serve as a competitor for thetransport of aspartate (6), glutamine (15), or

glutamate (3) by those systems believed to beprimarily responsible for the uptake of thelatter amino acids by E. coli. In this regard,unpublished experiments in this laboratoryhave confirmed these observations of the previ-ous workers with the strain of E. coli used in thisinvestigation. The general finding that excess

asparagine does not inhibit the transport ofthese related amino acids together with the

finding here that the related amino acids are noteffective inhibitors of asparagine transport(Table 2) argue strongly for the individuality ofthe high-affinity asparagine transport systemreported here.

ACKNOWLEDGMENTSThis research was supported by Public Health Service

Predoctoral Research Fellowship award GM 42291 from theNational Institute of General Medical Sciences awarded thesenior author and by Public Health Service grant CA-08390from the National Cancer Institute.

LITERATURE CITED

1. Cedar, H., and J. H. Schwartz. 1968. Production ofasparaginase 11 by Escherichia coli. J. Bacteriol.96:2043-2048.

2. Cedar, H., and J. H. Schwartz. 1969. The asparaginesynthetase of Escherichia coli. I. Biosynthetic role ofthe enzyme, purification, and characterization of thereaction products. J. Biol. Chem. 244:4112-4121.

3. Halpern, Y. S., and A. Evan-Shoshan. 1967. Properties ofthe glutamate transport system in Escherichia coli. J.Bacteriol. 93:1009-1016.

4. Holden, J. T., and J. M. Bunch. 1973. Asparaginetransport in Lactobacillus plantarum and Streptococ-cus faecalis. Biochim. Biophys. Acta 307:640-655.

5. Jackson, R. W., and J. A. DeMoss. 1965. Effects oftoluene on Escherichia coli. J. Bacteriol. 90:1420-1425.

6. Kay, W. W. 1971. Two aspartate transport systems inEscherichia coli. J. Biol. Chem. 246:7373-7382.

7. Koch, A. L. 1964. The role of permease in transport.Biochim. Biophys. Acta 79:177-200.

8. Lombardi, F. J., and H. R. Kaback. 1972. Mechanisms ofactive transport in isolated bacterial membrane vesi-cles. VIII. The transport of amino acids by membranesprepared from E. coli. J. Biol. Chem. 247:7844-7857.

9. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

10. Maaloe, O., and N. 0. Kjeldgarrd. 1966. In Control ofmacromolecular synthesis, p. 86-96. W. A. Benjamin,Inc., New York.

11. Monod, J., G. Cohen-Bazire, and M. Cohn. 1951. Thebiosynthesis of fB-galactosidase (lactase) by Escherichiacoli. Biochim. Biophys. Acta 7:585-599.

12. Oxender, D. L. 1972. Membrane transport. Annu. Rev.Biochem. 41:777-814.

13. Roberts, R. B., P. H. Adelson, D. B. Cowie, E. T. Bolton,and R. J. Britten. 1963. Studies of the biosynthesis inEscherichia coli. Carnegie Institution of Washington.Publication 607. Washington, D.C.

14. Slayman, C. W. 1973. The genetic control of membranetransport. Annu. Rev. Genet. 7:1-74.

15. Weiner, J. H., and L. A. Heppel. 1971. A binding proteinfor glutamine and its relation to active transport toactive transport in Escherichia coli. J. Biol. Chem.246:6933-6941.

16. Willis, R. C., and C. A. Woolfolk. 1974. Asparagineutilization in Escherichia coli. J. Bacteriol. 118:231-241.

VOL. 123, 1975

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