33Piochzem. J. (1977) 162,W33-38Printed in Great Britain

Substrate Specificity ofAmino Acid Transport in Sheep Erythrocytes

By JAMES D. YOUNG and J. CLIVE ELLORY*A.R.C. Institute ofAnimal Physiology, Babraham, Cambridge CB2 4AT, U.K.

(Received 19 July 1976)

The specificity of amino acid transport in normal (high-glutathione) sheep erythrocyteswas investigated by studying the interaction of various neutral and dibasic amino acids inboth competition and exchange experiments. Apparent K1 values were obtained foramino acids as inhibitors of L-alanine influx. Amino acids previously found to be trans-ported by high-glutathione cells at fast rates (L-cysteine, L-a-amino-n-butyrate) were themost effective inhibitors. D-Alanine and D-a-amino-n-butyrate were without effect. Of theremaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine andL-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux frompre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acidsthat inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine,D-a-amino-n-butyrate, L-threonine, L-asparagine, L-cx,fi-diaminopropionate, L-ormithine,L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux.L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitorypotency of L-alanine was not influenced by the replacement of Na+ in the incubationmedium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alaninebut not by D-alanine. It is concluded that these cells possess a highly selective stereo-specific amino acid-transport system. Although the optimum substrates are smallneutral amino acids, this system also has a significant affinity for dibasic amino acids.

Erythrocytes from Finnish Landrace sheep with aninherited deficiency of GSH (reduced glutathione)contain high intracellular concentrations of certainamino acids, notably ornithine and lysine (Ellory etal., 1972). These cells differ markedly from normalsheep erythrocytes in their permeability to someneutral and dibasic amino acids (Young et al., 1975).The decreased amino acid permeability is the resultofa membrane-transport defect, and the extent of thistransport lesion has been established by measuringthe relative uptake rates of a series of natural aminoacids by both normal (high-GSH) and GSH-deficient(low-GSH) erythrocytes (Young et al., 1976). Thesestudies strongly suggest that high-GSH sheeperythrocytes have a stereospecific transport systemfor neutral amino acids of intermediate size (thecysteine system), which is lacking in low-GSH cells.The permeability to the dibasic amino acids ornithineand lysine was also significantly different in the twotypes of erythrocyte. Since lysine uptake by high-GSH cells was markedly inhibited by L-alanine andL-a-amino-n-butyrate, but not by D-alanine, it wassuggested that these amino acids were also trans-ported by the cysteine system. The present investiga-tion studies the interaction of different amino acidsin both competition and exchange experiments tocharacterize further neutral and dibasic amino acid

* Present address: Department of Physiology, Univer-sity of Cambridge, Downing Street, Cambridge CB2 3EG,U.K.

Vol. 162

transport in sheep erythrocytes. Evidence is pre-sented which confirms the original findings that bothneutral and dibasic amino acids share a commontransport system in these cells. A preliminary reportof some of these results has already appeared (Elloryet al., 1976).

Materials and Methods

Whole blood was obtained from Finnish Landracesheep by jugular venepuncture into heparinizedcontainers. Animals were classified as to GSH-typeon the basis of erythrocyte GSH and amino acidconcentrations (Tucker & Kilgour, 1970; Ellory etal., 1972).

Materials

"C-labelled amino acids were purchased from TheRadiochemical Centre, Amersham, Bucks., U.K.Non-radioactive amino acids were obtained fromSigma (London) Chemical Co., London S.W.6, U.K.,except for L-alanine, L-cysteine, L-isoleucine, L-lysine,L-ornithine, L-threonine and L-serine (BDH Chemi-cals Ltd., Poole, Dorset, U.K.) and S-2-aminoethyl-L-cysteine and L-ac,f-diaminopropionic acid (Cal-biochem Ltd., Hereford, U.K.). When necessary,amino acid solutions were adjusted to the requiredpH with NaOH or HCI.

2

J. D. YOUNG AN; J. C, FL.+oRY

Amino acid-uptake stidiesAmino acid uptake was measured as previously

described (Young et al., 1976). All fluxes were carriedout at 25°C unless ptherwise stated; 25°C was chosenas a convenient low temperature for demonstratingkinetic effects, since lowering the temperatureresults in a marked increase in the apparent affinityof erythrocyte amino acid-transport systems (Hoare,1972b; Young ft al.? 1976). Iso-osmolality at variousamino acid concentrations was maintained byadjusting the concen,tration ofNaC in the incubationmedium. Choline replaced Nat in the Na+-depend-ence experiments,

Amino acid-e,fOlux studies

Washed erythrocytes were incubated at 4 haemato-crit of30% for 2h ;t 37°C in a medium (Young etal.,1976) containing 40mm-L-[U-14C]alanlne (0.02$5Ci/,umol). The cel4 were rapidly washed in 4x lOvol. ofice-cold incpbation medium by using an JEppendorf3200 micro-centrifuge (I5000g, lOs) and then re-suspended to give a haematocrit of 4%, L-Alanineefflux at 25"C was mepasured by mixing 0.5ml of cellsuspension with 0.5 ml of incubatiQA medium cot-taming the appropriate non-radioactive amino acid.At pre-determined time-intervals, incubations werestopped by rapidly sedimenting the cells (15000g20s). A sample (0.7ml) of supernaant was trans-ferred to 7ml of Unisolve (Koch-Light LaboratorisLtd, Colnbrook, Bucks., U.K.) and counted forradioactivity in a f-scintillation spectrometer withquench correction. The initial intracellular L-ahuueconcentration was measured by adding 0.25 ml of the4% cell suspension to 0.5ml of 0.5% (v/v) Triton

X-100, and deproteinizing the resulting lysate with0.25ml of 66 % (w/v) trl1oroaeetio acId. The pre-cipitate was removed by centrifugation (15000g,3Q0), and Q.9ml of the supernatant was added to 7mlofUnisolve and countod for radioactivity with quenchcorrection. Samples of the trichloroacetic acidextraets were also subjected to paper chromato-graphy (Winter & Christensen, 1965) to determinethe extent of cpnyerFion of ;-[U-'4C]alanine intoother soluble radioactive compounds. No metaboliteof L-alann was detetvd.

Effects qf amino acids on L-alanine uptake by highl-GSH erythrocytes

We have previously phown that the concentration-dependenc of L-alanine uptake by normal (high-GSH) erythrocytes is consistent with a single com-ponent conformiig to simple Michaelis-Mentenkinetics with an apparent Km pf 6.0mM and a Vmax.of2.85mmol/h per litre of cells at 25°C (Young et al.,1976). In the present series of inhibition studiesL-alanimne uptake was measured at two concentrations(5 and 10mM) in the presence of another amino acid(5-25mM). These data were analysed by the Dixon(1953) procedure to estimate apparent K, values. Fig. 1shows two typical amino arid inhibition patterns(L-e.tamino-n-butyr4tf, apparept K, 6.27m; and L-mthigni.nq, no i ibitioni measurab0l). To confirmthat the inhibition was of a simple competitive type,the eifqct of L-q,amino-nbptyrtt O L-llafnineuptake was investigpted over a wider range of i-alanipe c,pcentrationo3 a44 the data w-re analysedby the Dixon (1953) method and by th4e alternativ

2.o0(b)

I .5

l *, U p1.0

0.5

- I 25-5 0 5 15 25

[Amino acid] (mM)

Fig. 1. Effects ofL-c-amino-n-butyrate (a) and L-methionine (b) on L-alanine uptqke by high-GSHerytlirocytesExperimental XJetgiIs are deIscribd in the text. *, 5mM-L-alanine; 0, lOmM-L-alanine.

1977

-

C40

-q

34

AMNQ ACID1- NPORT IN S4IEEP ERYTHROCYTESS

Table 1. Effects of amino acids on L-alatiine influx andefflux in high-GSH erythrocytes

Apparent K1 values and initial L-alanine effluX rateswere determined as described in the text. In thr, effluxexperiments the initial intracellular L-larnin, con-centration was 9.2mmol/litre of cells, and extra-cellular amino acils were present at a concertjationof 50mM; values are means+S.E.M. of these obser-vations.

Apparent K, Stivalue of(MM) ef

-0.1 0.1 0.2 0.3 0.4

I /s (mm1)

Fig. 2. Effect ofL-a-amino-n-butyrate on L-alanine uptakeby high-GSH erythrocytes

The L-oc-amino-n-butyrate concentrations used were0 (0), 5 (o), 10 (-), 15 (o) and 25 (A) mM. See text forother experimental details. Analysis of these data bythe Dixon (1953) procedure gave an apparent KXvalue for L--amino-n-butyrate of 6.5mM.

L,?wegyer-Rur procedure (Fig. TTIe respltscomistjent with simgp1e t pitjw inhibi.tion

with iIppar4nt K1vaXle ofXI2~5.

Tabl@ 1 Iist8 tbW 4ppwrent &s yVu14s give] by a

Vrie.ty of ami acids ps ihb3tors ..-JnieI4pti~ke. The 4Minq acids previpp~ly fM,nd to betranpporte4 W higlSH pells gt high rates (e.g.

L4-cyatemeawd kr -f -b wqqwes4th mostffetqive iphibis Decrtgrg ksjn th

pgbh pf th4 gjd$idi cphi t inhi o theidchaina, c tiit 4pcion of aa rinIrxkedy 4e d the 4gqe o h,itis. The 1 e.

jphbitipn by D-R-n, jgo-ii-bu.tyt n4

-amin obutyrat eamphsized *tr4ruc.tur* r.,qpimeLnts at t4ep p-arbon atom. Sirilarly. the lac

i4ibition b y-amjno-ig-huav qfbuto4 ate

pimired t1 impotapew f the a-amnino Wup.y.yeiize and L-spriqe as stru-tt1 n1ou of

L-amnO-u-b tyrate were loth inhibitors of L-wuptaP. Cy4aine ws the nlqre effective pd

tbis may {e assoiated with the prwence of thergtiv.ely bukUY 4I1 gronp (cf. -OI gropp of serine).The prewce of e sulhu ator ne

resulted in a dramatic decrease in inhibitory effeivpnsp aed with the corresponding straight-chainamino acid (L-norleucine).

v ariety of dibic no acids were tested fortheirr effects on L-alaniqt ptake. L-2,4-tiamino-n-butyqAte was a surprisingly fgeCtive inhibitpr, n4results for ti4e homOloqOLls spes of dibasi aminO

ci4s parallld the data for straight-chain eutTralAi.iMA9 Si4-. Thu§ 4e4raina (P.X,ldmno

Vol. 162

Glycine * *

Proling $ *

L-Alanine t 167+4D-Alanine * 13±4Lx-Anino-n-lutyr e 6.5 125+1D-x-Amipo-n-buate * 19 2y-Amino-n-butyrate * *

a-Aminoisobutyrate * *

Sodi,um butyr,at,e * *

L-NQrvaline 8.5 1402L-'41rins 41.5 69±3L-Norleucfe 40.0 22 + 1L-Leucine * *

L-Isoleucine * *

L-Cycloleucine * *

L-Phenylalanine * *

L-Cysteine 7.0 143 ±4L-Senre 38.0 89+ 1-Threonine 23+2

L-Methionine *

L-Asparagine 22±2

L.a2jfi-1,a,@n ,inopropiptlate 60±: 1L-2>4-y4v-iz-bItyrte 23.Q 116±1L-Orpjthb. 57+3

L-Lysine * 42+1p-Lysinf **S-2-Aminoethyl-L-cysteine * 54±2L-.Argrnrne '*Cadavene * *

* NQ inhibition of ipfljix pr timul1tion of efflu:«rneasur~b1e.

t Tinder these experinntal corditions the apparentKm yalue for L-alanine influx was 6.0mM.

propionate) or incregsing (L-ornit.hiae, L-iyOse andm-2-aninoethyl-ri,ysteine) the length of tbh aminRacd side chain resulte in Ios& of iRhibitipp cpM.pared with L-2,4-diamino-n-kutyrate.

Effects of amino acids on L-alanine efflucxfrom high-GSH erythrocytes

Competition experiments suffer from the limitationthaat an analogue that inhibits the transport ofanother is not neces arily transportl by the same

rysteiR (we hristensep, 1975a). This sect

:imulation1.-alaninefl,ux (%)

35

J. tD. YOUNG AND J. C. ELLORY

_1.0 A

0 0.5

0 10 20 30 40Time (min)

Fig. 3. Time-course of L-alanine efflux from high-GSHerythrocytes in the presence and absence of extracellular

amino acidsL-Alanine efflux from pre-loaded cells was measuredas described in the text. Extracellular amino acidswere present at a concentration of 20mM.. , Control;A, L-alanine; V, D-alanine; *, L-a-amino-n-butyrate.The initial intracellular L-alanine concentration was8.8 mmol/litre of cells.

describes an alternative approach to the investigationof the structural specificity of the cysteine transportsystem.When certain amino acids were present in the

external medium, there was a marked stimulation ofL-alanine efflux. Fig. 3 shows the time-course ofL-alanine efflux in the presence ofextracellular D- andL-alanine and L-a-amino-n-butyrate. Efflux in thepresence of D-alanine was slightly greater than in thecontrol, and was considerably less than that observedin the presence of extracellular L-alanine and L-a-amino-n-butyrate. The relationship between theinitial rate of L-alanine efflux and the extracellularL-alanine concentration is shown in Fig. 4. Theexternal L-alanine concentration required to givehalf-maximal stimulation of efflux was 5.2mM. Sinceinitial rates of L-alanine efflux were measured in theseexperiments, and since the incubation haematocritwas only 2%, the observed stimulation of efflux isnot the result of inhibition of ['4C]alanine back-fluxby extracellular amino acid but is an example ofaccelerative exchange diffusion (Stein, 1967; Chris-tensen, 1975a). In these circumstances the stimulationof efflux is attributed to an influx of external aminoacid by the same transport system, resulting in a morerapid reorientation of the carrier than would other-wise occur. A similar effect has been observed forleucine transport by human erythrocytes (Hoare,1972a). In a control experiment L-[U-14C]alanineuptake (external concentration 10mM) was measuredin cells pre-loaded with non-radioactive L-alanine(13.4mmol/litre of cells) and compared with cells

1112.4 -

1.6-/

_0.8_ , , ,__ J

0 5 10 1 5 20

[L-Alanine] (mM)

Fig. 4. Stimulation of L-alanine efflux from high-GSHerythrocytes by extracellular L-alanine

Initial L-alanine efflux rates were measured in thepresence of various concentrations of extracellularL-alanine. The initial intracellular L-alanine con-centration was 10.6mmol/litre of cells. See the textfor methods.

preincubated in the absence of amino acid. There wasa 71 % increase in the initial influx rate in the L-alanine-loaded cells, demonstrating the symmetry ofthe transport system in this respect.Table 1 also shows the ability of various amino

acids to stimulate L-alanine efflux. A relatively highextracellular amino acid concentration (50mM) wasused in this series of experiments in an attempt todetect amino acids with very low, but still significant,affinities for the transport system. Amino acids in thiscategory could not be detected in the competitionexperiments. The amino acids that inhibited L-alanine influx also markedly stimulated L-alanineefflux, and the degree of stimulation strongly corre-lated with the estimated K1 values, amino acids withlow apparent K1 values giving the largest L-alanineefflux rates. Eight additional amino acids, namelyD-alanine, D-a-amino-n-butyrate, L-threonine, L-asparagine, L-a,fl-diaminopropionate, L-ornithine,L-lysine and S-2-aminoethyl-L-cysteine, also induceda significant stimulation of L-alanine efflux, demon-strating that these amino acids also interact with thecysteine system. The lack of effect of these aminoacids on L-alanine uptake (Table 1) probably reflectstheir low affinity for the transport system. Forexample the apparent Km value for L-lysine uptake byhigh-GSH erythrocytes has been estimated to be over100mM at 37°C (Young et al., 1976). The remainingamino acids did not significantly stimulate L-alanineefflux.These efflux data reinforce the conclusions reached

in the previous section about the structural specificityof the cysteine transport system. They also demon-strate that dibasic amino acids other than L-2,4-diamino-n-butyrate interact with this system. It issignificant that whereas L-lysine stimulated L-alanine

1977

16

AMINO ACID TRANSPORT IN SHEEP ERYTHROCYTES

Table 2. Effects of D-alanine and L-alanine on L-lysineuptake by high-GSH erythrocytes in the presence and

absence ofNa+L-Lysine uptake (extracellular concentration 0.2mM)was measured at 37°C by the method described in thetext for L-alanine. Values are means+S.E.M. of threedeterminations.

L-Lysine uptake(pmol/h per litre of cells)

135mM-Na+ 0.2nM-Na+

0 20 30 40 , 50

Time (min)

Fig. 5. Time-course of L-lysine efflux from high-GSHerythrocytes in the presence and absence of extracellular

amino acidsErythrocytes were incubated at a haematocrit of 30%/for 5h at 370C with 40mM-L-[U-14C]lysine (0.025pCi/pmol). Lysine efflux was measured by themethod described in the text for L-alanine. Extra-cellular amino acids were present at a concentrationof20mM. *, Control; A, L-alanine; V, D-alanine. Theinitial intracellular L-lysine concentration was10.4mmol/litre of cells.

Control+25mM-D-alanine+25mM-L-alanine

15.9+0.514.1±0.68.8±0.1

equivalent to a dibasic amino acid. To test whetherthis occurred in the present system, the effect of L-alanine on L-[U-14C]lysine uptake by high-GSH cellswas measured in both high- and low-Na+ media. Nodifference in inhibitory potency was observed whenthe Na+ concentration in the incubation medium waslowered from 135mM to 0.2mM (Table 2).

efflux, D-lysine was without effect. This agrees withthe stereospecificity observed for the neutral aminoacids. In addition the effiux results suggest that L-threonine and L-asparagine may also be transportedby the cysteine system. Each of these amino acidsshowed a threefold difference in uptake rate betweenhigh- and low-GSH cells (Young et al., 1976). Indeedthere was very good agreement between the group ofamino acids postulated to share the cysteine systemon the basis ofthe competition and efflux experiments,and those postulated to be transported by thissystem on the basis of permeability differencesbetween high- and low-GSH erythrocytes.

Effects ofD- andL-alanine on L-lysine effluxfrom high-GSH erythrocytes

L-Lysine but not D-lysine stimulated L-alanineefflux from cells pre-loaded with L-alanine. Fig. 5shows the effects of D- and L-alanine on L-[U-14C]-lysine efflux from erythrocytes pre-loaded with L-lysine. L-Alanine but not D-alanine produced amarked stimulation of efflux.

L-Alanine inhibition of L-lysine uptake by high-GSHerythrocytes: Na+-dependence

We have shown that L-alanine, L-a-amino-n-butyrate but not D-alanine markedly inhibit L-lysineinflux into high-GSH cells (Young et al., 1976).Christensen (1975a,b) has developed the concept that(neutral amino acid+Na+) can sometimes be

Vol. 162

Discussion

It is apparent from the present results that normalsheep erythrocytes possess a highly selective stereo-specific transport system for amino acids. Althoughthe optimum substrates are small neutral amino acidssuch as L-alanine, L-a-amino-n-butyrate and L-cysteine, it is clear that this system (the cysteinesystem) also has a significant affinity for a variety ofdibasic amino acids. The absence of this transportsystem from GSH-deficient erythrocytes is presum-ably responsible for the low GSH concentration, andmay also account for the presence of high concentra-tions of ornithine and lysine in these cells, since acomparison of the relative uptake rates of L-lysineand L-ornithine by normal and GSH-deficienterythrocytes (Young et al., 1976) suggests that thecysteine-transport system is a major route for thetransport of these dibasic amino acids in normalsheep erythrocytes. In this connexion it is note-worthy that whereas L-alanine and L-a-amino-n-butyrate but not D-alanine markedly inhibit L-lysineinflux into high-GSH cells (Young et al., 1976), theresidual L-lysine uptake by GSH-deficient erythro-cytes is insensitive to both L-alanine and L-a-amino-n-butyrate (J. D. Young & J. C. Ellory, unpublishedwork).When the kinetic parameters of the cysteine-

transport system were investigated, amino acidinflux was found to conform to simple Michaelis-Menten kinetics, with the apparent Km and Vmax.values showing a marked temperature-dependence(Young et al., 1976). The system also exhibited thephenomenon of accelerative exchange diffusion in

0.4a)

o 0.3tq)

& 0.2

I 0.1

L 16.1±0.115.3±0.18.4+0.2

37

38 J. Dl. YOUNG AN) J. C. ELLORY

both influx and efflux experiments, demonstratingthe symmetry of the transport mechanism in thisrespect. Although not investigated in such detail, thekinetic characteristics of this transport sysgtem insheep erythrocytes are superficially similar to thoseof the leucine-transport system in human erythro-cytes which has been investigated in some depth byHoare (I1972a,b).

Several conclusions can be made about thespecificity of the cysteine-transport system. It ishighly stereospecific. The presence of the ar-amino anda-carboxyl groups are essential for activity, and thereplacement of the a-hydrogen atom with a methylgroup similarly results in a loss ofactivity. In addition,the transport system prefers straight-chain aminoacids, and branching of the side chain or the presenceof an aromatic ring is not readily tolerated. Further,the length of the amino acid side chain is of critialimportance. Thus L-a-amino-n-butyate is a muchbetter substrate than either glycine or L-norleucine.The transport system has the ability to distinguishbetween L-Cysteine and L-serine on the one hand, andL-methionine and L-norleucine on the other, demon'strating a high degree of selectivity at two positionsalong the amino acid side chain. In contrast, thetransport system accepts the presence of a terminalamino group on the side chain. The bulkier guani-dinium group of arginine is not, however, tolerated.The acidic amtino acids L-aspartate and L-glUtamateare not transported.For a system with such a high substrate selectivity

in other respects, it is perhaps surprising that di-bsic amino acids are also transported. Severalother examples of dibasic and neutral amino acidssharing a transport system have been described(see, e.g., Christensen, 1975a). Chriatensen andco-workers have developed the attractive idea that(Na+±neutral amino acid) may substitute for adibasic amino acid in the cationic amino acidtransport system of rabbit reticulocytes and Ehrlichascites-tumour cells (Christensen et a., 1969;Thomas et al., 1971). Interestingly, in the presentcase, such a mechanism cantot be operating, since noNa+-dependence of neutral amino acid uptake can bedemonstrated. In addition, L-alanine inhibition ofL-lysine transport was not influenced by the absence ofNa+ from the incubation mnedium. Another possi-bility to be considered is the transport of dibasicamino acids as neutral species. However, the highpK of the lysine E-amino group makes only 0.1 % ofthe amino acid available in the uncharged form underour experimental conditions. Therefore, unless theamino acid-binding site has the ability to suppressdramatically protonation of the 8-amino group, thedibasic amino acids must be transported in theionized form.The amino acid specificity of the cysteine system is

unlike that of previously desribed amino acid-

transport systems in mammalian erythrocytes(Winter & Christensen, 1964, 1965; Antonioli &Christensen, 1969; Christensen & Antonioli, 1969;Gardner & Levy, 1972). In this respect it closelyresembles the alanine-serine-cysteine (ASC) systemdefined by Christensen and co-workers (Christensen,1969, 1975a,b). However, the 'ASC' system, which ispresent in rabbit reticulocytes but which has not beenfound in mature mammalian erythrocytes, differsfrom the present system in its Na+-dependence and inits apparent limitation to transport by exchange. It isintriguing to speculate that the present erythrocytesystem may represent a modified component of areticulocyte mechanism that has lost its Na+-dependence and has aitered exchange chavacteristics.Purther experiments to characterize amino acidtransport in reticulocytes from high- and low-GSHsheep may allow this point to be resolved.

This work was supported in part by a grant from theM.R.C. We thank Mrs. Sandra Mansfield and Mrs. SusanCarleton for excellent technical assistance.

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Bfol. 32, 1-20Christensen, H. N. (1975a) Biological Transport, 2nd

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1977