A Stereoselective Anomaly in Dicarboxylic Amino Acid Transport*

Using L-cysteate and tcysteinesulfinate as model substrates, we characterize here a transport system, both in cultured rat hepatocytes and human skin fibro- blasts, serving for the anions glutamate and aspartate, but not for the dipolar species glutamic and aspartic acids. This system appears to be accompanied by a second, lower affinity system for the anionic forms, which is also Na’-dependent; this lower affinity system applies at least to glutamate. These systems show the usual degree of preference for L- over D-glutamate and, in the fibroblast, for L- over DL-a-aminoadipate. D-AS- partate proved nearly as inhibitory to the uptake of L-cysteate or t-aspartate, however, as did L-aspartate itself, a comparison recalling a similar stereoselective anomaly discovered by Pall in Neurospora (Pall, M. (1976) Biochim Biophys. Acta 211, 513-520). We con- clude that this anomaly arises from the ability of the two substrate carboxylate groups to bond in the spatial order either a# for the L-isomer or &a for the D-isomer and also to bond in the order a,y for L-glutamate, but scarcely in the order y,a for D-glutamate. A major lack of inhibition by D-cysteate, which might be expected to bind like aspartate in the inverted order, shows,

Using L-cysteate and tcysteinesulfinate as model substrates, we characterize here a transport system, both in cultured rat hepatocytes and human skin fibroblasts, serving for the anions glutamate and aspartate, but not for the dipolar species glutamic and aspartic acids. This system appears to be accompanied by a second, lower affinity system for the anionic forms, which is also Na'-dependent; this lower affinity system applies at least to glutamate. These systems show the usual degree of preference for L-over D-glutamate and, in the fibroblast, for L-over DL-a-aminoadipate. D-ASpartate proved nearly as inhibitory to the uptake of Lcysteate or t-aspartate, however, as did L-aspartate itself, a comparison recalling a similar stereoselective anomaly discovered by Pall in Neurospora (Pall, M. (1976) Biochim Biophys. Acta 211, 513-520). We conclude that this anomaly arises from the ability of the two substrate carboxylate groups to bond in the spatial order either a# for the L-isomer or &a for the D-isomer and also to bond in the order a,y for L-glutamate, but scarcely in the order y,a for D-glutamate. A major lack of inhibition by D-cysteate, which might be expected to bind like aspartate in the inverted order, shows, however, that the two anionic groups are not recognized in identical manners by the two corresponding subsites, Precedent for a chemical difference in these two subsites is available from transport systems for neutral aand p-amino acids. A strong transport inhibition of the hepatocyte system by 3-aminoglutarate shows that an a,a relation between the amino group and either of the carboxylate groups of the anionic amino acid is not required. The above anomaly in stereoselectivity is compared with a corresponding one, applying to the reactions of aspartic acid and asparagine, versus glutamic acid and glutamine, with System L for neutral amino acid transport in the Ehrlich cell. A weak pHdependent inhibition of the uptake of anionic amino acids by cysteine can be associated with its unique mode of conversion to an anionic species.
The transport of the dicarboxylic amino acids across cellular membranes is marked by two interesting problems. Fist, they may be transported by systems for neutral amino acids, e.g. by the Na+-dependent System A and by Na+-independent System L (1, 2). (As a variant of this case, a neutral system might in theory also respond specifically to the o-carboxyl group as -COOH and, hence, reject ordinary neutral amino acids. The aspartatel-glutamic acid exchanging system of beef heart mitochondria appears to present such a feature for one of its two opposed fluxes (3).) Secondly, they may be transported by systems restricted to the anionic form of these amino acids, i.e. -OOC(CH2)nCH(NH3+)COO-, and not as glutamic or aspartic acid. In the fist case, one expects the transport to be enhanced by lowering the pH so as to convert the anionic form mainly to the zwitterion, HOOC(CHZ).CHINH~+)COO-, provided only that the cell or vesicle in question, and the transport system also, will tolerate the acidification needed. L-Glutamic acid uptake by System L of the Ehrlich cell increases roughly in the manner predicted by its titration curve, to maximize at about pH 4.2 (2). A parallel demonstration for System A uptake of glutamic acid has, however, been prevented by the sensitivity of that system to H'. Another test serves also to show that glutamic acid uptake by the Ehrlich cells is limited to the neutral systems: The unequivocally anionic amino acid cysteate, -O~SCHZCH(NH~+)COO-, introduced as a transport model by Pall (4), shows neither uptake itself nor inhibition of the uptake of glutamic acid or aspartic acid over a wide pH range. Other cells, including Neurospora (4), Escherichia coli (summary in Ref. 5, p. 531, mouse lymphocytes (6), and the rat hepatocytes and human skin fibroblasts to be reported here, show one or more transport systems specific to the anionic forms, in each case inhibited by cysteate and in some cases shown not to be stimulated by lowering the external pH. The present study is largely limited to transport systems of this second, anionic type.
The second interesting problem of dicarboxylic amino acid transport that concerns us here is the effect on transport by these two types of systems of the distance by which the second carboxylate group is separated from the fmt, i.e. in the selectivity among aspartic and glutamic acids and their higher homologs. We will show here that an apparently separate riddle concerns essentially the same problem, namely a difference in the selectivity between the D and L isomers of these amino acids, which we may restate as the sensitivity to the difference in the distance by which the two carboxyl groups are separated from the a-amino group in a given transport substrate molecule. An anomaly in the stereoselectivity in the series aspartic acid, glutamic acid, etc., has already been shown both for our first case of the opening paragraph, where the molecule is accepted as a neutral, dipolar ion (21, and for our second case, where it is accepted only as an anion (2, 4, 6). A new observation of the latter type has been reported (7) ' Terms such as aspartate and cysteate refer specifically to the anionic species of these amino acids. As usual, aspartic acid and the like may refer to the amino acid in general or, when the context indicates specifically, to the dipolar species without net charge.

Stereoselective Anomaly in Amino
Acid Transport 6055 but in a contrasting case the anomaly was inconspicuous (see Table 11 in Ref. 8). We investigate here new cases where the dicarboxylic amino acids must be anions, selecting the rat hepatocyte to represent an epithelial t h e and the human skin fibroblast to represent a mesenchymal tissue for contrast.

EXPERIMENTAL PROCEDURES
Celt Culture--Rat hepatocytes were isolated as previously described (9), seeded in collagen-treated 4-compartment ( 2 4 X 67 mm each) plastic trays (LUX), and cultured in Waymouth's medium (MB752/1) containing 0.2% bovine serum albumin, 5 pg/ml of sodium oleate, 0.41 m~ t-alanine, 0.53 m~ L-serine, 65.5 p g / d of penicillin, 5.8 , u g / m l of streptomycin, 28.4 pg/ml of gentamicin, and 600 microunits/ml of insulin. This medium was replaced after 4 h of culturing with the same medium lacking the insulin. Cells were used for transport assay 24 h after plating.
Human fibroblasts, obtained from skin biopsy explants as described previously (lo), were routinely grown in 10-cm diameter dishes (Costar) in Medium 199 containing 10% fetal calf serum. The conditions of culturing were: pH 7.4; atmosphere, 5% CO? in air; temperature, 37°C. For uptake experiments cells were seeded in 24-well plates (Costar) and used when cell density reached 25 f 3 pg of protein/cm*. All points out of this range were discarded. The culture medium was always renewed 24 h before the experiment.
Uptake Assay-Amino acid uptake was measured under conditions approaching initial entry rates (S min for fibrobfasts, 5 or 10 min for hepatocytes) as follows. After appropriate incubation in bicarbonatebuffered solutions (90 min in Earle's salt solution containing SO% dialyzed fetal calf serum for human fibroblasts and 30 min in Krebs-Ringer solution for hepatocytes) at pH 7.4 and 37 "C, cell layers were washed and incubated for the required time in the same salt solution containing the labeled amino acid under study and any other designated compounds. In some experiments, a medium in which choline replaced Na+ was used. During experiments in which the pH of the medium differed from 7.4, Tris(hydroxymethy1)aminomethane hydrochloride or e-aminocaproate was used as buffer. The incubations were terminated by rapidly rinsiig the cell layers twice with ice-cold saline. The soluble contents were extracted from the cells with 10% trichloroacetic acid and counted in a liquid scintillation spectrometer. Cells were dissolved in 1 N NaOH and assayed for protein by the method of Lowry et aZ. (11).
Calculations-In all cases, uptake was linear over the indicated time intervals. Kinetic parameters were determined by a computer (Hewlett-Packard 9845A) using the Marquardt's algorithm, a general procedure for least squares estimation of nonlinear parameters (12).
All uptake data are expressed in nanomoles or micromoles (as indicated) of tested amino acid/ml of intracellular water-min.
Materials-All sera, growth media, antibiotics, and trypsin solution were from Gibco, New York. ~-[2,3-~H]Aspartic acid (specific activity, 12 Ci/mmol) and ~-[G-~H]glutamic acid (27 Ci/mmol) were obtained from Amersham, Bucks, England. 2-(Methylamino)isobutyric acid was from ~~c h -E u r o~, Beerse, Belgium. Unlabeled Land D-cysteinesulfinic acids and L-homocysteic acid were generous gifts to one of US (G. C. G.) from Professor Ferdinand0 Palmieri, University of Bari, Bari, Italy. L-Cysteate and L-cysteinesulfinate were prepared synthetically from both L-cystine and ~-[3,3'-~H]cystine for the experiments at Michigan. L-and DL-Aminoadipic acids were obtained from ICN Pharmaceuticals, Cleveland. 3-Aminoglutaric acid (13) was the gift of Dr. Alton Meister, Department of Biochemistry, Cornell University Medical School, New York (to H. N. C.).' All other unlabeled natural or analogous amino acids were synthesized in the laboratory of one of us (H. N. C.) or purchased from Sigma.

RESULTS
Cysteic acid has been titrated potentiometrically and shown to have a pK1 value rather lower than that for taurine (14), perhaps about 1.5. Cysteinesulfinic and homocysteinesulfinic acids showed similar pK1 values, set at 1.50 and 1.66 (pK'2 = 2.38 and 2.60) at 0.2 M concentrations (15). We wiU Extract of the hepatocytes with 5% salicylic acid chromatographed on paper with a 3 5 mixture of 0.1 M sodium tartrate, pH 3.4, and 1propanol, and the position taken by the tritium label observed in relation to authentic samples of labeled cysteate (7 to 11 cm) and taurine (13 to 17 cm). therefore refer to cysteic acid as cysteate and cysteinesulfinic acid as cysteinesulfinate since, at the pH values studied, they can scarcely be present as other than anions, e.g. -OOSCH2CH(NH3+)COO-. We find that the hepatocyte rapidly converts cysteate to taurine. When presented to the cultured cells at 0.1 mM in a 1W1 volume ratio, % of the 3H carried by the labeled amino acid could be extracted from the cells after 1 min as unchanged cysteate and % could be extracted as taurine, as separated by paper c~o m a t o~a p h y . After 5 min the label was recovered mainly in taurine, only a trace remaining in cysteate (Fig. 1). Consistent with its usual behavior, taurine escaped only slowly from the hepatocyte, with a half-time of 20 to 30 min. Conversion to taurine can serve as a modest advantage in the study of the influx of labeled cysteate by providing a sink to decrease a possible complication from cysteate efflux. Cysteinesulfinic acid was also converted to taurine on a similar time schedule, We have not checked to see whether the fibroblasts are able to carry out these conversions. Table I shows that cysteate uptake by the rat hepatocyte was not inhibited by taurine, alanine, or glutamine, nor by the norbornane amino acid (BCH3), considered specific to System L, nor by 2-(methylamino)isobut~c acid, considered specific to System A (5). Table I1 shows in agreement and extension small or negligible inhibitory actions of several amino acids on aspartate uptake into the fibroblast. These results, when considered with the interactions to be presented below, confirm the validity of the use of cysteate and (by analogy) of cysteinesulfinate as model substrates for anionic amino acid transport systems in these cells.
The Naf-dependent uptake of the glutamate anion appeared to correspond to two widely separated components on kinetic plotting, both for the hepatocyte and for the fibroblast, the second component being minor and low affinity, with Km values well above 1 m~. The dichotomy was first observed in the hepatocyte, using both cysteate and cysteinesulfinate, and is shown in Fig. 2A for the latter amino acid. Evidence for separation of routes was not seen for aspartate uptake into the fibroblast (Fig. 2B) but could readily be discerned for glutamate uptake (Fig. 2 0 . The failure of the second component to yield evidence here for participation in aspartate uptake argues that it is a separate entity, and not merely a kinetic anomaly shown by the principal system. Other results in this paper concern the total Na+-dependent uptake but The abbreviations used are: BCH, the (*I aminoendo conformer of 2-amino(2,2,1)bicycloheptane-2-carboxylic acid MeAIB, 2-(methylaminofisobutyric acid. obtained at substrate levels too low to include a significant contribution by the second component. Fig. 3 shows that the Na+-dependent uptake of 0.01 mM aspartate by skin fibroblasts was little affected by the presence of 2 m M BCH or MeAIB, even at decreased pH values.

TABLE I
Lack of inhibition of rate of cysteate uptake into the rat hepatocyte by some neutral amino acids Uptake of tritium-labeled L-cysteate at 0.1 mM measured during 1 min at 37 "C and pH 7.4 from the bicarbonate buffered Krebs-Ringer medium with the inhibitor replacing part of the NaCl isoosmotically.  (0, left). The increase shows itself to be Na+-independent (0, center). This fraction was not inhibited by excess MeAIB (2 mM; A, left and center but was strongly inhibited by BCH (2 mM; W, left and center. Accordingly, it undoubtedly represents the contribution of System L, which accepts well the zwitterionic form of this substrate in the Ehrlich cell (2). The Na+-dependent component, as estimated by subtracting the Na+-independent component from the total uptake rate, was instead decreased by lowering the pH (0, right) but was not affected by MeAIB

FIG. 2. Kinetic parameters by Hofstee plots for the uptake of ['Hlcysteinesulfinate by hepatocytes (A), of ~-['H]aspartate (B), and of ~-['HIglutamate (C) in cultured human fibroblasts.
The transport assay was made during 5 min for the hepatocytes and 1 min for the fibroblasts, all at pH 7.4 and 37 "C and over the indicated ranges of values of u / [ q in the presence of either 142 mM Na+ (0, total uptake) or zero added Na+ (W, Na+-independent uptake). The raw data were corrected for the corresponding nonsaturable fraction according to Akedo and Christensen (17). The Na+dependent fraction (A) was then obtained by difference. The data were fitted by computer to two rectangular hyperbolas for the Na+dependent cysteinesulfiiate and glutamate uptake and to only one  m~. The following studies concern the predominating highaffinity transport system of the respective cultured cells. Fig. 5 shows the uptake of L-cysteate at 0.1 m~ into cultured hepatocytes at pH 7.4 and 37 "C under inhibition by various anionic amino acids, and Fig. 6 shows corresponding effects on the uptake of L-aspartate at 0.01 mM by the skin fibroblasts. L-Cysteinesulfinate showed a similar pattern of inhibition (data not included in Fig. 5). Table XI1 summarizes the Ki and maximal inhibition values derived from the data of these figures. Uptake by the fibroblast tended to half-saturate with aspartate (not shown in Table 111) and to be half-maximally inhibited by it at concentrations about one-tenth those re-  Inhibition of uptake rate of 0.1 mM f3H]cysteate (rat hepatocytes) and of 0.01 MM f3H]aspartate (human fibroblasts) by several analogs K, and the extrapolated rate for maximal inhibition (Imax) were are tabulated with 75% confidence intervals in all cases in which a calculated from the data shown in Figs. 5 and 6 , using nonlinear fitting was possible. In the remaining cases, progressive values on regression analysis to fit the best rectangular hyperbola and to coriterative analysis showed a continuous increase to very high values of respond to homogeneous inhibition. The later description of cys-K, (Ki + m ) , indicating that the competent agency is unreactive with teinesuffinate uptake by the hepatocyte and of glutamate uptake by the analog under study. The hepatocyte result with L-cysteinesulfithe fibroblast, as including a minor low affinity component was thus nate was for that amino acid at 10 m~. contrasts between the epithelial hepatocyte and the mesenchymal fibroblast include the presence in only the former of System N (9) and in only the latter, a transport system for cationic amino acids ( 18). The higher homolog of cysteate, namely homocysteate (Fig.  5, lower right; Fig. 6, lower right), produced little or no inhibition in either cell, in agreement with an unpublished observation by Kleinzeller for the freshly separated hepato-~y t e .~ D-Cysteate produced a much weaker inhibition than the &-isomer in both cell types (Fig. 5, upper left panel; Fig. 6, upper right). The Same distinction was seen for D-and Lglutamate (Fig. 5, lower left panel; Fig. 6, lower left; D-glutamate failing to cause perceptible inhibition even at 1 mM) and for D-and L-cysteinesulfhate (compared in the fibroblasts only, Fig. 6, upper center), whereas little difference was seen between the effect of D-and &-aspartate (Fig. 5, upper right;  Fig. 6, upper left). In the fibroblast, the X$ of DL-a-aminoadipate showed twice the value seen for the t-isomer (Fig. 6, lower center; Table 111), whereas L-a-aminopimelate failed to show inhibitory action (Fig. 6, lower right). The optically inactive structural isomer of glutamic acid, 3-aminoglutaric acid, proved essentially as effective an inhibitor of cysteate uptake by the hepatocyte as did glutamic acid, each in its anionic form (Fig. 5, lower right). DESCUSSION Pall (4) fist reported an anomaly in the stereoselectivity of an agency transporting cysteate into Neurospora mycelial pads, whereby n-aspartate was distinctly preferred to the Lisomer. Subsequently, Garcia-Sancho et al.
(2) discovered a corresponding anomaly between the same two amino acids but applying to the totally different transport system L, tested at pH values of about 4.2, low enough to convert glutamate and aspartate extensively to their neutral forms, NOW- The present anomaly corresponds in both cell types more closely to that described by Pall (4) in that each amino acid must be accepted in its anionic form, namely that predominating in the neutral pH range, and by the anion-accepting system. We may conclude that the receptor site for transport by the anion-accepting systems is constituted in such a way A. Kleinzeller, personal communication as cited in Ref. 5 (p. 62).

(CH2),-CH(NH,+)-COO-.
that the two-carbon difference in the separation of the aand y-carboxylate groups of glutamate is rather well discerned, whereas the one-carbon difference applying for aspartate is in our experiments largely neglected. From this circumstance we infer that D-aspartate is so reactive because it can be favorably received with its P-carboxylate falling mainly at the subsite normally recognizing the a-carboxylate group and its a-carboxylate accepted mainly at the subsite ordinarily receiving the &carboxylate. The high transport reactivity of "P-glutamate" f3-aminoglutarate; Ref. 13) shows that there is no requ~ement that the amino group and one of the carboxylate groups bear an a,a relation to each other and, hence, little or no indication of an obstacle to a "backward" binding of Daspartate. The stereoselectivity for L-cysteate over D-cysteate is also good: about 2% unrecognized contamination by the former could account for the reactivity seen in the two cell types for the D preparation. This result indicates through the close analogy between cysteate and aspartate that the two substrates each recognizing an anionic group do so in chemically different modes. Whereas the /3-sulfonate group on the substrate serves well as a surrogate for the w-carboxylate group, it does not so Serve for the a-carboxylate since Dcysteate is largely rejected even though at least at fwst approximation it should fit in an inverted position. This result extends a precedent already seen for neutral amino acid transport systems (Ref. 5, p. 59). Even an a-thiocarboxylate will not serve in place of the a-carboxylate in the neutral systems. Note also for the system transporting &alanine that the sulfonate group of taurine is accepted in place of the pcarboxylate group (5, 20). We cannot exclude of course that failure to accept the /3-sulfonate substitution arises in part from an inherent steric feature of that group, rather than a purely chemical difficulty in its binding.
The apparent unsuitab~ity of homocysteate as a test substrate for the low I(, system for anionic amino acids in either cell type (Figs. 5 and 6 ) does not exclude the possibility that this homolog may prove effective as a model substrate for the high K,, glutamate-preferring system (Fig. 2). Cysteate and homocysteate each has proved to be a selective inhibitor for a system, one for aspartate and one for g l u~m a t e , respectively, in the hepatoma cell line, HTC5 ' M, Makowske and R. N. Christensen, unpublished results.

~t e~e o s e~e~t i~e A n o m a~~ in Amino Acid Transport 6059
The anomaly in the stereoselectivity of the L-system of the Ehrlich cell in the transport of the w-protonated forms, namely glutamic and aspartic acids at low pH (2), bears a provocative relation to that just discussed. (The L system apparently also transports aspartic acid into the fibroblast at decreased pH (Fig. 3, central panel).) In this case the two carboxyl groups of the substrate are no longer alike since one is protonated and the other is charged. Furthermore, we have a priori no reason for expecting a second carboxyl g r o u p -r e c o~~i n g structure to serve as a part of the transport acceptor site of System L. Therefore, we cannot logically insist that D-aSpartiC acid binds in an inverted position in that case, even after a secondary proton transfer on binding. Furthermore, the same anomaly is seen on comparing the stereoselectivity for glutamine and asparagine, for which an interchange of terminal side chain structures during transport is excluded. This behavior of glutamine and asparagine and of glutamic and aspartic acids appears to signal the presence of a receptor structure positioned in space so as to sense in a favorable or an unfavorable way the presence and configuration of a side chain group common to these substrates, perhaps the carbonyl group. Although we accept the idea that D-aspartate combines in an inverted way at the site for the anionic system, nevertheless we note as a caution that a different explanation is needed for the anomalous behavior of 0-asparagine with System L.
Where tested, Na' dependency has been a usual finding for membrane transport systems by which aspartate and glutamate move in the form of those anions and, in some cases as summarized elsewhere (Ref. 5, pp. 82-83), two sodium ions have been reported to migrate with each anionic molecule. Renal brush border membranes appear, however, to take up glutamate with only one sodium ion since the uptake process is electrically neutral (20). Here too, both D-and L-aspartate, but not n-glutamate, are taken up.
In both the hepatocyte (see Fig. 4 in Ref. 22) and the skin fibroblast ( Table TI), inhibition of anionic amino acid transport was shown by cysteine. Cysteine inhibition has also been observed for the glutamate transport system in the mouse lymphocyte (in Ref. 6, see fmal footnote). In the present work (data not shown), this inhibition rises with pH in a manner consistent with reaction by the anionic form, -SCH2CH-(NHa+)COO-, which appears to be present already at pH 7.3 to the extent of about 2% of the total cysteine present, and to increase in abundance, along with the presumably inert anion, HSCH&H(NH2)COO-, as the pH is raised (21). Considered, however, as to their making a con~bution to cysteine transport, the anionic systems may apparently be neglected except at unphysiologically high pH values (22). Whether cysteine levels rise high enough to give this amino acid a regulatory function for anionic amino acid movements may also be questioned.
Our experiments have not yet shown differences in glutamic acid transport of a kind we might expect from the great functional differences between the hepatocyte and a peripheral tissue in handling this amino acid.