ARCHIVES

OF BIOCHEMISTRY

Mechanism

AND

173,448-462

BIOPHYSICS

(1976)

of Glutamate-Aspartate Mitochondrial Inner

MARC E. TISCHLER,

JAMES

Translocation Membrane1

Across the

PACHENCE, JOHN R. WILLIAMSON,2 F. LA NOUE3

AND KATHRYN Johnson

Research

Foundation,

University

of P&nsylvaniu,

Philadelphia

Pennsylvania

19174

Received July 18, 1975 In order to study the mechanism of the glutamate-aspartate translocator, rat liver mitochondria were loaded with either glutamate or aspartate. In the presence of ascorbate plus tetramethyl-p-phenylenediamine as an electron donor at the third energy conservation site, exchange of external glutamate for matrix aspartate is highly favored over the reverse exchange. In the absence of an energy source, although the asymmetry of the exchange rates is much smaller, it is still observable. Further studies have shown that the proton uptake accompanying influx of glutamate in exchange for aspartate efflux occurs by protonation of a group on the carrier (pK = 7.9) at the external side of the inner mitochondrial membrane, followed by deprotonation at the matrix surface. It is postulated that glutamate binds to the protonated form of the carrier and aspartate to the deprotonated form. Because of the alkaline pK, aspartate efflux is inhibited with increased matrix [H+l due to limitation of the availability of deprotonated carrier for aspartate binding. For the reverse exchange, aspartate uptake is inhibited by increasing external [H+]. Thus the rate of aspartate uptake by mitochondria is apparently impeded both by a proton motive force (Ap) unfavorable to entry of ions with net negative charge ss well as by the small proportion of deprotonated carrier at the outer surface of the membrane. This conclusion is further illustrated by inhibition of the aspartate-aspartate exchange with increased [H+l and by addition of an energy source. The glutamateglutamate exchange, however, showed a slight stimulation by increased [H+l and was unaffected by the energy state. The model initially proposed for the carrier, in which a neutral glutamate-carrier complex exchanges for a negatively charged aspartate-carrier complex, is tested further. Glutamate uptake was noncompetitively inhibited by external aspartate, which indicates that aspartate and glutamate bind to separate forms of the carrier. Intramitochrondrial glutamate at a concentration of 18 mM, however, had no effect on aspartate efflux. Arrhenius plots for the glutamate-aspartate and aspartate-glutamate exchanges were linear over the range of temperatures tested (l-35% and 5-25”C, respectively) and provided an average value of 14.3 kcal/mol for the energy of activation. In addition, there appear to be two pools, exchangeable and nonexchangeable, of matrix aspartate available to the translocator, since extramitochondrial radiolabeled aspartate can equilibrate only with unlabeled matrix aspartate at low aspartate loading (l-2 nmol of aspartate/mg of protein). The physiological significance of the data is discussed. The exchange of extramitochondrial glutamate for intramitochondrial aspartate is 100% electrogenic due to the stoichi-

ometric uptake of protons during the process (1). The driving force for mitochondrial anion transport may be provided by a pro-

1 Supported by grants No. HE-14461, AM-15120, GM-22158, and AA-00292 from the U.S. Public Health Service and Grant No. AHA-72-739 from the American Heart Association. * To whom correspendence should be addressed.

3 William Daniel Stroud Established Investigator of the American Heart Association. Present address: Department of Physiology, Hershey Medical Center, Hershey, Pa 17033. 448

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

GLUTAMATE-ASPARTATE

ton pump as proposed in Mitchell’s chemiosmotic hypothesis (2). According to this theory, the oxidation-reduction reactions of the mitochondrial electron transport chain generates a proton motive force (Ap) created by the extrusion of protons from the mitochondria to the cytosol. The proton motive force comprises two components: an electrical potential gradient (A$), negative inside, and a pH gradient (ApH), alkaline inside, where Ap(mV) = A$ - (2.3 RTIF) ApH; A$ = $in - &,,t; and ApH = pH1, - pH,,,. Electrogenic glutamate-aspartate exchange is characterized by the net movement of electrons from the mitochondria to the cytosol and is driven by the proton motive force. The thermodynamics for the exchange, as described in the appendix following the paper, predicts that aspartate transport should be influenced by the A+ while glutamate should be affected by the ApH due to the accompanying movement of protons. Although the ApH should theoretically influence the exchange, the AI,!I, being the major component of the proton motive force (3), is predicted to provide the major driving force for the exchange. Support for these findings has been obtained by the demonstration of energy-dependent aspartate eMux in rat heart (4-8) and liver (4, 9) mitochondria as well as by data presented in this paper. Although under experimental conditions, in the absence of an energy source, the glutamate-aspartate exchange causes accumulation of protons in the matrix (l), the normal emux of protons during respiration will maintain the Ap. The glutamate-aspartate exchange is an integral part of the malate-aspartate cycle (lo), which facilitates the transport of reducing equivalents from the cytosolic to the mitochondrial compartment in both liver (11, 12) and heart (13). Existing data indicate, however, that the malate-aspartate cycle is not involved in the efflux of mitochondrial reducing equivalents to the cytosol in the intact cell because of the asymmetry of the glutamate-aspartate exchange (13-15). The latter process is energy requiring, and several special mechanisms have been proposed to provide a net transport of NADH from mitochondria to

ANTIPORTER

449

cytosol in liver (15-17). Bremer and Davis (14), on the other hand, have shown that under energized conditions the reconstituted malate-aspartate cycle is slowly reversible, but only after inhibiting mitochondrial NADH oxidation and using uncoupled mitochondria can a rapid reversal be achieved. Bremer and Davis concluded, therefore, in agreement with other workers, that a mechanism other than the malate-aspartate cycle is required to facilitate the efIlux of mitochondrial reducing equivalents. A model for the glutamate-aspartate antiporter has been proposed which presumes binding of glutamate or aspartate to a carrier protein in the inner mitochondrial membrane (1). Since the monovalent anions of glutamate and aspartate are the predominant species at neutral pH, it is reasonable to assume that both amino acids will bind to the carrier in this form. Previous data have suggested that the form of the carrier specific for glutamate is protonated so that the glutamate-carrier complex is therefore a neutral species, while the aspartate-carrier complex, being deprotonated, carries a single negative charge. The pH profile of the exchange as well as the binding of glutamate or aspartate to the carrier would then be governed by the pK group on the carrier undergoing protonation and deprotonation. In the present paper a further investigation of the exchange system has been undertaken. Studies on the effects of pH on the carrier have provided an apparent pK value for the carrier group undergoing protonation-deprotonation as well as a better understanding of the transport process. Another important aspect of the model is the reversibility of the exchange. Evidence presented in this paper together with that previously published suggests that under energized conditions the exchange is asymmetric (1, 5, 8, 14, 18). Thus, uptake of glutamate by the mitochondria in exchange for aspartate is greatly favored over the reverse process. Unlike the adenine nucleotide exchange (19), which is also electrogenic (20), the asymmetry of the glutamate-aspartate ex-

450

TISCHLER

change is not fully eliminated by collapse of the proton motive force by addition of uncoupling agents. MATERIALS

AND

METHODS

Preparation and incubation of mitochondriu. Rat liver mitochondria were prepared from fed, male Sprague-Dawley rats weighing 200-300 g (21), and isolation media containing mannitol (225 mM), sucrose (75 mM) and EDTA (0.1 mM) were used. Rotenone-inhibited, glutamate-loaded mitochondria were prepared as described previously (9). Transamination of externally added oxaloacetate with the accumulated glutamate then yields aspartateloaded mitochondria (9). Incubations were carried out at various temperatures and pH’s and in the buffer mixtures described in the figure legends. When measurements of matrix contents of anions were required, O.&ml samples of the incubation mixture were taken, and the mitochondria were rapidly centrifuged through silicone oil (22). Our modification of this technique is described fully in a recent publication (8). Microcentrifuge tubes for the separation were obtained from Fisher Scientific Co. and were fitted with a sleeve for use in the Eppendorf microcentrifuge. Assay procedures. Glutamate and aspartate were assayed fluorimetrically and spectrophotometrically by enzymatic technique (23). When aminooxyacetate was present in the samples, they were analyzed for aspartate content on a Beckman Model 121 automatic amino acid analyzer. Protein was determined by the biuret procedure (29). Measurements of proton movements associated with the transport of aspartate were performed in an expanded scale piI meter, made at the Johnson Research Foundation, connected to a Texas Instrument Company recorder. The medium used was lightly buffered (1 mM) to permit medium proton changes to be readily observed. This procedure is described in a recent publication (1). Oxygen was measured polarographically with a Clark electrode. Radioactivities were assayed with an Intertechnique liquid scintillation spectrometer using Handiflour, a liquid scintillation cocktail for aqueous samples obtained from Mallinckrodt Chemical Works, St. Louis, MO. The intramitochondrial pH was determined by measuring the distribution of [2-14C]DM04 across the inner mitochondrial membrane by the procedure of Addanki et al. (25) modified by the use of [YXsucrose and 4 Abbreviations used: DMO, 5,5’-dimethyloxazolidine-2,4-dione; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; PIPES, piperazineN,N’-bis(B-ethane sulfonic acid); MOPS, morpholinopropane sulfonic acid; TMPD, N,N,N,‘N’-tetramethyl-p-phenylenediamine; ATPase, adenosine triphosphatase.

ET AL. 3Hz0 to measure the extramitochondrial and total pellet water, respectively. Materials. m-Cycloserine was obtained from Regis Chemical Co., Chicago, Ill. The diethyl ester of butylmalonate was obtained from Fluka A.G., Buchs, Switzerland, and hydrolyzed to produce 2-nbutylmalonate, sodium salt. The uncoupler FCCP was generously supplied by Dr. Peter Heytler of the Research Division, E.I. DuPont de Nemours and CO., Wilmington, Del. All radioactive chemicals were purchased from New England Nuclear Corp. Other biochemical supplies such as PIPES, MOPS, dextran (molecular weight 40,000) and sucrose were purchased from Sigma Chemical Co. Enzymes were purchased from Boehringer Mannheim Corp. or Sigma Chemical Co. Aminooxyacetate and TMPD were obtained from Eastman Organic Chemicals. Mannitol for the mitochondrial preparations was purchased from Baker Chemical Co. because of its low calcium content. RESULTS

Asymmetry Antiporter

of the Glutamate-Aspartate

LaNoue and Williamson (5), Azzi et al. (26) and Brand and Chappell (27) have demonstrated that aspartate uptake readily occurs with uncoupled heart, liver and brain mitochondria, respectively. Contrary to the above data, Meijer et al. (18) obtained no ef&x of glutamate from uncoupled mitochondria preloaded with glutamate upon addition of aspartate. It should be noted, however, that in their experiment the intramitochondrial glutamate level of the uncoupled mitochondria was much lower than that of the control. To resolve the question of the reversibility of the exchange and to quantitate the asymmetry of the glutamate-aspartate antiporter, exchange rates were measured by using glutamate or aspartate-loaded mitochondria. The data of Fig. 1 illustrate, as expected (1, 4-8, 14), that the exchange of external glutamate for internal aspartate is stimulated by the addition of ascorbate plus TMPD, which presumably acts via the generation of a proton motive force across the membrane (2). The ‘uptake of added to glutamate[U-14Claspartate, loaded mitochondria, demonstrates that the reverse exchange is decreased by providing a source of energy (Fig. 11, in agreement with previous observations (14, 18). However, even with uncoupled mitochon-

GLUTAMATE-ASPARTATE

OF 30/ $0 sb-;io Ok%Gmj

120

Seconds 0, Inc”bollan

FIG. 1. Effect of energy state on the glutamateaspartate and aspartate-glutamate exchanges. Rat liver mitochondria (5.2 mg of protein/ml) loaded with glutamate as described under Materials and Methods were added at 28°C to an incubation mixture containing 130 mM KCl, 0.01 mM rotenone, 6% dextran, 25 &i of n-[l-3H]mannit.ol and 10 mM MOPS, pH 7.5. Oxaloacetate (3 mM) was also present for the glutamate-aspartate exchange incubations. At 45 s after the mitochondrial addition, 5 mM m-cycloserine was added to all incubations whereas ascorbate (5 mM) plus TMPD (0.2 mM) was only added when a mitochondrial energy source was required. Samples were taken after a 1-min incubation time for separation and were assayed for external glutamate (0.2 mM) and matrix aspartate (26 nmol/mg of protein) in the glutamate-aspartate incubation and matrix glutamate (20 nmol/mg of protein) in the aspartate-glutamate incubation. Immediately following this sampling, 10 mM glutamate was added for the glutamate-aspartate exchange and 10 mM (W-labeled; 10 &i) aspartate was added for the aspartate-glutamate exchange. Samples were then taken at 30, 60, and 120 s afterwards for separation through silicone oil. The glutamate-aspartate exchange was measured as nanomoles of aspartate per milligram of protein appearing in the medium, while the uptake of radiolabeled aspartate into the matrix, also expressed as nanomoles per milligram of protein, gave the results for the other exchange. See text footnote 4 for abbreviations.

dria, the glutamate-aspartate exchange was faster than the aspartate-glutamate exchange. Although the uptake of aspartate by mitochondria has been previously demonstrated (26-28), it must be noted that the mitochondria were uncoupled in these experiments. In order to illustrate the effect of proton motive force on the asparta@,,,s The subscripts “out” and “in” used here and throughout the paper refer to the substrate location, extra- and intramitochondrial, respectively.

ANTIPORTER

451

glutamatei, exchange,5 the experimental technique of Azzi et al. (26) was utilized. With uncoupled, rotenone-inhibited mitochondria, the oxidation of intramitochondrial NAD(P)H was followed by double beam spectrophotometry (340-373 nm). In Fig. 2, trace B shows results similar to those obtained previously by Azzi et al. (26). ARer reduction of intramitochondrial NAD(P) by addition of cy-ketoglutarate plus malonate, aspartate addition resulted in the oxidation of NAD(P)H, which was further stimulated by addition of glutamate. In trace C, it is seen that aminooxyacetate, an inhibitor of both the cytosolic and mitochondrial aspartate aminotransferases (29, 30), abolishes the NADH oxidation observed in trace B. However, DLa transaminase inhibitor cycloserine, which cannot penetrate the mitochondrial membrane (30, 31) is without significant effect (trace D). Thus aspartate enters the mitochondria and is converted by transamination to oxaloacetate, which in turn oxidizes NADH via malate dehydrogenase. The uptake of aspartate can be prevented (trace A), however, by absorption of the uncoupler FCCP onto added bovine serum albumin (4) followed by generation of the proton motive force by addition of ascorbate plus TMPD. Under these condition, the oxidation of NAD(P)H is not observable. The absence of any aspartate uptake after glutamate addition, however, is in apparent disagreement with the data presented in Fig. 1 where it is shown that a low rate of aspartate-glutamate exchange occurs in the presence of ascorbate plus TMPD. The discrepancy is probably due to the fact that the rate of aspartate uptake is so slow that the reduction of pyridine dinucleotides by the added substrates continues at a faster rate than their rate of oxidation, thereby preventing a net oxidation from being observable with the spectrophotometric technique. In further experiments the aspartateaspartate and glutamate-glutamate exchanges were studied in order to determine which transported anion was most affected by an energy source. Figure 3A depicts the results of [2,3-3Hlaspartate addition to mitochondria preloaded with as-

452

TISCHLER ET AL. ASZChJte Ammxyacetate

o-Ketoglutarate

D

t o-Keloqlutarate

FIG. 2. Mitochondria (2.8 mg of protein/ml) were added to a cuvette containing 130 mM KCl, 2.3 PM FCCP, 10 mM KH2POI, and 20 mM Tris-HCl, pH 7.4, at 28°C. After 2 min, 0.01 mre rotenone was added followed by 1 mM cy-ketoglutarate. The other additions as indicated were 1 mM malonate, 1 mM aspartate, 1 mM glutamate, 1% bovine serum albumin (BSA), 5 mM ascorbate + 0.2 mM TMPD (added as mitochondrial energy source), 5 mM nn-cycloserine, and 5 mre aminooxyacetate. The reduction and oxidation of mitochondrial pyridine nucleotides was followed by double-beam spectrophotometry at 340-373 nm. See text footnote 4 for abbreviations.

partate. Measurement of radiolabeled aspartate uptake shows an inhibition under energized conditions as with the aspartam-glutamate exchange (cf. Fig. 1). When mitochondria were preloaded with 12,3-3Hlaspartate and the efflux of radiolabeled aspartate measured, identical results were obtained (data not shown). The uptake of [3-3Hlglutamate in exchange for intramitochondrial glutamate (Fig. 3B), however, shows no energy effect. Thus, aspartate transport is influenced by the membrane potential, with aspartate efilux being inhibited and influx being stimulated by collapse of the potential. It may be noted from Fig. 3 that, prior to equilibration of either external radiolabeled glutamate or aspartate with the internal amino acid, the rate of exchange levels off. Further experiments dealing with the phenomena were performed on the aspartateaspartate exchange. The data in Fig. 4 show that, although at higher levels of aspartate loading (7.5-17.5 nmol/mg of protein) the exchange is incomplete, external radiolabeled aspartate will equilibrate with matrix aspartate at lower levels of loading (1.5 nmol/mg of protein) but only in the absence of an energy source. However, in the presence of an energy source,

the level of loading still affects the extent of exchange. Thus apparently two pools of amino acids are available to the translocator, one which can readily exchange with the extramitochondrial amino acid and the other which cannot. As a result, incomplete equilibration occurs at higher levels of loading due to a higher amino acid concentration in the nonexchangeable compartment. Further studies on this phenomena and the effect of energy state are presently in progress. The above data show that much of the asymmetry of the glutamate-aspartate translocator is brought about by the presence of the proton motive force across the membrane. Thus after 30 s of incubation with ascorbate plus TMPD the glutamateaspartate exchange rate was 40 times faster than the aspartate-glutamate exchange. On the other hand, in the absence of ascorbate plus TMPD the rate was only fourfold faster. An additional study shows that the glutamate-aspartate exchange is favored over the aspartate-aspartate (Fig. 5A) and glutamate-glutamate (Fig. 5B) exchanges by 7- and E-fold, respectively, in the absence of energy. It may be concluded, therefore, that despite the obvious influence of the proton motive force a cer-

GLUTAMATE-ASPARTATE

tain residual asymmetry is characteristic of the carrier itself. This asymmetry is also apparent in studies on the effect of temperature on the activity of the carrier in the absence of a source of energy. An Arrhenius plot of the data (Fig. 6) produces a straight line for the efflux or uptake of aspartate in exchange for glutamate over the temperature ranges indicated. Although the energies of activation for the exchanges are similar (15.1 and 13.4 kcabmol, respecAspartate-Asportate Exchange

Glutamate-Glutamate Exchange

Seconds Of lncubatlon

FIG. 3. Effect of energy state on the aspartateaspartate and glutamate-glutamate exchanges. (A), Mitochondria (7.2 mg of protein/ml) loaded with glutamate were added to the same incubation mixture at 28”C, as in Fig. 1, with 2 mM oxalacetate, 5 mM ascorbate + 0.2 mM TMPD (added as a mitochondrial energy source), and no D-[l-3H]mannitol. After a 60-s incubation, 5 mM nL-cycloserine plus 5 mM butylmalonate were added and 30 s later 19 $i of 2 mM [2,3-3Hlaspartate was added. Measurement of matrix aspartate prior to the addition at 90 s of aspartate, as well as in all subsequent samples, showed that it remained constant at 28 nmol/mg of protein with ascrobate + TMPD present and 18 nmol/mg of protein in the absence. (B), Mitochondria (8.3 mg of protein/ml) loaded with glutamate were added to the same initial medium as in (A) with no oxaloacetate present and with 10 mM PIPES (pH 6.8) substituted for the MOPS. After 60 s incubation 10 &i of 5 mM [3-3H]glutamate was added. The external glutamate concentration prior to this addition was 0.1 mM, and matrix glutamate, which remained constant to the end of the experiment, was measured to be 20 nmol/mg of protein. Samples to measure the exchange were taken for separation at the times indicated after addition of radiolabeled glutamate or aspartate, and the uptake of radiolabeled amino acid was measured. [“C]sucrose was added to permit sucrose space measurements after samples for the extent of exchange were taken. See text footnote 4 for abbreviations.

453

ANTIPORTER

tively), the characteristic asymmetry of the carrier is evident since the rate of glutamate,,,aspartatei, exchange is twice as fast as the reverse process. The energy of activation varies with substrate concentration (32); hence measurements should be made at saturating levels of substrate. Unfortunately this is not technically feasible with isolated mitochondria, and previous measurements of Eact on other translocators (32-38) have been determined in a manner similar to the present experiments. Specificity Carrier

of the

Glutamate-Aspartate

At present few amino acids have been shown to penetrate the mitochondria via carrier-mediated transport. In addition to the studies with glutamate and aspartate, saturation kinetics have been demonstrated for leucine (39), ornithine (401, and arginine (41). The possibility that other amino acids undergo exchange transport was investigated by measuring the efflux of aspartate. or glutamate from loaded mitochondria after the addition of various amino acids. Of those amino acids and carboxylic acids tested at 5 mM concentration with glutamate-loaded mitochondria, which included alanine, P-hydroxyaspartate, asparagine, arginine, cY-ketoglutarate, pyruvate, succinate, threonine, serine, valine, phenylalanine, leucine, isoleucine, glytine, acetylaspartate, acetylglutamate, acetylglutamine, cY-aminoadipate, proline, lysine, ornithine and citrulline (all amino acids were of the L-form), only L-alloy-hydroxyglutamate showed any appreciable exchange (3.5 nmol min-’ (mg of prot&n)-l) at 5°C. Based on Q1,,of about 2 for the carrier (see Fig. 6) the rate would be about 14 nmol min-’ (mg of protein)-’ at 25”C, which is similar to the rates obtained for the glutamate-glutamate exchange (see Figs. 3 and 5). y-Hydroxyglutamate also exchanges with matrix asparate as evidenced by the observation of proton uptake upon its addition to aspartate-loaded mitochondria. However, the exchange was slower than with glutamate since the rate of proton uptake for the glutamate control

TISCHLER

ET AL. II3 ‘Amrbole Matrix

semnds

(+TMPDl AwmJte

klnoles/mg

potem)

Smmds

4. Effect of matrix aspartate levels on the extent of the aspartate-aspartate exchange. Glutamate-loaded mitochondria (8 mg of protein/ml) were incubated in a buffer containing 120 mM KC1 and 15 mM MOPS, pH 7.2 at 25°C. To obtain three different levels of matrix aspartate loading, the above medium was divided into three equal portions to which was added 52, or 1 mM oxaloacetate to convert matrix glutamate to aspartate. Aminooxyacetate (5 mM) was then added 120, 40 or 5 s later, respectively, to yield high, intermediate, and low loading. The three portions of mitochondria were then washed and collected. The aspartate-loaded mitochondria (5.9-6.8 mg of protein/ml) were then incubated at 6°C in 120 mM KCl, 8% dextran, 0.01 mM rotenone, and 15 mpRMOPS, pH 7.5, in the absence (A) or presence (B) of ascorbate (5 mM1 plus TMPD (0.2 mM). Prior to the addition of 1.5 rnru (2,3-3H-labeled; 30 &i) aspartate, a 0.5-ml sample was removed for analysis of matrix aspartate. The aspartate loading is shown and remained constant throughout the incubation. Samples were taken at the times indicated after radiolabeled aspartate addition and exchange was measured by the uptake of 12,3W]aspartate. The percentage of aspartate loading exchanged equals nanomoles of aspartate exchanged per nanomole of matrix aspartate times 100. See text footnote 4 for abbreviations. FIG.

was about threefold greater.6 Of the other amino acids tested for exchange with aspartate, only alanine (5 mM) initiated aspartate efIlux from aspartate-loaded mitochondria. An exchange rate of 1.8 nmol min-’ (mg of protein)-l was obtained at 2o”C, while glutamate (5 mM) exchanged at a rate of 14.4 nmol min-’ (mg of protein)-’ under the same conditions. Since the mitochondrial alanine aminotransferase comprises only 8% of the total cellular homogenate activity (42), alanine may diffuse out of the mitochondria as a neutral amino acid at physiological pH instead of being metabolized following its uptake. Effect of pH on the Glutamate-Aspartate Antiporter Previous data obtained with heart (43) and liver (44, 45) mitochondria indicate that aspartate efflux is inhibited at acid pH (6.0-6.5). To quantitate this effect, the 6 Tischler, M. E., and La Noue, K. F. (1974) unpublished observations.

initial linear rate of aspartate efflux was measured at different pH values. Because interpretation of pH effects on the transport system is complicated by the pH gradient (alkaline inside) across the membrane generated by electron transport (251, both the external and internal pH were varied and measured. Measurements were made at three different internal pH values for each external pH. The ApH across the mitochondrial membrane was diminished by addition of either phosphate or nigeritin to the medium. Phosphate causes acidification of the matrix through the exchange of external phosphate for hydroxyl ions (46-48). Nigericin, a cyclic polypeptide with a free carboxyl group, binds K+ at physiological pH to form a neutral complex (49, 50). The uncharged complex, being permeable to lipid membranes, facilitates the exchange of protons between the medium and intramitochondrial K+. This results in a decreased pH gradient (51). In Fig. 7A the flux of aspartate is plotted

GLUTAMATE-ASPARTATE

versus external pH. Three different curves are obtained, each corresponding to a different set of internal pH values at the same external pH. However, when the same aspartate flux rates are plotted versus the internal pH (Fig. 7B), a single curve (solid line) is obtained. The plot correlates well with that derived from the Henderson-Hasselbach equation (dashed line) assuming a pK of 7.93 and a maximal flux of 71.2 nmol min-’ (mg of protein)-‘. Thus the data show that the glutamate,,taspartatei, exchange is inhibited at acid pH and that the pH effect occurs on the matrix side of the membrane. In addition, the similarity of the pH profile to the theoretical pH titration curve indicates that the pK of the carrier group undergoing protonation and deprotonation is about 7.9. The pH profile for the reverse exchange was also examined. In this instance the mitochondria were deenergized by addition of FCCP, an uncoupler which permits proton equilibration across the membrane

455

ANTIPORTER

and thus a collapse of the proton chemical potential (2, 52). The data in Fig. 8 demonstrate that the effect of pH on aspartate uptake (closed circles) is identical to the effect of matrix pH on aspartate efYlux (open circles). These results suggest that the pH effect is not restricted to a particular side of the membrane but instead is caused by a limited availability of deprotonated carrier for aspartate binding. This notion is supported by studies on the effect of external pH on the aspartate-aspartarte and glutamate-glutamate exchanges with uncoupled mitochondria. Whereas acid pH inhibits the former exchange (Fig. 9A), the glutamate-glutamate exchange is stimulated slightly with decreasing pH (Fig. 9B). Substrate Inhibition partate Carrier

of the Glutamate-As-

The reversibility of glutamate-aspartate exchange in uncoupled mitochondria indicates that both substrates can bind to the carrier on either side of the membrane.

0

30

60

FIG. 5. Comparison of the glutamate-aspartate exchange with the aspartate-aspartate (A) and glutamate-glutamate (B) exchanges. Mitochondria (5.6 mg of protein/ml) loaded with glutamate, as described under Materials and Methods, were added to the initial incubation mixture used in Fig. 1. Oxaloacetate (1 mM) for aspartate loading was added to those incubations in which the glutamate-aspartatc and aspartate-aspartate exchanges were measured. After 30 a, m-cycloserine (5 mM) was added to all incubations and 30 s later a sample was taken for separation to assay the extent of the initial aspartate or glutamate loading. In (A), the aspartate loading was 20 nmol/mg of protein for both exchanges, with the level remaining constant throughout the measurement of the aspartate-aspartate exchange. To initiate the exchange measurements 10 mM glutamate was added at 60 s for the glutamate-aspartate exchange and 10 mM (“C-labeled, 10 &i) aspartate was added for the aspartate-aspartate exchange. The external substrate concentrations prior to these additions were 0.2 and 0.3 mM for glutamate and aspartate, respectively. In (B), the aspartate loading was 34 nmobmg of protein for the glutamate-aspartate exchange, while the glutamate loading for the glutamateglutamate exchange was 36 nmol/mg of protein, the latter remaining constant to the end of the incubation. To initiate the exchanges 2.5 mM [W-labeled, 2.6 &i (for the glutamate-glutamate exchange)] glutamate was added at 60 a. In (A) and (B), samples were taken at the times indicated after glutamate or aspartate addition. The extent of the glutamate-aspartate exchange was determined from the appearance of aspartate in the medium while the other exchanges were determined from the uptake of radiolabeled glutamate or aspartate.

456 4.0 ^ k 5 e

32

n .

TISCHLE :R ET AL. Exchange Glutamate -Aspartate EA= 15.1 KC&L. Mole-’ H

Asportate-Glutamate E,= 134 KCAL Mole-l

External PH

I/T x 10s OK-’

6. Influence of temperature on the glutamate-aepartate and aspartate-glutamate exchanges. To initiate aspartate loading (7-12 nmoll mg of protein) for the glutamate-aspartate exchange, oxaloacetate (1 rn@ was added to an incubation medium containing 130 mM KCl, 0.01 mM rotenone, 8% dextran, 1 &i of [W]sucrose, glutamate-loaded mitochondria (6.1 mg of protein/ml), 8 PM FCCP and 10 mM PIPES, pH 7.2, at various temperatures. After 60 s, aminooxyacetate (5 mM) was added, followed 30 s later by 4 mM glutamate to initiate the exchange. The reverse exchange was examined with glutamate-loaded (30-33 nmol/mg of protein) mitochondria (8.6 mg of protein/ml) in the above medium at pH 7.5 and various temperatures. Aspartate (5 mr& was added to initiate the exchange. The medium appearance and matrix disappearance of glutamate or aspartate were measured in samples taken at varied times. The data were plotted versus time and the rates of exchange Fed in the Arrhenius plots were determined from the average linear rates plotted at each temperature. See text footnote 4 for abbreviations. FIG.

Thus, it was of interest to test the inhibitory effects of the substrates on the glutamate-aspartate exchange. The inhibitory effect of aspartate was studied by measuring the initial linear rate of proton uptake (Fig. 10A) following the addition of glutamate to aspartate-loaded rat liver mitochondria. The results, depicted as either a Michaelis-Menten plot (Fig. 10B) or a double-reciprocal plot (Fig. lOC>, suggest that the inhibition is noncompetitive (Ki = 4 mn&. This is further evidenced by a Dixon plot (Fig. 10D) of the data. The inset, in which the slope of the line at each glutamate concentration is replotted against the reciprocal of the glutamate concentra-

lnternol pli

FIG. 7. Effect of medium and matrix pH on the glutamate-aspartate exchange. The buffer used in the control (O-O) and nigericin (A-A) incubations consisted of 100 mM KCl, 10 mM PIPES, 10 mM Tris-HCl, 6% dextran, 20 mM sucrose, 1 mM oxaloacetate, 5 mM m-cycloserine, 5 mM ascorbate, 0.2 mM TMPD, 0.01 mM rotenone, 1 &i of [2W]DMO and, where added, 20.6 ng of nigericin/mg mitochondrial protein. Where phosphate (10 rnM) was used (O-O), only the 20 mM sucrose was omitted. Glutamate was added 2 min after the incubation was begun with mitochondria (4.9 mg of protein/ml), and samples were taken for mitochondrial separation. Flux of aspartate was determined from the linear rate of aspartate appearance in the medium. Mitochondrial spaces were determined in parallel runs. The dashed line in (B) is explained in the text. The temperature was 28°C. See text footnote 4 for abbreviations.

lntema, B-4 Or External And Internal pH k-1

8. Comparison of pH effects on the aspartate-glutamate and glutamate-aspartate exchanges. The data for the glutamate-aspartate exchange (O-O) are taken from Fig. 6B. The reverse exhange was studied with glutamate-loaded (15-25 nmol/mg of protein) mitochondria (4.5 mg of protein/ml) in a medium containing 115 mM KCl, 8% dextran, 0.01 mM rotenone 5 mM m-cycloserine, 9 PM FCCP, 3 PCi of [“Clsucrose, 15 mM Tris-HCI and 15 rn~ MOPS at various pH values and a ternperattire of 10°C. After a 90-s incubation, 5.2 mM aspar@te was added followed by removal of samples for mitochondrial separation. Glutamate efflux is the average value obtained from the linear medium appearance and matrix disappearance of glutamate. See text footnote 4 for abbreviations. FIG.

GLUTAMATE-ASPARTATE Asaartate-Aspartate Exchange

Glutamate-Glutamate Exchange pH 70 pH 75 pH 6.0 pH 6.5

24

46 0 24 Seconds Of lncubatm

40

FIG. 9. Influence of pH on the aspartate-aspartate and glutamate-glutamate exchanges. The aspartate-aspartate exchange in (A) was studied at 6°C with aspartate-loaded (8.5-9.5 nmol/mg of protein) mitochondria (7.6 mg of protein/ml) in the medium used for Fig. 7 except for no [Wlsucrose and 8 PM FCCP. nn-Cycloserine (5 mM) and butylmalonate (5 mM) were added 3 min into the incubation, aspartate (2,3-3H-labeled; 2 mM; 3 &i) was added 30 s later, and the uptake of radolabeled aspartate was determined at the times shown after aspartate addition. The glutamate-glutamate exchange was measured similarly in the same time intervals at the indicated pII’s. Glutamate-loaded (13-15 nmol/mg of protein) mitochondria (4.2 mg of protein/ml) were incubated as above at 28°C except that FCCP was 5 PM. After 60 s, 2 mM (3-3H-labeled; 6.6 &i) glutamate was added. In both (A) and (B), Wlsucrose was added 60 s after aspartate or glutamate for sucrose space determination.

tion, indicates that the inhibition is noncompetitive (53, 54). Kinetic data obtained for rat heart mitochondria by studying glutamate-dependent respiration (55) yielded an aspartate Ki = 3.5 f 0.8 mM and a glutamate K, = 6.4 mM. The inhibition was also noncompetitive. Interestingly a comparison of the kinetic data for heart and liver mitochondria indicates that the glutamate-aspartate carrier is similar in the two tissues. This is implied by calculation of the ratio of external glutamate K,:external aspartate Ki which is the same for liver (K,Xi = 1.77) and heart (K,KK, = 1.88) mitochondria. The effect of internal glutamate on aspartate transport was studied by measuring the increase of aspartate in the medium as a result of glutamate-aspartate exchange at a low (5.8 mM) and a high (17.7 mM) concentration of intramitochondrial glutamate. Intramitochondrial glu-

ANTIPORTER

tamate was varied by allowing matrix glutamate in the control mitochondria to be oxidized to NH, and cu-ketoglutarate (1) by a prolonged (11-min) preincubation in the presence of oxalacetate and aminooxyacetate. The results, plotted as the double reciprocal of aspartate eMux against matrix aspartate concentration (Fig. ll), show that a threefold increase of intramitochondrial glutamate had no effect on the rate of aspartate eMux. Thus glutamate binding to the carrier on the matrix side of the membrane appears to be so weak that no inhibition can be observed over the

Gltimwte

fl; -8 -4 0 4 8 12 I rmw x 102 GlUtomofe

(mM)

-4 0 4 8 Av.ntote

12 CM)

10. External aspartate inhibition of glutamate uptake via the glutamate-aspartate exchange in rat liver mitochondria. Mitochondria (5.2 mg of protein/ml) loaded with glutamate were added to a lightly buffered medium at 28°C containing 130 mM KCl, 0.01 mM rotenone, 5 mM m-cycloserine, varied aspartate concentrations and 1 mM MOPS, pH 7.2, for the measurement of proton uptake as described under Materials and Methods. Oxaloacetate (3 mM) was added to permit aspartate loading, and different concentrations of glutamate were added after 90 s. The data, presented as a Michaelis-Menten (B), a Lineweaver-Burk (C), and a Dixon (D) plot, were taken from the initial linear rate of proton uptake corrected for drift of the pH. A typical proton uptake measurement is shown in (A) for 16 mM glutamate in the presence of 3.9 mM aspartate. The inset in(D) is a replot of the slope of each line (in D) versus the reciprocal of the glutamate concentration for that line. See text footnote 4 for abbreviations used. FIG.

458

TISCHLER

0’

002

004

MATRIX

006

0.08

ASPARTATE

0.1

0 12

0.14

CONCENTRATION

0.16

018

(mM)-’

FIG. 11. Effect of matrix glutamate on the eSlux of aspartate. Glutamate-loaded mitochondria (5.8 mg of protein/ml) were incubated in a buffer containing 150 mM KCl, sH20 (1 &i), 6% dextran and 20 mM MOPS (pH 7.2) at 10°C. The experiment was begun with the addition of oxaloacetate (1 mM). After 1.5 min aminooxyacetate acid (5 rnM1 was added. In two experiments (control) more oxaloacetate (4 mM) was added at 3 min followed by ascorbate (5 mM) plus TMPD (0.2 mM) at 14 min. At 14.5 min [Wlsucrose was added, followed 0.5 min later by 2.5 mbf glutamate to initiate aspartate efflux. Samples were taken for rapid separation by centrifugation immediately prior to glutamate addition and at 15, 20,60 and 90 s afterwards. In two other experiments ascorbate plus TMPD were added at 1.6 min after initiation of the incubation and [Wlsucrose at 1.8 min. External glutamate (2.5 mnr) was added at 2 min to initiate the efllux of aspartate. Samples were taken as in the control. Aspartate levels in the supematant solution were measured with an amino acid analyzer and those in the mitochondrial fraction were measured enzymatically. Fluxes shown are the rates of aspartate appearance in the medium at 0, 30 and 60 s after glutamate addition. The aspartate concentrations shown are the amounts in the matrix divided by the matrix volume. See text footnote 4 for abbreviations.

range of intramitochondrial concentrations used.

glutamate

DISCUSSION

Previous studies have shown that the exchange of extramitochondrial glutamate for matrix aspartate on the glutamateaspartate carrier is accompanied by the stoichiometric uptake of protons (1). The exchange is electrically asymmetric and results in the accumulation of protons within the matrix in the absence of an energy source (11, though under physiological conditions the outwardly directed proton pump (2) will balance the movement of

ET AL. protons. Data presented in this paper show that transport is stimulated at alkaline pH’s (Fig. 7) despite the fact that proton uptake accompanies the exchange. Measurement of the exchange of external glutamate for internal aspartate, under conditions where the medium and matrix pH were varied independently, showed that flux rates were a function of the matrix rather than the medium pH (Fig. 7). From these data the conclusion can be drawn that aspartate binding requires deprotonation of the acid-base group on the matrix side of the membrane. It seems reasonable to assume that the same group binds the proton when external glutamate is bound to the carrier and transports the proton across the membrane for release into the matrix prior to aspartate binding. Data obtained on the pH dependence of the other exchanges catalyzed by the aspartate carrier tend to support this conclusion. Thus the reverse exchange of internal glutamate for external aspartate has a very similar pH profile, with the pH-sensitive site located on the outer surface of the membrane (Fig. 8) where aspartate binds to available deprotonated carrier. The aspartate-aspartate exchange is also stimulated at alkaline pH’s (Fig. 9A), whereas the glutamate-glutamate exchange is slightly stimulated by lowering the pH (Fig. 9B). The fact that the glutamate-glutamate exchange has a very different pH profile from the exchanges which involve aspartale, indicates that glutamate anions only exchange on the protonated carrier. The pK value obtained from the profile of the flux rates is 7.9 (Figs. 7, 81, a value which is quite different from any of the glutamate pK’s and implies that negatively charged glutamate molecules are binding to a protonated carrier rather than neutral molecules to a deprotonated carrier. As predicted from the thermodynamics for the exchange of glutamate for aspartam (see Appendix following the paper), the glutamate-glutamate exchange is not influenced by the membrane potential (Fig. 3B). On the other hand, the thermodynamics do predict that glutamate (plus a proton) flux should be influenced by the

GLUTAMATE-ASPARTATE

ApH. Physiologically, however, the ApH probably has little effect, because of its low value under both State 3 and State 4 conditions (3) and because of the alkaline pK which confers a large matrix-pH sensitivity on the exchange. The insensitivity of the glutamate-glutamate exchange to the influence of the membrane potential (Fig. 3B) indicates some interesting characteristics of the carrier. The data imply that glutamate and protons move in a concerted manner through the electrical potential gradient of the membrane. These results seems incompatible with the fixed, gated pore model proposed by Singer (561, since binding of negatively charged glutamate to a site located within a channel or pore of a membrane, across which an electrical potential gradient has been generated, should be very sensitive to the potential gradient. The data show that aspartate on the outer surface of the membrane inhibits glutamate entry noncompetitively (Fig. 10). This type of inhibition occurs (54) when substrate (glutamate) and inhibitor (aspartate) interact with enzyme (carrier) forms separated by reversible steps. Thus aspartate binds to a form of the carrier different from that to which glutamate binds. The ability of the carrier to bind either glutamate or aspartate specificially probably results from a conformational change of a single binding site induced by protonation or deprotonation of the carrier. Aspartate may inhibit by binding to available deprotonated carrier, causing a shift of the protonation equilibrium towards deprotonation and resulting in a decreased availability of protonated carrier for glutamate binding. Although a threefold increase of internal glutamate had no effect on aspartate efflux (Fig. ll), it must be considered that glutamate may still be a noncompetitive inhibitor of aspartate efflux. If the affinity of glutamate for the protonated carrier is much lower than that of aspartate for the deprotonated carrier, no inhibition might be observable under the conditions obtained in the experiment. That glutamate binding is weaker than aspartate binding is suggested by the glu-

ANTIPORTER

459

tamate K,:aspartate Ki ratio of about 2 obtained for liver (cf. Fig. 10) and heart (55) mitochondria. A difference in the binding of the substrates to their respective carrier forms could have important physiological significance. Since matrix glutamate appears to have little effect on aspartate eMux, control of the glutamate-aspartate exchange, in this manner, would seem to be of little consequence. On the other hand, variations of extramitochondrial aspartate could play an important regulatory role. Thus high concentrations of cytosolic aspartate could inhibit the carrier and decrease flux of aspartate out of the mitochondria, whereas.10~ cytosolic aspartate levels would permit more rapid aspartate efflux. If the binding of glutamate to the carrier is indeed poor, as suggested above, then kinetic regulation of aspartate efYlux could also be obtained through large changes of the cytosolic glutamate concentration. A model for glutamate-aspartate exchange can be envisioned whereby in the presence of a large protonmotive force the exchange would be essentially irreversible. External glutamate would bind to the protonated translocator at a binding site exposed to the cytosolic surface of the inner membrane, forming a neutral complex. Glutamate as a neutral species could then be readily transported against the A$, negative inside, and with the ApH alkaline inside. The proton binding site upon being exposed to the more alkaline matrix pH would be deprotonated, thus facilitating the dissociation of glutamate and the subsequent binding of aspartate at the inner surface. The transport of aspartate, as a monovalent anionic species, could then be driven by the AI/J. At the other surface of the inner membrane, the more acidic pH would favor protonation and thus dissociation of aspartate from the translocator. Although deprotonated carrier is always available for external aspartate binding, the charged aspartate species cannot readily be transported against the membrane potential and irreversibility of the exchange results. A phenomenon which has only recently

460

TISCHLER

been observed will complicate interpretation of further kinetic data. Ongoing kinetic experiments show that different results are obtained in the presence and absence of aminooxyacetic acid, an inhibitor of the mitochondrial aspartate aminotransferase (28, 29). Since the cytosolic enzyme, which is present in the mitochondrial preparations, is inhibited in all experiments with either aminooxyacetic acid or DIrcycloserine, the data suggest that possible interaction exists between the carrier and the mitochondrial enzyme. Interestingly, Klingenberg (33) initially proposed that a membrane-bound aspartate aminotransferase was exclusively involved in aspartate transport, although the antiporter characteristics exclude this possibility. Another carrier-enzyme interaction has also been reported by Vignais et al. (57) between the adenine nucleotide carrier and the mitochondrial F,-ATPase, thus lending credence to our observation. Further investigation into the significance of this interaction, the kinetics of the carrier, and the compartment&ion of the aspartate and glutamate in the mitochondria are in progress. REFERENCES 1. LA NOUE, K. F., AND TISCHLER, M. E. (1974) J. Biol Chem. 249, 7522-7526. 2. MITCHELL, P. (1966) in Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Research Report No. 66/l, May 1966, Glynn Research Ltd., Bodmin, Cornwall. 3. MITCHELL, P., AND MOYLE, J. (1968)Eur. J. Biothem. 4, 530-539. 4. LA NOUE, K. F., BRYLA, J., AND BASSETT, D. J. B. (1974) J. Biol. Chem. 249, 7514-7521. 5. LA NOUE, K. F., AND WILLIAMSON, J. R. (1971) Metabolism 20, 119-140. 6. LA NOUE, K. F., AND HEMINGTON, J. G. (1973) Fed. Proc. 32, 557. 7. LA Nom, K. F., AND BRYLA, J. (1971) Fed. PFOC. 30, 1238. 8. LA NOUE, K. F., WA~AJTYS, E. I., AND WILLIAMBON, J. R. (1973) J. Biol. Chem. 248, 7171-7183. 9. LA NOUE, K. F., MEIJER, A. J., AND BROUWER, A. (1974) Arch. Biochem. Biophys. 161, 544550. 10. BOR~T, P. (1963) in Functionelle und Morphologische Organisation der Zelle (Karlson, P., ed.), pp. 137-158, Springer-Verlag, Berlin. 11. WILLIAMSON, J. R., JAKOB, A., AND REFINO, C. (1971) J. Biol. Chem. 246, 7632-7641.

ET

AL.

12. BERRY, M. M., KUN, E., AND WERNER, H. V. (1973) Eur. J. Biochem. 33, 407-417. 13. SAFER, B., AND WILLIAMSON, J. R. (1973) J. Biol. Chem. 248, 2570-2579. 14. BREMER, J., AND DAVIS, E. J. (1975) Biochim. Biophys. Acta 376, 387-397. 15. BERRY, M. M. (1971) Biochem. Biophys. Res. Commun. 44, 1449-1456. 16. MEIJER, A. J., AND WILLIAMSON, J. R. (1974) Biochem. Biophys. Res. Commun. 29, 146-152. 17. ROGNSTAD, R., AND KATZ, J. (1972) J. Biol. Chem. 247, 6047-6054. 18. MEIJER, A. J., BROUWER, A., REIJNWUD, D. J., HOEK, J. B., AND TAGER, J. M. (1972) Biochim. Biophys. Actu 283, 421-429. 19. KLINGENBERG, M., AND PFAFF, E. (1965) in Regulation of Metabolic Processes in Mitochondria (Tager, J. M., Papa, S., Quagliariello, E., and Slater, E. C., eds.), pp. 180-201, Elsevier, Amsterdam. M., WULF, R., HELDT, H. W., AND 20. KLINGENBERG, PFAFF, E. (1969) in Mitochondrial Structure and Function: Vol. 17, Proc. FEBS (Ernster, L., and Drahota, Z., eds.), pp. 59-77, Academic Press, New York. 21. SCHNEIDER, W. C., AND HOGEBOOM, G. H. (1950) J. Biol. Chem. 183, 123-128. 22. HARRIS, E. J., AND VAN DAM, K. (1968) Biochem. J. 106, 759-766. 23. WILLIAMSON, J. R., AND CORKEY, B. E. (1969) Methods Enzymol. 13, 434-513. 24. LAYNE, E. (1957) Methods Eznymol. 3, 447-454. 25. ADDANKI, S., CAHILL, F. D., AND SOTOS, J. F. (1968) Anal. Biochem. 25, 17-29. 26. Azzr, A,, CHAPPELL, J. B., AND ROBINSON, B. H. (1967) Biochem. Biophys. Res. Commun. 29, 148-152. 27. BRAND, M. D., AND CHAPPELL, J. B. (1974) Biothem. J. 140, 205-210. 28. SOLING, H.-D., AND SECK, A. (1975) FEBS Lett. 51, 52-59. 29. HOPPER, S., AND SEGAL, H. L. (1962) J. Biol. Chem. 237, 3189-3195. 30. WILLIAMSON, J. R., MEIJER, A. J., AND OHKAWA, K. (1974) in Regulation of Hepatic Metabolism, Alfred Benxon Symposium VI (Lundquist, F., Tygstrup, N., and Thaysen, J. H., eds.), pp. 457-479, Munksgaard, Copenhagen. 31. AZARKH, R. M., BRAUNBHTEIN, A. E., PASKHINA, T. S., AND T’ING-SEN, H. (1960) Biochemistry (USSR) 25, 741-748. 32. BRAY, H. G., AND WHITE, K. (1966) Kinetics and Thermodynamics in Biochemistry, p. 318, Academic Press, New York. 33. BRADFORD, N. M., AND MCGIVAN, J. D. (1973). Biochem. J. 134, 1023-1029. 34. KLINGENBERG, M. (1970) Essays Biochem. 6, llS159. 35. MEYER, J., AND VIGNAIS, P. M. (1973) Biochim.

GLUTAMATE-ASPARTATE Biophys. Acta 325, 375-384. 36. PALMIERI, F., QIJAGLIARIELU), E., AND KLINGENBERG, M. (1972) Eur. J. Biochem. 29, 408416. 37. PALMIERI, F., STIPANI, I., QUAGLIARIELLO, E., AND KLINGENBERG, M. (1972)Eur. J. Biochem. 26, 587-594. 38. QUAGLIARIELLO, E., PALMIERI, F., PREZIOSO, G., AND KLINGENBERG, M. (1969) FEBS Lett. 4, 251-254. 39. BUCHANAN, J., POPOVITCH, J. R., AND TAPLEY, D. F. (1969) Biochim. Biophys. Acta 173, 532539. 40. GAMBLE, J. G., AND LEHNINGER, A. L. (1973) J. Biol. Chem. 248, 610-618. 41. KELLER, D. M. (1968) Biochim. Biophys. Acta 153, 113-123. 42. SWICK, R. W., BARNSTEIN, P. L., ANDSTANGE, J. L. (1965) J. Biol. Chem. 240, 3334-3340. 43. WILLIAMSON, J. R., SAFER, B., LA NOUE, K. F., SMITH, C. M., AND WA~AJTYS, E. (1973) in Rate Control of Biological Processes, Symp. 27, Sot. Exp. Biol. (Davies, D. D., ed.), pp. 241-281, Cambridge University Press, Cambridge. 44. HEMINGTON, J. G. (1973) Fed. Proc. 32, 557. 45. LA NOUE, K. F., AND ZIMMERMAN, U.-J. P. (1974) Fed. Proc. 33, 1360.

461

ANTIPOR’I’ER

46. HOEK, J. B., LOFRUMENTO, N. E., MEIJER, A. J., AND TAGER, J. M. (1971) B&him. Biophys. Acta 226, 297-308. 47. MCGIVAN, J. D., AND KLINGENLIERG, M. (1971) Eur. J. Biochem. 20, 392-399. 48. PAPA, S., LOFRUMENTO, N. E., LOGLIBCI, M., AND QUAGLIARIELLO, E. (1969) B&him. Biophys. Acta 189, 311-314. 49. STEINRAUF, L. K., AND PINKERTON, M. (1968) Biochem. Biophys. Res. Commun. 33,29-31. 50. PRESSMAN, B. C. (1968) Fed. Proc. 27,1283-1288. 51. COCKRELL, R. S., HARRIS, E. J., AND PRE((SMAN, B. C. (1967) Nature (London) 215, 1487-1488. 52. MITCHELL, P., AND MOYLE, J. (1965) Nature (London) 208, 147-151. 53. CLELAND, W. W. (1967)Annu. Rev. Biochem. 36, 77-112. 54. CLELAND, W. W. (1963) Biochim. Biophys. Acta 67, 188-196. 55. LA NOUE, K. F., AND TISCHLER, M. E. (1976) in Regulation of Metabolism in Isolated Liver Cells (Tager, J. M., Siiling, H.-D., and Williamson, J. R., eds.), in press, North-Holland, Amsterdam. 56. SINGER, S. J. (1974) Annu. Rev. Biochem. 43, 805-833. 57. VIGNAIS, P. V., VIGNAIS, P. M., AND DOUSSIERE, J. (1975) Biochim. Biophys. Acta 376, 219-230.

APPENDIX The Driving

Biochemistry

Force for Glutamate-Aspartate HAGAI ROTTENBERG Department, Tel Aviv University,

The driving force for carrier-mediated anion exchange can be evaluated by taking into consideration the thermodynamic driving forces of the two species which are transported by the carrier. Net transport by an exchange carrier should vanish when the difference of the electrochemical potential gradient for the transported species is zero. Thus the carrier-mediated exchange transport would reach equilibrium when A,& - A& = 0. PI It is assumed here that transport by the exchange carrier is completely coupled; i.e., net transport of one species in one direction must always be coupled to an equivalent transport of the second species in the opposite direction. A more general analysis of the energetics of carrier ex-

Exchange

Tel-Aviv,

Israel

changes is given in a recent review by Rottenberg (1). Several types of exchanges, both electrogenie and electroneutral, have been previously described by Klingenberg (2). Although the exchange of ADPHi-, against ATPHi::, as discussed by Klingenberg, is electrogenic, it does not result in net proton transport. Thus the electrogenic exchange of an anion with an undissociated acid, which results in net proton transport, was not discussed. For the glutamate-aspartate exchange we assume (3) that the monovalent aspartate anion (asp-) exchanges against a neutral glutamate molecule (glu H). Thus at equilibrium AL-

= ApQIUH,

or expressed more explicitly,

PI

462

TISCHLER

RT In ([asp-]&asp-I,,,]

- FAQ =

RT ln (klu Hldklu

HIout),

[31

where A$ = &, - I,&. Since at physiological pH most of the glutamate molecules exist in the monovalent anion form and recalling that

[glu Ml = KJdu-1 [ H+l,

[41 Eq. [3] can be rewritten in terms of the monovalent glutamate anion concentration.

RT In ([asp-ld[aw-lout) - FA* = RT In ([glu-]iJH+]J

PI

klu-lout[H+lout). Rearranging

Eq. 151 yields the result

RT In ([asp-li,[glu-l,ut/[asp-l,,, [glu-Ii,

= FAQ - 2.3 RTApH,

WI

where ApH = pHi, - PI&,,,, and Fhrj, - 2.3 RTApH = A fin. Equation [6] indicates that the driving force for the glutamate-aspartate exchange is provided by the asp- and gluconcentration gradients, as well as the electrochemical potential difference of the protons, A &, (or in Mitchell terminology

ET

AL.

(41, the proton motive force, where Ap= -A&IF). Thus even when the ion concentration gradient term approaches zero, the existence of Ap can still drive the exchange. In energized mitochondria A ,kH is quite high and negative (5) and therefore asp- may be driven out against its own concentration gradient while neutral glutamate (glu H) should be driven in. Hence the exchange process is observed to be asymmetric (cf. Figs. 1-4). Exactly the same result (Equation [Sl) is obtained if we consider the molecular mechanism of the exchange to be asp- against glu- plus Hf. This is expected, since thermodynamics deals with net macroscopic processes, and the transport of glu- plus H+ is thermodynamically indistinguishable from the transport of neutral glutamate (glu H). REFERENCES 1. ROTTENBERG, H. (1976) in International Review of Cytology (Danielli, J. F., ed.), in press. 2. KLINGENLIERG, M. (1970) Essays Biochem. 6,119159. 3. LA NOUE, K. F., AND TISCHLER, M. E. (1974) J. Bid. Chem. 249, 7522-7526. 4. MITCHELL, P., AND MOYLE, J. (1969) Eur. J. Biothem. 7, 471-484. 5. ROTTENBERG, H. (1975) J. Bioenerg., 7, 63-76 (1975).