Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.

When Na+ was equilibrated across the membrane, countertransport stimulation of L-["Hlalanine uptake occurred in the presence of accumulated unlabeled L-alanine, Z-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent K,,, valuls for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process.
Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ > internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent K,,, for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interiornegative membrane potential, such as the presence of the permeant anions NOs-and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal > K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.
* This investigation was conducted during tenure of Grant 5F32CA05174.02 &om the United States National Cancer Institute, National Institutes of Health. This is the second paper in a series on transport mechanisms in mammalian cells. Ref. -16 is the preceding paper in this series.

Investigations
of energy transduction and active transport across such diverse biological membranes as mitochondrial, chloroplast, bacterial, intestinal, and tumor cell membranes have focused on the role of electrostatic and chemical concentration gradients (l-5). For example, Riggs et al. (1) and Crane (2) have proposed a "Na+ gradient hypothesis" in which Na+ gradients generated by active Na+ extrusion via the (Na+ + K+)-ATPase system are utilized to drive Na+-dependent active transport in certain cases (6)(7)(8). Similarly, respirationcoupled transport in Escherichia coli has been interpreted (4,5,9) in terms of Mitchell's chemiosmotic hypothesis (3) which proposes that proton gradients generated by proton extrusion via the respiratory chain can be utilized to drive active transport of a variety of solutes. In these conceptually parallel hypotheses, active transport is coupled to metabolism by means of an electrochemical ion potential across the membrane, the sum of two thermodynamic components, an electrical term which is the membrane potential and a chemical component.
The  (17) with 2 liters of Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Cell lines were checked frequently for absence of mycoplasma contamination.
Membrane Vesicle Preparation -Cells (5 to 9 x 10y cells from 5 x 9,500-cm" culture vessels) were washed twice with phosphatebuffered saline (50 mM potassium phosphate (pH 7.51, 5 rnM KCl, and 0.15 M NaCl) at room temperature, harvested by scraping with a rubber blade, and collected in 1 liter of phosphate-buffered saline. The cell suspension was centrifuged at 4,000 x g for 10 min and then resuspended in 500 ml of phosphate-buffered saline, filtered through cheesecloth, and centrifuged again at 4,000 x g for 10 min. Subsequent steps were carried out at 2-4". The cell pellet was washed in 500 ml of 0.25 M sucrose, 0.01 M Tris/HCl (pH 7.51, and 0.2 mM MgCl,. total cellular protein. Preparations from Balb/c and-Swiss mouse fibroblasts differed in their iso-Abu transport specific activity (16).
For the preparation of purified plasma membrane vesicles the method of Quinlan and Hochstadt (18) was used, starting from 5 to 9 x lo9 SV40-transformed Balb/c 3T3 cells washed with phosphatebuffered saline as above. From 1 g of homogenate protein a yield of 10 to 17 mg of plasma membrane vesicles was obtained (Table I).
Marker Enzyme Assays -5'-Nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.51, used as a marker for plasma membrane, was assayed at 37" by the method of Avruch and Wallach (19)  was removed, diluted with 0.25 M sucrose, 0.01 M Tris/ HCl, pH 7.5, and centrifuged at 100,000 x g for 45 min. The pellet was suspended in 0.25 M sucrose, 0.01 M Tris/HCl, pH 7.4, by passage through a fine gauge needle. Vesicles were stored in l-ml aliquots in liquid nitrogen. Vesicles were not frozen for reuse after thawing from storage. These preparations contained 39 to 68% of the total 5'nucleotidase activity, 20% of the total NADH oxidase activity, 1.5% of the total succinate-cytochrome c reductase activity, and 11% of were carried out at 21" in lOO-~1 volumes containing 40 to 250 pg of membrane vesicle protein, 0.125 M sucrose, 10 mM Trisiphosphate (pH 7.41, 5 mM MgCl,, and other additions as indicated. Uptake was terminated by addition of 5 ml of 0.8 M NaCl, 0.01 M Tris/HCl, pH 7.5 (wash buffer), at 2", immediate filtration through a 0.2-pm or 0.45-/*rn nitrocellulose filter (Schleicher and Schuell, 2.5 cm diameter), and filtration of an additional 5 ml of cold wash buffer. Dilution, filtration, and wash steps were carried out within 20 s without letting the filter become dry. Nonspecific absorption was determined by dilution of the membranes with wash buffer before addition of radioactive substrate, followed by filtration and wash steps. Radioactivity of dried filters was measured by scintillation counting in toluene/Liquifluor (New England Nuclear Corp.).
Analysis of Accumulated Radioactivity -After incubation of vesicles with radioactive amino acids and collection of vesicles by the filtration assay, accumulated radioactivity was eluted with 1 ml of boiling H,O for 5 min and concentrated by lyophilization.
Thin layer chromatography of samples plus 0.3 pmol of unlabeled amino acid was carried out using polyethyleneimine-cellulose plates (Machery-Nagel) in 67% ethanol. Distribution of radioactivity was analyzed by scintillation counting of portions of the chromatogram. Materials -Ouabain, valinomycin, gramicidin D, iso-Abu, antimycin A, oligomycin, and NADH were from Sigma. Dextran 110 was from Pharmacia.
[ were not found optimal either for maximal solute retention or estimation of initial rates of uptake. Based on results shown in Figs. 1 and 2, an incubation temperature of 21" and the use of cold (2") buffers for collection of vesicles on filters were chosen for the standard assay conditions used in experiments to be described here. Fig. lA shows that during Na+ gradient-stimulated iso-Abu uptake the time for half-filling of vesicles was about 45 s shorter at 37" than at 21". The same quasi-steady state accu-supernat&t was determined by scintillation counting 'in Aquasol (New England Nuclear Corp.). NADH oxidase (NADH:acceptor oxidoreductase; EC 1.6.99.31, a marker for endoplasmic reticulum and outer mitochondrial membrane (20, 211, was assayed at 37" as described previously (19) by measuring oxidation of 0.14 mM NADH in the presence of 1.3 mM potassium ferricyanide at 340 nm. Succinatecytochrome c reductase (EC 1.3.99.1) activity, a marker for the inner mitochondrial membrane (21, 221, was measured at 37" in l-ml volumes using 45 PM cytochrome c (type VI, Sigma), 2 m&f potassium cyanide, and 50 mM sodium succinate in phosphate-buffered saline by recording the change in absorbance at 550 nm (19). (Na+, K+, Mg'+)-dependent ATPase activity (ATP phosphohydrolase; EC 3.6.1.31, a marker for plasma membrane (23, 24), was determined at 37" in loo-k1 volumes containing 100 mM TrisiHCl (pH 7.51, 2 rnM MgSO,, 2 mM ATP, 15 mM KCl, 60 mM NaCl, and 0.1 mM ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The reaction was terminated by addition of 100 ~1 of 10% perchloric acid. Inorganic phosphate released was determined by the method of Ames (25) with the modification that tubes were centrifuged at 2,000 x g for 20 min to remove precipitated protein before reading absorbance at 820 nm. (Na+, K+)-dependent ATPase activity was estimated as the increase in initial rate of ATP hydrolysis relative to that of incubation mixtures lacking KCl. Membranes preincubated 20 min with 1 mM ouabain in the complete mixture showed the same rate of ATP hydrolysis as observed in mixtures lacking KCl

Amino
Acid Transport in Plasma Membrane Vesicles mulation (15 min) was achieved at 21 and 37" but was de-accumulated iso-Abu, has been presented earlier using this creased at 2" (Fig. IA).
Similar effects of temperature were observed on iso-Abu efflux in the incubation mixture.
After iso-Abu was accumulated at 21" (Fig. 1B) or 37" (not shown) in the presence of a Na+ gradient, efflux was measured after temperature shiR and dilution to a lo-fold-decreased iso-Abu concentration. Iso-Abu efflux was increased at 37" and decreased at 2" relative to the rate at 21".
The temperature and composition of the dilution and wash steps of the filtration assay were chosen to maximize solute retention.
Almost no leakage was observed when dilution and wash in 0.8 M NaCl, 0.01 M Tris/HCl, pH 7.5 (wash buffer) were carried out at 2" as shown in Fig. 24. Loss of vesicle contents was extensive at 37". Use of hypotonic wash buffers caused loss of accumulated iso-Abu. This stimulation was specific for substrates of this trafisport carrier.
Internal iso-Abu was much more effective in stimulating alanine uptake by exchange diffusion than leucine, glytine, or methionine (not shown). Additional evidence inconsistent with binding to fixed sites, the osmotic sensitivity of  Membrane fractions were isolated from 9 x 10y Balb SV3T3 cells (1 g of protein) by a modification of the procedure of Quinlan and Hochstadt (18) and assayed within 72 h without freezing for marker enzyme activities and transport as described under "Experimental Procedures." The initial rate of Na+ gradient-stimulated iso-Abu uptake was assayed 30 s after 0.17 mM ['4Cliso-Abu (91 fmol/cpm) and 50 rnM NaSCN were added. Results are the average of duplicate determinations with a range of IT 15%. Data were corrected for Na+independent uptake determined using 50 mM KSCN instead of NaSCN. Specific activities for Na+-dependent iso-Abu uptake in the nuclear and crude mitochondrial fractions were below the level of detection. Numbers in parentheses refer to per cent distribution of these activities relative to the homogenate. ["Cliso-Abu as substrate were diluted 50-fold in 0.8 M NaCl, 0.01 M Tris, pH 7.4 (wash buffer), for the indicated times before filtration. Then filters were washed with 5 ml of wash buffer at 2". Efflux in wash buffer at 21" (O), 2" (W), and 37" (A) after uptake at 21". Efflux in wash buffer at 21" (01, 2" (Cl), and 37" (A) after uptake at 37". B, linearity of initial rate of ["Cliso-Abu uptake with amount of vesicle protein under assay conditions described in Fig. IA  ment of Na+ gradient-stimulated iso-Abu uptake specific activity with that observed for 5'-nucleotidase activity, a marker enzyme bound to plasma membrane fractions (19, 29) and resolvable in multiple forms (30). The plasma membranes fractionated after dextran 110 discontinuous gradients contained 20% of the 5'nucleotidase activity, 21-fold increased in specific activity with respect to the homogenate, and 20% of the Na+ gradient-stimulated iso-Abu uptake activity, 9.2-fold increased in specific activity.
The crude endoplasmic reticulum fraction contained only 1.4% of the total transport activity, 5.5fold reduced in transport specific activity compared with the homogenate and 50-fold reduced with respect to the plasma membrane fraction.
No Na+ gradient-stimulated transport activity could be detected in the nuclear or mitochondrial fractions but these fractions contained Na+-independent iso-Abu transport activity. For the transport experiments described below, unless otherwise noted, a purified mixed vesicle population (16, 31) contaminated 20% by endoplasmic reticulum and 1 to 2% by mitochondria as determined by marker enzymes for these organelles was used. Purified plasma membrane vesicles had an internal space of 2 &mg of protein accessible to 34methyl-n-[ 1-;'H]glucose and that of purified mixed vesicles was 0.8 to 1.1 pl/mg. Both types of preparations showed the same degree of Na+ gradient stimulation of alanine uptake. Na+ Gradient-coupled Concentrative Amino Acid Transport- Fig.   4 shows that both the initial rate and "steady state" of uptake of alanine, glycine, glutamine, methionine, and iso-Abu were stimulated by artificial imposition of a Na+ gradient (external Na+ > internal Na+) across the membrane.
This was achieved by adding Na+ together with the amino acid to the solution at zero time. Maximal accumulation driven by 50 mM NaCl added externally was 6-to 'I-fold for these amino'acids. Leucine uptake showed a lesser degree of stimulation by a Na+ gradient.
When 50 mM NaSCN was used to generate a Na+ gradient, both the initial rate and steady state of uptake were 30 fmol/cpm. Substrate and Na+, choline, or K+ salts were added at zero time. A, 50 rnM NaSCN; 0, 50 mM NaCl; 0, 50 mM choline chloride substituted for Na+; a, 1 pg of monensin in 1% Me$O and 50 mM NaCl; 0, vesicles were incubated with Na+ 15 min before uptake was measured at 50 mM external NaCl; v, 50 mM NaSCN plus 1 wg of monensin in 1% Me&SO; n , 50 mM KSCN. Control experiments (not shown) showed 1% MeSO had no effect on uptake increased compared with 50 mM NaCl. When the sodium gradient was dissipated by addition of monensin, an ionophore which promotes a Na+/H+ exchange (32), both the initial rate and steady state of transport of these amino acids were decreased, although still slightly increased over the Na+-independent rate. Similarly, previous incubation of vesicles with Na+ (Fig. 40) decreased iso-Abu uptake to the level observed after addition of monensin.
Within the time interval shown, iso-Abu or alanine accumulation did not decrease (overshoot) although gradual dissipation of the Na+ gradient would be expected during incubation.
Addition of monensin at 15 min (not shown) produced efflux of accumulated alanine which indicates that the Na+ gradient was not completely dissipated at 15 min. Table II compares the ability of electrochemical gradients of several ions to stimulate the initial rate and extent of iso-Abu uptake. Na+ was the only cation effective in stimulating both parameters of iso-Abu uptake. The initial rate and extent of Na+ gradient stimulation of uptake was markedly affected by the anion present. SCN-and NOR-, which are more permeant than Cl-to biological membranes (33, 341, stimulated Na+ gradient-driven accumulation with respect to Cll whereas the relatively impermeant anion SO,'-caused decreased accumulation (Table II). Na+ gradient-stimulated amino acid transport could be dissociated from endogenous (Na+ + K+)-ATPase activity of the vesicles. Table III shows that 1 mM ouabain, a specific inhibitor of the (Na+ + K+)-ATPase system (35), did not inhibit Na+ gradient-stimulated alanine uptake in the presence of concentrations of ATP, KCl, MgCIZ, and NaCl required for optimal expression of (Na+ + K+)-ATPase activity, yet was effective in completely inhibiting K+-dependent ATP hydrolysis assayed simultaneously in the same vesicle preparation. Addition of 5 mM KC1 and 2 mM ATP, both substrates of the (Na+ + K+)-ATPase system, did not stimulate Na+ gradient-coupled alanine transport.

Concentration Dependence of Na+ Gradient
Uptake Stimulation -Na+ gradients decreased the apparent Michaelis constant (K,,,) for the initial rate of L-ala&e transport with smaller effects on the maximal velocity (V,,,) in the presence of a permeant anion. From inverse reciprocal Lineweaver-Burke plots shown in Fig. 5, an apparentK,,, = 10 2 2 mM and I',,,,, = 20 nmol/min/mg were calculated for uptake in the presence of 50 mM KSCN. Values of apparent K,,, = 1.2 mM and V,,,, = 14.9 nmol/min/mg were observed using a 50 mM NaSCN gradient. At decreased Na+ gradients but at constant SCN-concentration, the K, value increased to 1.7 mM using 10 mM NaSCN plus 40 mM KSCN and V,,,,, was 14.9 nmol/ min/mg; the K,, was 2.4 mM using 1 mM NaSCN plus 50 mM KSCN and V,,, was 11.9 nmol/min/mg. Similar effects of Na+ on the apparent K,,, and V,,,,, for L-glutamine and iso-Abu uptake were observed (not shown).

K+-specific electrogenic
ionophore (36), caused a marked transient increase in Na+ gradient-stimulated accumulation of Lalanine. No stimulation of uptake under these conditions was observed in the absence of Na+ (data not shown). Fig. 8 shows that this stimulation was dependent on valinomycin concen-Both the initial rate (Fig. 6A) and steady state accumulation (Fig. 6B) of L-alanine increase as a function of increasing NaSCN gradients at constant SCN-concentration.
After addition of monensin to dissipate Na+ gradients, the initial rate of alanine uptake maintained a decreased degree of stimulation as a function of Na+ concentration, but saturation became evident at NaSCN concentrations above 50 mM with halfmaximal stimulation at 10 mM NaSCN. By contrast, Fig. 6I3 shows that monensin prevented Na+ stimulation of steady state accumulation at all Na+ concentrations. The inset in Fig.  6I3, shows that an alternate plot of the same data converted to logarithmic form after subtraction of Na+-independent uptake gave a straight line with a slope of 0.7.

Effect of Zonophores
on Na+ Gradient-stimulated Uptake - Fig. 7 shows that creation of a potassium diffusion potential (internal K+ > external K+) in the presence of valinomycin, a activity Aliquots of the same batch of vesicles from Balb SV 3T3 were assayed simultaneously for ouabain sensitivity of alanine uptake and (Na+ + K+)-ATPase activity. The initial rate of ATP hydrolysis was assayed using 50 kg of vesicles from Balb SV3T3 as described under "Experimental Procedures." Incubation mixtures for alanine uptake contained the same additions used for assay of ATP hydrolysis and 120 pg of vesicles with the inclusion of 0.125 M sucrose and 0.2 rnM L-[2,3-"Hlalanine (39 fmol/cpm). Transport was estimated by the filtration assay at the indicated times. Where indicated, membranes were preincubated 30 min with ouabain. Transport specific activity" at: Initial rate of ATP hydroly-

tration,
with maximal response at 20 pg/ml of valinomycin and no stimulation observed at higher concentrations. No effect on Na+ gradient-driven alanine uptake was observed in K+-loaded vesicles in the absence of valinomycin or with valinomycin in the absence of K+ gradients (Fig. 7). Addition of nigericin, an ionophore which catalyzes a nonelectrogenic K+/ H+ exchange (37) Fig. 8 shows that addition of nigericin to K+-loaded vesicles slightly decreased the initial rate of Na+ gradient-stimulated uptake. Gramicidin D, which creates a channel for several cations including Na+, K+, and H+ in biological membranes (381, decreased Na+ gradient-stimulated uptake in Kc-loaded vesicles (Fig. 8). Interactions among Neutral Amino Acids for Na+-dependent Transport -Substrate specificity properties of Na+ gradient-stimulated neutral amino acid transport suggested that transport activity assayed in vesicles represents the corresponding in uivo plasma membrane uptake system. Table IV  summarizes the effect of addition of several unlabeled amino acids in 25-fold excess on the initial rates of uptake of labeled 0.2 mM alanine, methionine, leucine, and iso-Abu. Alanine and iso-Abu uptake showed a similar pattern of inhibition by the unlabeled amino acids tested, which suggests they share a common carrier for transport into vesicles with the specificity properties described for the "A system" in Ehrlich ascites cells (39)(40)(41). Leucine uptake showed the amino acid specificity described for the "L system" (39, 40). Methionine uptake was inhibited by both categories of amino acids. No significant inhibition of L-alanine or iso-Abu, uptake by n-amino acids tested was observed, which suggests uptake is stereospecific. By contrast, L-methionine and L-leucine uptakes were inhibited by n-methionine and n-leucine. About 15% of total alanine and methionine uptake activity, 20% of iso-Abu uptake, and 50% of leucine uptake was Na+-independent.
% control 1OOb loo *  37  90  75  85  26  74  22  28  42  42  38  30  78  71  71  79  79  68  62  90  82  51  37  36  59  48  40  28  53  32  62  31  38  34  50  45  47  34  8  23  33  36  81  28  46  31  32 same extent as unlabeled L-alanine, which suggests that the "ASC system" (42) does not contribute appreciably to alanine transport in vesicles measured at this pH. Glycine, which is transported by a separate Na+-dependent carrier system in some cells (40,43), showed minimal effectiveness in inhibiting uptake of these amino acids, although its uptake was markedly stimulated by a Na+ gradient. Effect of pH-pH profiles for Nat-stimulated alanine and iso-Abu transport were almost identical with those obtained for in uiuo uptake in Ehrlich ascites cells (39). When the initial rate of Na+-stimulated transport into vesicles was measured as a function of external pH, uptake of both iso-Abu (Fig. 9A) and alanine (Fig. 9B) was optimal at pH 7.4. The ratio of activity at pH 7.4 to that of pH 6 was 2.3 for alanine uptake, and 6.3 for iso-Abu uptake. The initial rate of Na+-independent alanine uptake did not vary appreciably over this range.
Effect of Inhibitors -Several uncouplers and inhibitors of oxidative phosphorylation tested for effect on Na+-stimulated iso-Abu uptake (Table V) had minimal or no effect. Na+stimulated alanine uptake (not shown) was not inhibited by addition of 10 mM sodium arsenate or 50 pg/ml of oligomycin.
The lack of inhibition by proton conductors, such as car-bony1 cyanide p-trifluoromethoxyphenylhydrazone and 2,4-di- (40 fmolicpm) and 50 mM NaSCN. The Na+-independent rate, determined using 5C mM choline chloride in A or 50 mM KSCN (open symbols) in B has been subtracted. Results are averaged from duplicates. pH was measured at room temperature in each incubation mixture minus vesicles.
nitrophenol, suggests that H+ gradients are not coupled to this transport system as has been proposed (44).

DISCUSSION
Active neutral amino acid transport in vesicles was unequivocally linked to an electrochemical Na+ gradient. When a Na+ gradient (external Na+ > internal Na+) was artificially imposed across the membrane amino acid uptake against its concentration gradient occurred. Although in whole cells this Na+ asymmetry is provided by active Na+ extrusion via the (Na+ + K+)-ATPase transport system (451, endogenous (Na+ + K+)-ATPase activity of vesicles was not directly involved, in confirmation of previous hypotheses (l-3, 6). Also, evidence that K+ gradients, Na+/K+ antiport, or proton gradients do not directly contribute to this transport mechanism was obtained. Uptake was decreased when Na+ was equilibrated across the membrane by previous incubation with Na+ (12,13,15,16,27). This was not due to Donnan equilibrium effects or vesicle volume changes since equilibration with other cations such as K+ did not affect uptake, and monensin, an ionophore which dissipates a Na+ gradient by Na+/H+ exchange (32), blocked concentrative uptake. Addition of ionophores for other cations by themselves did not affect uptake. I have shown previously (16) that the direction of accumulation of iso-Abu was reversibly dictated by the direction of the Na+ gradient such that iso-Abu accumulated trans to the side of the membrane with increased Na+ concentration.
The affinity of alanine, iso-Abu, and glutamine for transport into vesicles was increased in the presence of Na+ with minimal changes in V,,,. Although monensin decreased the stimulation of the initial rate of alanine uptake as a function of Na+ gradient, it did not abolish it. This suggests that the presence of Na+ in the absence of a Na+ gradient can modify the carrier to increase its affinity for the amino acid, as proposed in the Na+ gradient hypothesis (1, 2, 6). Evidence for an electrogenic mechanism of Na+-dependent amino acid uptake was obtained.
Uptake was stimulated by conditions expected to create an interior-negative membrane predicts that transport involves movement of a positively charged complex across the membrane without a direct coupling of charge compensation to achieve electroneutrality to the transport process. Significantly, this implies that in the presence of Na+ an additional driving force for concentrative amino acid uptake can be provided by other electrogenic membrane processes.
Thus the net force driving concentrative amino acid uptake is the electrochemical potential of Na+ across the membrane, pXLp;,+, which is the sum of electrical and chemical thermodynamic parameters.
acids, it follows that the latter acids should have the "D" configuration at C-5 (5-n-hydroxy-6,8,11,14-eicosatetraenoic The line should read acid) and at C-8 (8-n-hydroxy-9,11,14-eicosatrienoic acid). 5.0 mM Na 0.168 23.8 142 Vol. 252 (1977) 1990-1997Vol. 252 (1977  The concentration of [Wliso-Abu was written as 1.1 mM instead of 0.11 mM. The correct line should read: The extraction buffer also contained 0.3 M ammonium sulfate. The sentence should read: salts listed below and 0.11 rnM [l-'%]iso-Abu (61 fmol/cpm) to 140 PLg The pellet was suspended with a glass rod and finally a glass We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriate places where the article to be corrected originally appeared. Authors are urged to introduce these corrections int,o any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.