A Cycle of Deprotonation and Reprotonation Energizing Amino-Acid Transport? (Diamino Acids/H+ Cotransport/Proton Gradients/Plasma Membrane/Ehrlich Cell)

Proc. Nat. Acad. Sci. USA Vol. 72, No. 1, pp. 23-27, January 1975

A Cycle of Deprotonation and Reprotonation Energizing Amino-Acid Transport? (diamino acids/H+ cotransport/proton gradients/plasma membrane/Ehrlich cell)

HALVOR N. CHRISTENSEN AND MARY E. HANDLOGTEN Department of Biological Chemistry, University of Michigan, Ann Arbor, Mich. 48104 Communicated by J. L. Oncley, September 23, 1974

ABSTRACT Although lowering the pK2 of neutral Uptake of the lower homologs of lysine and ornithine amino acids only weakens their concentrative uptake by Ehrlich cells, the same change greatly enhances uptake of By chance we encountered in 1952 behavior revealing that diamino acids. This effect does not arise merely from the data of Table 1 and Fig. 1 do not tell the whole story of the putting the distal amino group in its uncharged form, but role of dissociation in amino-acid transport (18). The struc- depends on an enhanced deprotonation of the a-amino group. Parallel effects are seen for the transport system tural feature responsible for this behavior was a nearness of a for basic amino acids, for which the assignment of pK second amino group to the a-amino group. In the homologous values within the membrane is less ambiguous. To explain series of a,-diamino acids composed of lysine, ornithine, the paradoxical advantages of having the a-amino group 2,4-diaminobutyric acid (A2bu), and 2,3-diaminopropionic protonated yet readily deprotonated, we propose that a acid, we observed an increase in the rate of proton withdrawn from that group is pumped over an abrupt uptake intramembrane interval to energize amino-acid transport. and intensity of accumulation as the chain is shortened to four carbons or less (Fig. 3). The most intensely accumulated Although a driving of amino-acid uptake by cotransport with is A2bu, both for Ehrlich ascites tumor cells and for rat liver Na+ has long been known (1-4), this has recently been shown (18). Almost all of the endogenous levels of K+ and Na+ not to be the obligatory mode of energization (5). Further- could be displaced from the Ehrlich cell by this organic cation, more, Na+-independent systems can unambiguously produce its uptake along with Cl- having in the meantime caused the uphill transport (5). Therefore, we have long given attention cells to swell to two to four times their normal volume during to suggestive effects of modifying the H+-dissociation of the several hours. Gradients as high as 180 mAM could be obtained amino acids as clues to a more fundamental mode of energiza- when uptake occurred from hypertonic solution, thus mini- tion (ref. 5; ref. 6, pp. 91-92; refs. 7-9). mizing simultaneous water transfer. Table 1 illustrates that a systematic lowering of pK2' of The principal difference among these diamino acids lies not neutral amino acids (in this case by introducing one, two, or in the rates at which they are accumulated by the cationic three fluorine atoms) decreases both the initial rate of uptake amino-acid system, for which their Km values are relatively and the steady-state distribution attained with Ehrlich low, but in their reactivities at high Km values with ascites tumor cells. Fig. 1 shows a corresponding effect of the systems for neutral amino acids. When we contrasted the presence of two fluorine atoms in the amino-acid molecule: concentration dependence of the uptake of lysine and A2bu by the pH optimum for uptake is lowered sharply. These results Ehrlich cells, we saw similar hyperbolic portions for the curves indicate that the amino acid is accepted for uptake by the at low levels, representing uptake by the cationic system, system in question with the amino group in the form RNH3+ inhibitable by homoarginine. At higher levels we noted a rather than RNH2. component that was difficult to saturate, much larger for This result did not prove, however, that the recognition A2bu than for lysine. An inhibition analysis showed that this site at the interior surface of the membrane has the same component for A2bu and for diaminopropionate occurs by preference. In 1958 we considered, using Fig. 2, whether a System A and is Na+-dependent (19). This was a surprising difference in the state of protonation preferred at the two result, because that system ordinarily has neutral amino surfaces might produce co- or countertransport of H+ with acids for its substrates (20). the amino acid (3). The figure shows a hydrogen ion left We begin in this way to encounter a series of paradoxes. behind when the amino acid combines with the external Even though A2bu, pK2 = 8.4, should be cationic to the receptor site, and a new hydrogen ion taken up by the amino extent of 91% at pH 7.4, most of its uptake at high levels acid on entering the cytoplasm. This scheme produces a occurs by a neutral amino-acid system. Of the 9% that is countertransport between H+ and the amino acid, which was present in dipolar form in water solution at that pH, a major in 1958 an arbitrary choice over cotransport. Possibilities of part takes the form of the a,-y-dipolar ion, by analogy not a this kind have subsequently become more important through substrate for a transport system for a-amino acids. The the development by Mitchell of his chemiosmotic hypothesis values for pK2' for the homologous series of Fig. 3 in order of for the intermediation of hydrogen ion gradients in the process decreasing chain length, are 9.5, 8.7, 8.4, and 6.8. The initial of energy transduction by the mitochondrial and chloroplast rates of uptake increase as pK2' decreases, just the reverse membrane (11, 12). The observation of cotransport of H+ of the order shown in Table 1 for monoamnino acids in the same with amino acids (see for example refs. 8 and 13) and with transport system. sugars (14-16) has accelerated the extension of these ideas to The latter paradox becomes not quite so implausible when the plasma membrane (see ref. 17). one considers that for transport these diamino acids need to lose a proton to convert their cationic form to a dipolar species. Abbreviation: A2bu, L-2,4-diaminobutyric acid. One might expect that A2bu could become as good a substrate 23 Downloaded by guest on September 28, 2021 24 Biochemistry: Christensen and Handlogten Proc. Nat. Acad. Sci. USA 72 (1975)

? 1.6 0.2 TABLE 1. Effect of 3-fluoro substitution on transport of

x 2-aminoisobutyric acid into the Ehrlich cell D 1.2 (scaleatright Distribution ratio co 0.8 - (scale at left) 0.1 for Ehrlich cell 750-0 pK'/ 0) Substrate (250) at 1 min at 30 min a 0.4 Aminoisobutyric acid, 1 mM 10.21 0.85 27.7 E E 0~ Fluoroaminoisobutyric acid, :: 6 7 8 1 mM 8.58 0.95 13.0 PHex Difluoroaminoisobutyric acid, FIG. 1. Effect of the presence of two fluorine atoms in 2- 3mM 7.40 0.17 3.6 aminoisobutyric acid on the pH optimum for the rate of uptake Trifluoroaminoisobutyric acid, by Ehrlich ascites tumor cells. Uptake observed during 2 min at 1 mM 5.94 0.27 0.74 370 from Krebs-Ringer phosphate solution, adjusted to give the final pH shown. Presumably, the increasing quantity of this Difluoroaminoisobutyric acid is the symmetrical difluoro amino acid in its dipolar form compensates in part for the de- derivative, prepared from 1,5-difluoro-3-pentanone by the same crease in rate that would otherwise occur below pH 7.4. *, 0.2 procedure used for the monofluoro derivative. At 30 min, its mM difluoroaminoisobutyric acid; 0, 0.2 mM aminoisobutyric distribution ratio was at the maximum value. In trifluoroiso- acid. butyric acid, the fluorine atoms are all on the same carbon atom. The transport disadvantage of the fluoro analogs applies also to for System A as homoserine, once it has lost a proton, given renal resorption and hepatic uptake (10). only that this proton be lost from the distal amino group. But now the paradox reappears: A2bu is far more strongly stabilization of a species unsuitable for transport by the accumulated by the Ehrlich cell than is homoserine (Fig. 3). inner receptor site is not shared by the outer receptor site. Why should these diamino acids act as "super-substrates," To show that the two opposed transport receptor sites given their special difficulty in assuming the preferred struc- place the substrate in dissimilar electrostatic environments ture for uptake? Even though A2bu can assume this structure would not provide a sufficient explanation of the flux asym- more readily than ornithine, which can assume it more readily metry, however, unless we add an energy input to maintain the than lysine, all of these amino acids should be at a handicap environmental difference, whatever it is. Under the simple in comparison with homoserine or other ordinary neutral model implied here, energy would have to be applied steadily amino acids. Clearly, the ability of the amino acids to assume to keep changing the environment of the site for entry into the statically their a,a-dipolar form by no means provides a full membrane to an environment suitable for release into the explanation of the effects of changes in the pK values. cytoplasm. Let us examine, then, the possibilities introduced along with We early began to attribute the contrast in transport a second amino group. We may propose, first of all, that the among the diamino acids to the inductive interactions be- receptor site for entry provides a microenvironment for the tween the two amino groups. In lysine, the separation between side chain such that the distal amino group of the diamino them is great enough so that the presence of each amino acid is stabilized in its deprotonated form, given that its group has little effect on the pK of the other, so that the ob- tendency to retain the proton is not too great. The large served pK' values lie close to the intrinsic pK values, namely, gradients of A2bu that can be generated correspond to a large at 9.53 for the a- and at 10.94 for the C-amino group (21). asymmetry between influx and efflux, retained even at high As the two groups are brought nearer together, this interac- loads. We may seek provisionally to account for this phe- tion is increasingly transmitted through the carbon chain. nomenon by asserting that the behavior shown by the recep- The result is that both pK2 and pK3 are lowered, and the tor site for entry, whereby the acceptable species of the amino disparity in their internal environments in the molecule acid presumably is stabilized, is not shared by the internal decreases somewhat. It becomes easier then for the order of receptor site serving for exodus; or alternatively, that a their dissociations to be reversed, given only a modest change in the external environment. The lowering of the values of R-CR'-COO- R-CR'-C=O + X+ + H+ pK2 and pK3 has thus made the dissociation of the amino R"-NH2+ R"-N 0 group particularly sensitive to its immediate environment.

x Numerous cases are known where the sequence of dissocia- Outside tion of a substance can be inverted by the addition of a mis- cible organic solvent, e.g., dioxane, to an aqueous solution. Inside I As a result of these interactions, whether we focus our atten- R-CR'-COO H R-CR'- C=O 0 of OH- + + X+ 2 tion on one or the other of the two amino groups A2bu, R"-N that group may be seen as environmentally sensitive to a heightened degree. The question may then be asked, is the x to the environmental FIG. 2. Scheme to show the amino acid accepted in its de- transport system particularly responsive amino or of both? protonated form for entry into the membrane; and subsequently sensitivity of the a- or of the distal group, taking up a hydrogen ion from the cytoplasm on its release into Other diamino acids showing strong flux asymmetry the cell. Reproduced with permission from Riggs, T. R., Walker, L. M. & Christensen, H. N. (1958) J. Biol. Chem. 233, 1479- Before facing this question, let us note that the phenomenon 1484. is not restricted to the transport system (A) implicated up to Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) De- and Re-protonation Cycle Energizing Transport 25

this point. By placing two methyl groups on the distal amino 40 A group of diaminopropionic acid, the branched-chain amino acid, azaleucine, pK = 6.8, is obtained. This diamino acid

also proved to undergo transport largely as a neutral amino 20 acid, the reactivity decreasing as the pH is lowered, around .co an apparent midpoint of pH 5.8 even though pK2 in free solution at 250 is 6.8 (22). As a predictable consequence of the 20- branching (19), this transport occurs largely by Na+-independent System for which L, the amino acid shows a pronounced 30 60 90 120 flux asymmetry. Minutes The replacement of a methylene group in the side chain of FIG. 3. Comparison of time course of uptake of the homol- lysine with a sulfur atom to yield thialysine, pK2 = 8.4, also ogous diamino acids (1 mM) in the presence of 10 mM\I homo- leads to a "gradient-sensing" transport substrate (5, 9). In arginine. Contrast between uptake of A2bu and homoserine (each this case uptake is especially fast both by the Na+-dependent 10 mM) in media in which Li+ replaces Na+. A2pr, 2,3-diaminopropionic acid. The four longer curves show the abrupt intensi- and the Na+-independent routes. Other diamino acids fication of uptake of the diamino acids by neutral systems (not strongly accumulated by Na+-independent System L are inhibited by homoarginine) when the chain length falls below 5 4-amino-4-carboxyl-1-methylpiperidine, pK2 = 7.2, and cis- carbon atoms. The shorter curves contrast accumulation of A2bu 1,4-diaminocyclohexanet arboxylic acid, pK2 = 8.7. The and homoserine, both somewhat handicapped by substitution ordinary substrates of System L are concentrated only rather of Li+ for Na+ to restrict homoserine uptake to System A (see weakly. Hence, these analogs serv3 to show that the presence text). of a second amino group with a low pK2 increases trapping of the energy accessible to the Na+-independent system. chain has on The mutual effects the probability of the dissociation observation that the trans isomer of the last-named of each. Similarly, the sulfur atom in thialysine lies amino acid is much more weakly accumulated than the cis midway between the amino groups, and the unsaturated links intro- isomer (8) brought attention back to an idea entertained for duced into lysine will affect both dissociations. Therefore, we some years. In all the diamino acids showing the phenomenon tested next the effects of structural features designed discussed so far, the two amino groups in each molecule can specifically to favor the deprotonation of the distal amino group. For come together, for example, to share a proton (as in Fig. 14 of example, the pK2 = 6.0 of histidine is well known to ref. 19). This behavior would not be expected in water solution pertain largely to the imidazole group. This amino acid, at 3 mM, was (23), but it could be facilitated in some membrane environment. concentrated only 1.45-fold from Na+-free medium containing excess homoarginine to block the cationic We have recently found, however, that 2,6-diamino4- system. Canavanine and canaline, pK2 = 7.4 and 4.3, respectively, were concen- hexynoic acid (24), pK' = 8.4, can also generate rather high trated only 3- and 6-fold, respectively, via System gradients across the plasma membrane of the Ehrlich cell, A, and m-aminophenylglycine, pK2 = 3.6, was not concentrated principally by System A. In this compound the two amino at groups can scarcely all from a Na+-free medium. These tests need extension to approach each other. Nevertheless, di- diamino acids for which pK2 applies mainly aminohexynoic acid in the DL form generates gradients as high to the distal amino group and yet falls in the range 7-8.4. Several of the strongly as 100 to one, taking into account near failure of the D isomer to accumulated be taken up (Fig. 4). Rather strong gradients are also generated diamino acids show through chemical shifts by proton magnetic resonance for trans-4,5-dehydrolysine. These results appear to mean that that pK2 in D20 solution concerns the alpha more than the group; a the amino groups do not need to come together. This conclu- distal amino but small preference in the other direction may serve sion probably applies to both Systems A and L, since both well also. The results so far no systems participate substantially in the strong offer encouragement, however, for the idea that what accumulation is needed for strong uptake is to get of diaminohexynoate. If one of these systems were only weakly merely the side chain into concentrative, exodus by it would deplete the gradient generated by the other. Apparently, therefore, we need to look for another explanation in the topography provided by System L for the discrimination between the cis and trans 4) 20 isomers of diaminocyclohexanecarboxylic acid. The high reactivity and high stereospecificity for dehydrolysine and S 158 0 E similar diamino acids also point to a preference as to the posi- E tion taken at the receptor site by the second amino group. 1<1 05 1

Does more specific facilitation of deprotonation of the 05 distal amino group enhance uptake? 0.2 mM

o 0 30 60 90 If the neutral transport systems merely C need to direct the E Minutes deprotonation inherent in a low pK2 toward the distal amino group, then we should be able to assist the process by introducing structural features more specifically enhancing that FIG. 4. Strong accumulation of 2,6-diamino-4-hexynoic acid by Ehrlich cells. Partition coefficients as as 100 could dissociation. Note that the structural changes selected so far high be calculated for the L isomer. At 10 m-M, a gradient of 84 mMI was for lowering pK2 have all acted on both amino groups. That is, obtained at 120 min. Of the uptake from 3 mMI solution, 55% bringing the two amino groups closer together on the carbon was blocked by 2-(methylamino)-isobutyric acid in excess. Downloaded by guest on September 28, 2021 26 Biochemistry: Christensen and Handlogten Proc. Nat. Acad. Sci. USA 72 (1975) and high degrees of accumulation (8). Arginine, pK2' = 9.04, 1 10 0 and homoarginine are accumulated by only 2- or 3-fold, except 0 8 at relatively low substrate levels (Fig. 5). Their uptake may in 0 6 fact be entirely passive, since the transmembrane potential -S gradient is sufficient to account for a 2-fold accumulation. The analogous 1-amino-4-guanidine-cyclohexanecarboxylic acid, cc = 0 2 0 o pK2 9.3, also showed only weak accumulation, whereas .E g-guanidinium-L-alanine, pK2 = 7.8, was concentrated much U 2 4 6 8 more strongly under similar conditions. Incidentally, these [Amino acid],,, mM compounds, along with others already discussed, showed no FIG. 5. Comparison of the extent of accumulation by Ehrlich measurable binding to undialyzable components of an un- cells in 3 hr of model substrates of the cationic amino-acid system diluted cytolysate prepared by freezing and thawing Ehrlich Ly +, L-homoarginine (0) and e-N-methylhomoarginine (0), cells. with that of 4-amino-1-guanylpiperidine-4-carboxylic acid (A) These results show that the association between low values and ,3-guanidino-L-alanine (star). Uptake was at 370 from Krebs- for pK2 and high degrees of accumulation extends also to this Ringer bicarbonate medium at pH 7.4. Lysine and arginine ac- transport system. What is especially significant is that it is cumulation resembles that of homoarginine. highly unlikely that pK2 for these compounds applies to the guanidinium group, to which we assign instead the pK3 an uncharged form. On the contrary, they draw attention back higher than 12 noted on titrating. Even in special environ- to the probability that the a-amino group needs to participate ments of the membrane, pK2 probably applies almost exclu- in a deprotonation for optimal energy trapping. sively to the a-amino group. Attention is thus directed even Evidence from the cationic amino-acid transport system more emphatically to the indications that a ready deprotonation of the a-amino group gives a special energy-trapping So far we have dealt with the paradoxical transport of diamino ability to amino acids for transport, provided that a second acids by neutral systems. Let us now consider transport by basic group is present on the side chain. Note also that the the cationic system. If we make the pK2 of the distal amino ordinary substrates of the neutral transport systems carry no group high enough, as in lysine, we can minimize transport by amino group on their side chains. Hence, the only amino group neutral systems and maximize that by System Ly+, the basic on those molecules whose traffic in protons can figure in the system. Better yet, by using a guanidinium group for the distal energization of transport is the a-amino group. basic structure, the selectivity for System Ly+ can be made unequivocal and nearly complete. Discussion In preparing 4-amino-1-guanylpiperidine-4-carboxylic acid Table 1 and Fig. 1 indicate that a proton should be present on as a model, metabolism-resisting substrate for System Ly+ the a-amino group if a neutral amino acid is to be accepted for (25), we unintentionally obtained a low value for pK2 of 8.0, transport. Also for the cationic system, it is clear that the

H H

7NH+3 I NH RCH ,-yopls R-CH < =: R-CH / 3 Coo I N~~coo~

external Barrier Zone of H Barrier cytoplasm solution #1 pumping; #1 mediated mediated passage mediated rpassage of may favor passage of zwitterion RCH (NH2) COO zwitterion with H+ impelled separately

FIG. 6. Scheme to show one form of our proposal as to how pumping of a separated proton may produce a structure-dependent energization of amino-acid transport of neutral or cationic amino acids. The amino acid is shown being accepted from the external solution in an a, a-zwitterionic form, net charge zero, or if we are discussing cationic System Ly +, net charge plus one. The mediate-d passage of the amino acid through the membrane brings it to a zone of internal H + pumping (represented as barrier 2) across which passage of the amino-acid molecule is largely limited to the form with the a-proton absent. This zone could lie within an oligomeric pro- tein assembly. The gradient attributed here to a proton pump could instead be a gradient of polarization or of electron distribution sufficient to cause the a-amino group to yield a proton over this spatial interval. Facilitation of the reversible removal of the proton by side chain structures plays a role in determining the flux asymmetry. Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) De- and Re-protonation Cycle Energizing Transport 27

a-amino group needs to be protonated; the initial rates of We do not suggest that this mode of coupling between proton transport of the two arginine analogs of low pK2 showed no flows and the flows of organic metabolites is universal since decrease as the pH was lowered from 8 to 5.5, whether for no chemical basis is obvious for a parallel mode of coupling of entry or exodus. Nevertheless, we have considered evidence flows between simple sugars and the hydrogen ion. here that the readiness of departure of the proton from that group (perhaps to another position on the same molecule) This research received support from Grant HD01233, National gives transport an intense directional asymmetry. The situa- Institute for Child Health and Development, U.S. Public Health tion seems to say that the uphill process finds advantages both Service. in the protonated and in the deprotonated state of the a- amino group. Since the amino-acid molecule can scarcely 1. Christensen, H. N., Riggs, T. R. & Ray, N. E. (1952) J. Biol. Chem. 194, 41-51. present these two states simultaneously, our interpretation 2. Christensen, H. N. & Riggs, T. R. (1952) J. Bidl. Chem. (Fig. 6) is that it probably does so sequentially. First the 194, 57-68. amino acid must be accepted in the proton-bearing form, in 3. Riggs, T., R., Walker, L. M. & Christensen, H. N. (1958) advance of the rate-limiting event, to yield the kinetics J. Biol. Chem. 233, 1479-1484. reflected by 1 and 4. Heinz, E. (Od.) (1972) Na-linked Transport of Organic Solutes Table Fig. 1. Ultimately, the amino-acid (Springer, Berlin), pp. 1-98. molecule must again be released into the cytoplasm carrying 5. Christensen, H. N., de Cespedes, C., Handlogten, A. E. & the same proton load, since we can detect so far no cotransport Ronquist, G. (1974) Ann. N.Y. Acad. Sci. 227, 355-379. of H+ with these diamino acids across the plasma membrane 6. Christensen, H. N. (1955) in Amino Acid Metabolism, eds. of the Ehrlich cells. Furthermore, this cell shows only McElroy, W. D., & Glass, B. (Johns Hopkins Press, Balti- small more, Md.), pp. 63-106. transmembrane gradients of H + or the electrical potential (4). 7. Christensen, H. N. (1974) Biological Transport (W. A. [It is an important additional circumstance that another class Benjamin, New York), 2nd ed., pp. 176-196. of unusual substrates for System L, illustrated by a,a- 8. Christensen, H. N. (1973) J. Bioenerg. 4, 31-61. diethylglycine, does cause H+ uptake during its unusually 9. Christensen, H. N., de Cespedes, C., Handlogten, M. E. & Ronquist, G. (1973) "Reviews on biomembranes," Biochim. concentrative entry into the cell, and that lowering the ex- Biophys Acta 300, 487-522. ternal pH stimulates the accumulation of ordinary substrates 10. Christensen, H. N. & Oxender, D. L. (1963) Biochim. of System L (5, 8, 9).] Somewhere between the binding and Biophys. Acta 74, 386-391. release events, we suggest that a proton is reversibly disso- 11. Mitchell, P. (1961) Nature 191, 144-148. ciated from the a-amino group, and subjected to proton 12. Mitchell, P. (1970) in Membrane and Ion Transport, ed. Bittar, E. E. (Wiley-Interscience, London), Vol. 1, pp. 192- pumping (Fig. 6). We suggest further that this process repre- 256. sents the fundamental mode of coupling of the driving force 13. Eddy, A. A. & Nowacki, J. A. (1971) Biochem. J. 122, 701- in the membrane to uphill amino-acid transport. 711. This idea gives the selected diaminio acids importance not 14. West, I. C. (1970) Biochem. Biophys. Res. Commun. 41, 655- merely as probes may 661. that sense and report differences in 15. West, I. C. .& Mitchell; P. (1972) J. Bioenerg. 3, 445-462. hydrogen ion availability at separated points along the trans- 16. Kashket, K. R. & Wilson, T. H. (1972) Biochem. Biophys. port pathway through the plasma membrane, as suggested Res. Commun. 49, 615-620. earlier (5). The concept implies, in addition, that the structure 17. Harold, F. M. (1972) Bacteriol. Rev. 36, 172-230. of these diamino acids intensifies the availability of H+ from 18. Christensen, H..N., Riggs, T. R., Fischer,; H. & Palatine, I. M. (1952) J. Biol. Chem. 198, 1-15, 17-22. the amino acid itself, thus pointing to the way in which the 19. Christensen, H. N. & Liang, M. (1966) J. Biol. Chem. 241, directional thrust may be given to the amino-acid molecule 5542-5551. within the membrane. 20. Oxender, D. L. & Christensen, H. N. (1963) J; Biol. Chem. The observed similarity in the responses of Systems A and L 238, 3686-3699. 21. Bradbury, J. N. & Brown, L. R. (1973) Eur. J. Biochem. to a lowering of pK2 indicates that both of these systems, as 40, 565-576. well as System Ly+, probably participate in an intramem- 22. Christensen, H. N. (1964) J. Biol. Chem. 239, 3584-3589. brane linkage of amino acid and H+ movements. Although, in 23. Hine, J., Via, F. H. & Jensen, J. H. (1971) J. Org. Chem. contrast to System L, we have seen no evidence for linked 36, 2626-2629. movements of H+ and amino acids in System A, neither have 24. Davis, A. L., Rodney, L., Maul, S., Cook, D. E. & McCord, T. J. (1964) Arch. Biochem. Biophys. 104, 238-240. we sufficient evidence for any other way in which the thrust is 25. Christensen, H. N. & Cullen, A. M. (1973) Biochim. Biophys. universally applied to amino acid flow through that system. Acta 298, 932-950. Downloaded by guest on September 28, 2021