Proc. Natl. Acad. Sci. USA Vol. 73, No. 6, pp. 1892-1896, June 1976 Biochemistry The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles (flow dialysis/membrane potential/energy transduction/lipophilic cations/weak acids) SOFIA RAMOS, SHIMON SCHULDINER*, AND H. RONALD KABACK The Roche Institute of Molecular Biology, Nutley, New Jersey 07110 Communicated by B. L. Horecker, March 17, 1976

ABSTRACT Membrane vesicles isolated from E. coli gen- presence of valinomycin), a respiration-dependent membrane erate a trans-membrane proton gradient of 2 pH units under potential (AI, interior negative) of approximately -75 mV in appropriate conditions when assayed by flow dialysis. Using E. coli membrane vesicles has been documented (6, 13, 14). the distribution of weak acids to measure the proton gradient (ApH) and the distribution of the lipophilic cation triphenyl- Moreover it has been shown that the potential causes the ap- methylphosphonium to measure the electrical potential across pearance of high affinity binding sites for dansyl- and azido- the membrane (AI), the vesicles are shown to generate an phenylgalactosides on the outer surface of the membrane (4, electrochemical proton gradient (AiH+) of approximately -180 15) and that the potential is partially dissipated as a result of mV at pH 5.5 in the presence of ascorbate and phenazine lactose accumulation (6). Although these findings provide ev- methosulfate, the major component of which is a ApH of about idence for the chemiosmotic hypothesis, it has also been dem- -110 mV. As external pH is increased, ApH decreases, reaching o at pH 7.5 and above, while AI remains at about -75 mV and onstrated (6, 16) that vesicles are able to accumulate lactose and internal pH remains at pH 7.5. Moreover, the ability of various other substrates to intravesicular concentrations which are electron donors to drive transport is correlated with their ability 100-fold or greater than those of the external medium. To to generate A4H+. In addition, ApH and Ad can be varied re- sustain concentration gradients of this magnitude, a membrane ciprocally in the presence of valinomycin and nigericin. These potential of at least -120 mV is required. This observation, in data and others (manuscript in preparation) provide convincing addition so numerous negative attempts to establish the exis- support for the role of chemiosmotic phenomena in active tence of a transmembrane pH gradient (1, 6) has left some transport. doubt as to the quantitative relationship between AILH+ and Despite apparently contradictory initial observations (1-3), an solute accumulation. [In the apparent absence of a transmem- increasing accumulation of experimental evidence (4-6) indi- brane pH gradient, it is also not immediately clear how anionic cates that chemiosmotic phenomena, as postulated by Mitchell solutes are accumulated, by the chemiosmotic mechanism. (7-10), play a central role in respiration-linked active transport suggested for this process (10)]. in Escherichia coli membrane vesicles. It now seems clearly Recently, Padan et al. (17) have shown that intact E. coli established that membrane vesicles prepared by the techniques generate a ApH (interior alkaline), and that the magnitude of developed in this laboratory retain the same orientation as the the ApH is very dependent upon external pH, exhibiting a membrane in the intact cell (see ref. 11 for a summary of the maximal value of about 2 pH units at pH 6.0 or below. In ad- evidence), and that oxidation of electron donors which drive dition, Rottenberg (18) has utilized acetate to determine ApH transport in the vesicles results in the generation of a trans- in mitochondria, and suggested that this weak acid might be membrane electrical potential (interior negative) by means of more useful than 5,5'-dimethyloxazolidine-2,4-dione (DMO) electrogenic proton extrusion (6, 12-14). The potential is pos- (19) in certain systems because it might be less permeant (H. tulated to drive solute accumulation via facilitated diffusion Rottenberg; personal communication). of positively charged substrates such as lysine or via coupled The results presented in this paper were obtained by means movements of protons with neutral substrates such as lactose of flow dialysis (20), a technique which allows a rapid, contin- or proline (i.e., "symport"). uous determination of changes in the concentration of solutes According to the chemiosmotic hypothesis, the total driving in the external medium without manipulation of the vesicles. force generated by proton extrusion is the electrochemical Using this technique, it is demonstrated that E. coli membrane potential of protons across the membrane (A,4H+) (7-10). This vesicles generate a large proton gradient under appropriate thermodynamic entity is composed of an electrical and a conditions. In addition, we have shown that ApH and AI can chemical parameter according to the following relationship: be manipulated reciprocally by the ionophores valinomycin and nigericin. The results to be presented, and others (S. Ramos 2.3RTA and H. R. Kaback, manuscript in preparation) which will be A =H+= A - F ApH [1] discussed, leave little doubt as to the primary role of chemios- motic phenomena in respiration-dependent active transport. where Ai represents the electrical potential across the mem- brane, and ApH is the chemical difference in proton concen- METHODS tration across the membrane (2.3RT/F is equal to 58.8 mV at Growth of Cells and Preparation of Membrane Vesicles. room temperature). E. coli ML 308-225 (i-z-y+a+) was grown on minimal me- Through the use of lipophilic cations and rubidium (in the dium A with 1.0% sodium succinate (hexahydrate), and membrane vesicles were prepared as described (21, 22). Vesicles Abbreviations: DMO, 5,.5'-dimethyloxazolidine-2,4-dione; TPMP', M and triphenylmethylphosphonium (bromide salt); PMS, phenazine were suspended in 0.1 potassium phosphate (pH 6.6) methosulfate; CCCP, carbonylcyanide m-chlorophenylhydrazone. stored in liquid nitrogen. * Present address: Department of Molecular Biology, Hadassah For studies at various pH's, membrane suspensions containing Medical School, Hebrew University, Jerusalem, Israel. about 4 mg of protein per ml were removed from storage, 1892 Downloaded by guest on October 2, 2021 Biochemistry: Ramos et al. Proc. Natl. Acad. Sci. USA 73 (1976) 1893 thawed rapidly at 460, diluted at least 10-fold with 0.1 M po- tassium phosphate buffer at the desired pH, and incubated for 10 min at 25°. The suspension was centrifuged at 40,000 X g for 30 min, and the pellet resuspended and washed once in a similar volume of the same buffer. The final pellet was then resuspended to an appropriate protein concentration in 0.1 M potassium phosphate at the same pH. 0

Transport Assays. Filtration assays (6, 23) were carried out x on Millipore Cellotate filters (0.5 ,um pore size). Electron donors ., and isotopically labeled solutes were used as described. Flow 0. 15 dialysis was performed as described (24) except that the ap- paratus was modified so that the upper chamber was com- pletely open to the atmosphere, and the reaction mixture was gassed with oxygen. The upper and lower chambers were separated by Spectrapor 1 dialysis tubing (600-8000 molecular weight cut-off; Fisher Scientific), and both chambers were stirred by means of magnetic bars. Membrane vesicles sus- FRACTION NUMBER pended in 0.05 M potassium phosphate at a given pH containing FIG. 1. Ascorbate-PMS-dependent acetate uptake by E. coli ML 0.'01 M magnesium sulfate were added to the upper chamber 308-225 membrane vesicles as determined by flow dialysis. The assay (total volume 0.8 ml), and electron donors, isotopically labeled shown was carried out at pH 5.5 as described in Methods with sodium solutes, and ionophores were added as indicated. Potassium [3H]acetate (685 mCi/mmol) at a final concentration of 18 gM and phosphate (0.05 M at the same pH as the buffer in the upper E. coli ML 308-225 membrane vesicles at a final concentration of 2.5 mg ofprotein per ml in the upper chamber. As indicated by the arrows, chamber) was pumped from the lower chamber at a rate of 6.0 ascorbate and phenazine methosulfate (ASC/PMS), valinomycin ml/min with a Pharmacia pump (model P3). Fractions of about (VAL), and nigericin (NIG) were added to the upper chamber at final 1.7 ml were collected and assayed for radioactivity by liquid concentrations of 20 mM and 0.1 mM, 1 ,uM and 1 gM, respectively scintillation spectrometry. Since phenazine methosulfate (PMS) (closed symbols). Open symbols were obtained from an identical ex- causes about 5.5% quenching of tritium under the conditions periment carried out in the absence of ascorbate and PMS. The data described, control experiments were carried out in the absence have been corrected for a control performed in the absence of mem- of membrane vesicles, and the data were corrected appro- brane vesicles as described in Methods. priately. appears in the dialysate, increasing linearly for about 2 min, and Determination of ApH. ApH.was determined by measuring reaching a maximum which then decreases at a slow and con- the accumulation of acetate, propionate, butyrate or DMO by stant rate (open symbols). When ascorbate and PMS are added flow dialysis unless otherwise stated. Data were quantitated to the upper chamber (closed symbols), the vesicles accumulate assuming that dialysis rates obtained after addition of nigericin acetate, and its concentration in the dialysate decreases mark- (Fig. 1) represent 0 ApH. edly to about 60% of the level observed in the absence of elec- Determination of AI. The electrical potential across the tron donor. Addition of valinomycin, an ionophore which membrane (Au') was determined by measuring the accumu- specifically increases the potassium permeability of the mem- lation of [3H]triphenylmethylphosphonium (TPMP+) by either brane (27), causes the vesicles to accumulate more acetate, and filtration (6) or flow dialysis. its concentration in the dialysate decreases still further to about Calculations. Concentration gradients for solutes taken up 50% of the control level. Finally, when nigericin is added, ac- by the vesicles were calculated using a value of 2.2 ,ul of intra- etate is released from the vesicles, and the external concentra- vesicular fluid per mg of membrane protein (25). Internal pH tion returns to the control level, an observation which is con- was calculated as described by Waddel and Butler (19), and sistent with the notion that nigericin catalyzes an electrically ApH was determined by difference. The electrical potential neutral exchange of potassium for protons, and thus collapses (A') was calculated from the Nernst equation (A' = 58.8 log ApH (28). [TPMP+]n/[TPMP+]out) using steady-state concentration values Using the equilibrium concentration of acetate observed in obtained from TPMP+ uptake experiments. The proton elec- the dialysate after addition of ascorbate-PMS (i.e., fraction 25), trochemical gradient (AUH+) was calculated by substituting it can be calculated that the vesicles take up approximately 1.8 calculated values for A* and ApH into equation (1). nmol of acetate per mg of membrane protein. Making the Protein Determinations were carried out as described (26). calculations described in Methods, this value represents an Chemicals. [3H]Triphenylmethylphosphonium bromide was internal pH of 7.5. Since the external pH is 5.5, it is clear that prepared by the Isotope Synthesis Group at Hoffmann-La the vesicles generate a ApH (interior alkaline) of approximately Roche, Inc. under the direction of Dr. Arnold Liebman as de- 2 units under these conditions. scribed (6). Other isotopically labeled materials were purchased Previous attempts to observe a ApH in this in vitro system from New England Nuclear and Amersham-Searle. Vali- with DMO and standard assay techniques have been negative nomycin and carbonylcyanide m-chlorophenylhydrazone (1, 6). These observations are documented in Table 1 where it (CCCP) were obtained from Calbiochem. Nigericin was the it shown that DMO is not accumulated to any extent when generous gift of Dr. J. Berger of Hoffmann-La Roche, Inc. uptake is assayed by filtration or rapid centrifugation. With flow dialysis, however, the ApH observed with DMO is similar RESULTS to that obtained with acetate. Moreover similar ApH values are obtained with propionate and butyrate, whereas filtration Determination of ApH gives values which are increasingly lower for propionate and Fig. 1 depicts a typical flow dialysis experiment carried out as butyrate relative to acetate. Presumably, the low values ob- described in Methods. Shortly after [3H]acetate is added to the tained by filtration and centrifugation are due to the high upper chamber containing membrane vesicles, radioactivity passive permeability of the vesicle membrane to weak acids Downloaded by guest on October 2, 2021 1894 Biochemistry: Ramos et al. Proc. Natl. Acad. Sci. USA 73 (1976) Table 1. Uptake of different weak acids by E. coli ML 308-225 membrane vesicles as determined by various techniques Uptake assayed by: Flow dialy- Filtrationt Centrifuga- Acid sis* (mV) (mV) tiont (mV) z 0. Acetate -114 (3.9) -23.4 (1.1) -46 (0.5) Propionate -110 (3.2) -3.3 (0.5) n.d. E z Butyrate -118 (4.3) -2 (0.4) n.d. w9 DMO -127 (6.9) 0 (0) 0 (0) Values given in parentheses represent uptake of the weak acids in nmol/mg of membrane protein; n.d., not determined. * Flow dialysis was performed as described in Methods and in Fig. 1 using sodium [3H]acetate (685 mCi/mmol), sodium [1-'4C]pro- pionate (53 mCi/mmol), sodium [1-14C]butyrate (25.8 mCi/ mmol), or [2-14C]DMO (11 mCi/mmol) at final concentrations of 37.5, 37.5, 37.5, and 200 gM, respectively. Internal pH was calculated as described by Waddel and Butler (19). The pK EXTERNAL pH values used in the calculations were as follows: acetate, 4.75; propionate, 4.87; butyrate, 4.81; and DMO, 6.3. ApH was cal- FIG. 2. Effect ofexternal pH on ApH, internal pH, /v, and AjH+. culated and converted to mV as described in Methods. ApH values (0-0) were calculated from flow dialysis experiments t Filtration assays were performed as described previously (21, 23) carried out with [3H]acetate at the pH values given as described in and in Methods with the same isotopically labeled weak acids as Fig. 1 and Methods. Internal pH (v-v) was calculated from values given above (except that sodium [1,2-14C]acetate at a specific for acetate uptake determined at each pH as described in Methods. activity of 57 mCi/mmol was used in place of sodium [3H]acetate) AdI values (0-*) were calculated from flow dialysis experiments at final concentrations of 200 ,uM. A value of zero indicates that carried out with [3H]TPMP+ (1.33 Ci/mmol) at a final concentration the experimental value was insignificantly different from a con- of 24MuM as described in Fig. 1 and Methods, and from filtration as- trol sample that has been diluted 20-fold before addition of says. The filtration assays were carried out after 5 and 10 min incu- radioactive solute and filtered and washed (23). Attempts were bations with ascorbate (20 mM) and PMS (0.1 mM) performed as also made to carry out filtration assays without washing the described previously (6) except that [3H]TPMP+ (1.33 Ci/dimol) was samples on the filters. Results obtained in this fashion were so used at a final concentration of 24 ,M. Similar results were obtained variable that the experiments were not reproducible. using flow dialysis and filtration assays. A4H+ values (A-A) were t Centrifugation assays were carried out in the following manner: calculated from ApH and A'I as described in Methods. aliquots of membrane vesicles (50 ,l containing 0.2-0.4 mg of membrane protein) were diluted to a final volume of 100 Al con- taining (in final concentrations) 0.05 M potassium phosphate Effect of external pH on ApH, A', internal pH, and (pH 5.5) and 0.01 M magnesium sulfate. The samples were in- AAH+ cubated in an oxygen atmosphere at 250 for about 30 sec, and ascorbate and PMS were added to final concentrations of 20 mM It is apparent from Fig. 2 that ApH varies markedly with ex- and 0.1 mM, respectively. Immediately thereafter, sodium ternal pH, as reported by Padan et al. (17) for intact cells. From [3H]acetate (685 mCi/mmol) or [2-14C]DMO (11 mCi/mmol) pH 5.0 to 5.5, ApH remains almost constant at -114 to -113 were added to final concentrations of 37.5 and 200 AM, respec- mV; above pH 5.5, ApH decreases drastically, and is negligible tively. The incubations were continued for 5 min, at which time at pH 7.5 and above. Since ascorbate-PMS-dependent the samples were transferred as rapidly as possible to centrifuge tubes by means of a Hamilton syringe (5-10 sec). Each sample [14C]methylamine uptake is not observed at pH 7.5 or above was then centrifuged in a Beckman Airfuge at about 160,000 x g (data not shown), ApH does not become reversed in the vesicles for about 15 sec (a total of approximately 2 min was required at high external pH as reported for intact cells (17). Signifi- from the time the tubes were placed into the centrifuge until they cantly, despite marked variation in ApH as a function of ex- were removed). The supernatants were discarded, and the pellets ternal pH, internal pH remains essentially constant at pH 7.5, were resuspended in 100 ,A water and aliquots (70 Ml) were assayed for radioactivity. Values were corrected for a control sample and AI ranges from a low of about -65 mV at pH 5.0 to a high treated identically in the presence of 1 MM nigericin and 5 AM of about -75 mV at pH 7.0. As a result of these individual valinomycin. variations, AOH+ exhibits a maximum value of -180 mV at pH 5.5 (-110 mV ApH + -70 mV AI) which decreases to a con- (filtration) and to the sensitivity of ApH to oxygen tension stant value of about -75 mV at pH 7.5 and above (O ApH + (centrifugation). In any case, it should be emphasized that such -75 mV A'i). differences are not observed with TPMP+ or lactose which are accumulated to the same extent when assayed by filtration or Effect of various electron donors on ApH, AI, and flow dialysis. Finally, ApH values obtained by flow dialysis are constant over at least a 100-fold range of weak acid concen- Although there is little relationship between the ability of the trations and over a range of membrane protein concentrations vesicles to oxidize an electron donor and the ability of that from 1 to 3 mg/ml;.and none of the weak acids utilized is me- electron donor to drive active transport, a qualitative correlation tabolized by the vesiclest. exists between the ability of various electron donors to drive transport and their ability to generate a AI (interior negative) t Membrane vesicles were incubated for 10 min with each of the weak (6). The data presented in Table 2 corroborate the latter oh- acids as described in Table 1. Aliquots of the reaction mixtures were servation and demonstrate further that a similar relationship chromatographed on thin-layer chromatography plates coated with ascorbate-PMS silica gel G and radioautographed as described previously (29), except exists with respect to ApH and AAH+. Clearly, that the solvent system used was water-saturated ethyl ether/ and D-lactate produce maximal relative values for each pa- ammonium hydroxide (7:1, vol/vol). rameter, whereas succinate and especially NADH produce Downloaded by guest on October 2, 2021 Biochemistry: Ramos et al. Proc. Natl. Acad. Sci. USA 73 (1976) 1895 Table 2. Effect of various ides atpH 5.5 or 7.5, ascorbate-PMS-dependent TPMP+ uptake electron donors on ApH, A4', and 4LH+ decreases to approximately 10% of the control value at 2.5-5 AM valinomycin (Fig. 3A), corresponding to a decrease in A' (mV) from about -70 mV to -20 mV (insert). Strikingly, there is an Electron donor ApH* AV 4 increase in acetate uptake to about 130% of the control value at 1 MM valinomycin [an increase in ApH of about 10 mV (in- Ascorbate-PMS -115 -74 -189 sert)] which remains constant,at higher valinomycin concen- D-Lactate -102 -70 -172 trations. The effect of valinomycin on the total driving force Succinate 0 -64 -64 at pH 5.5 is relatively small, producing only about a 20% loss NADH 0 0 0 at 5 MM valinomycin [from a AMH+ of about -190 mV to-150 NADH + Q1 -59 -62 -131 mV (insert)]. As shown in Fig. 3B, nigericin induces effects which are * ApH was calculated from flow dialysis experiments carried out opposite to those of valinomycin. Acetate uptake decreases with sodium [6H]acetate (685 mCi/mmol) at a final concentration to of i8 AM as described in Methods and in Fig. 1. Ascorbate and 0 as the nigericin concentration is increased from 0 to 0.1 AM PMS, lithium-D-lactate, sodium succinate, NADH (sodium salt), [from a ApH of-110 mV to 0 (insert)], whereas TPMP+ uptake and ubiquinone-1 (Q1) were used at final concentrations of 20 mM increases almost 2.5-fold over the same concentration range and 0.1 mM, 20,mM, 20 mM, 5 mM, and 0.08 mM, respectively. [from a AI of about -60 mV to -90 mV (insert)]. Despite t A'I was determined from filtration assays performed after 5 and marked effect on ApH and A+, 0.1 MM nigericin produces 1G min incubations as described previously (6) except that only [3H]TPMP+ (1.33 Ci/mmol) was used at a final concentration of a 45% decrease in the total driving force at pH 5.5 [from a AMH+ 24 AM. Electron donors were used at the concentrations given of -170 mV to -90 mV (insert)]. It is also apparent that this above, and the data were corrected f6r the amount of TPMP+ ionophore has no effect on TPMP+ uptake at pH 7.5, a finding taken up in the absence of exogenous electron donors. which is consistent with the absence of a ApH at this external t AUH + was calculated as described in Methods. pH (see Fig. 2). The proton conductor CCCP inhibits both TPMP+ and ac- much weaker effects. Moreover, when ubiquinone-1 is added etate uptake at pH 5.5 over a concentration range from 0 to 1 to the vesicles, NADH generates much higher values. This ob- MM CCCP, and diminishes the total driving force at pH 5.5 by servation is highly significant in that NADH oxidation under approximately 60% (a decrease in AAH+ from -170 mV to these conditions drives transport in the presence of ubiqui- about -65 mV at 1 MM CCCP) (Fig. SC). It is also noteworthy none-i, but not in its absence (11). that CCCP is significantly more effective at pH 5.5 relative to pH 7.5, and that at pH 5.5, ApH (acetate uptake) is inhibited Effect of valinomycin, nigericin, and CCCP on 4pH, more markedly than A' (TPMP+ uptake). The extent of in- AI, and AMH+ hibition of AAH+ observed at 1 AM CCCP is similar to that ob- As increasing concentrations of valinomycin are added to ves- served with many of the vesicular transport systems (30).

FIG. 3. Effect of valinomycin (A), nigericin (B), and CCCP (C) on ApH, ASip, and UH+. ApH was determined by flow dialysis in the presence of acetate and ascorbate-PMS at pH 5.5, and the indicated concentrations ofvalinomycin, nigericin, or CCCP as described in Fig. 1 and ii Methods. Steady-state levels of TPMP+ accumulation (A+) were determined at pH 5.5 (A-A) and pH 7.5 (v-v) in the presence ofgiven concentrations of valinomycin, nigericin, or CCCP by filtration assays as described in Fig. 2. The effect of the inhibitors on the total driving force at pH 5.5 (AiH+, 0-0) was calculated from ApH and AI at each inhibitor concentration. The percentage of the total driving force remaining at each inhibitor concentration is the percentage of AAH+ measured in the absence of inhibitors. The following control values (100%) were obtained in the absence ofinhibitors (nmol/mg ofmembrane protein): acetate uptake 1.6; TPMP+ uptake, 0.80 (at pH 5.5) and 0.91 (at pH 7.5). The mV values plotted in the inserts were calculated from the experimental values presented. Downloaded by guest on October 2, 2021 1896 Biochemistry: Ramos et al. Proc. Natl. Acad. Sci. USA 73 (1976) DISCUSSION 1. Kaback, H. R. (1972) Biochim. Blophys. Acta 265,367-416. that E. ML 308-225 membrane 2. Kaback, H. R. & Hong, J.-s. (1973) in CRC Critical Reviews in These results demonstrate coli Microbiology, eds. Laskin, A. I. & Lechevalier, H. (CRC Press, vesicles generate a considerable ApH when assayed under the Ohio), Vol. 2, pp. 333-376. proper conditions. When considered together with certain 3. Lombardi, F. J., Reeves, J. P., Short, S. A. & Kaback, H. R. (1974) previous observations (1, 6) it seems eminently clear that some Ann. N.Y. Acad. Sci. 227,312-327. of the apparent shortcomings of the chemiosmotic hypothesis 4. Kaback, H. R. (1974) Science 186,882-892. are resolved. To wit, AIH+ is thermodynamically sufficient to 5. Patel, L., Schuldiner, S. & Kaback, H. R. (1975) Proc. Natl. Acad. account for the magnitude of solute accumulation by the ves- Sci. USA 72,3387-3391. icles at pH 6.6, where most of the transport studies have been 6. Schuldiner, S. & Kaback, H. R. (1975) Biochemistry 14,5451- carried out. 5461. The findings also have an immediate bearing on the question 7. Mitchell, P. (1967) Nature 191, 144-148. the 8. Mitchell, P. (1966) Biol. Rev. Cambridge Philos. Soc. 47,445-502. of the polarity of bacterial membrane vesicles prepared by 9. Mitchell, P. (1973) J. Bioenerg. 4,63-91. methods developed in this laboratory (21, 22). Since the AaSH+ 10. Harold, F. M. (1972) Bacteriol. Rev. 36, 172-230. established by the vesicles is at least as high as that reported for 11. Stroobant, P. & Kaback, H. R. (1975) Proc. Natl. Acad. Sci. USA intact E. colh (17), it seems extremely unlikely that a significant 72,3970-3974. number of the structures can be inverted or seriously damaged. 12. Reeves, J. P. (1971) Biochem. Biophys. Res. Commun. 45, Finally, this work has provided a framework within which 931-936. to test other more specific aspects of the relationship between 13. Hirata, H., Altendorf, K. & Harold, F. M. (1973) Proc. Natl. Acad. A4H+ and active transport. Although space limitations preclude Sci. USA 70,1804-1808. Chem. a detailed presentation or discussion of the data (S. Ramos and 14. Altendorf, K., Hirata, H. & Harold, F. M. (1975) J. Biol. H. R. Kaback, manuscript in preparation), it should be obvious 250, 1405-1412. titrations of various transport 15. Schuldiner, S., Rudnick, G., Weil, R. & Kaback, H. R. (1976) that valinomycin and nigericin Trends in Biochemical Sciences, 1, 41-45. activities at pH 5.5 and pH 7.5 should yield considerable insight 16. Lombardi, F. J. & Kaback, H. R. (1973) J. Biol. Chem. 247, into the coupling between individual components of AAH+ and 7844-7857. the accumulation of particular solutes. Such studies have been 17. Padan, E., Zilberstein, D. & Rottenberg, H. (1976) Eur. J. Bio- performed with many vesicular transport systems, and it is clear chem., 63,533-541. that the transport systems fall into two groups when assayed at 18. Rottenberg, H. (1975) J. Bioenerg. 7,61-74. pH 5.5, i.e., those that are driven by ASH+ and those that are 19. Waddel, W. J. & Butler, T. C. (1959) J. Clin. Invest. 38,720-729. driven by ApH. It is also significant that each of the transport 20. Colowick, S. P. & Womack, F. C. (1969) J. Biol. Chem. 244, systems exhibits considerable activity at pH 7.5 where 774-777. AAH+ 21. Kaback, H. R. (1971) in Methods in Enzymology, ed. Jakoby, a Ai component, and that valinomycin is composed solely of W. B. (Academic Press, New York, New York), Vol XXII, pp. or nigericin inhibits, stimulates, or produces no effect de- 99-120. pending upon the external pH. These effects are particularly 22. Short, S. A., Kaback, H. R. & Kohn, L. D. (1975) J. Biol. Chem. striking with respect to the active transport of organic anions. 250,4291-4296. With glucose-6-P, for example, there is dramatic stimulation 23. Kaback, H. R. (1974) in Methods in Enzymology, eds. Fleischer, of accumulation by valinomycin at pH 5.5, and dramatic in- S. & Packer, L. (Academic Press, New York, New York), Vol. hibition at pH 7.5. Nigericin, on the other hand, inhibits glu- XXXI, pp. 698-709. cose-6-P accumulation at pH 5.5, but has no effect at pH 7.5. 24. Schuldiner, S., Weil, R. & Kaback, H. R. (1976) Proc. Natl. Acad. Thus, it is clear that anion transport is driven by ApH at pH 5.5 Sci. USA 73, 109-112. 25. Kaback, H. R. & Barnes, E M., Jr. (1971) J. Biol. Chem. 246, or 7.5. by A*I at pH 5523-5531. 26. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. We are indebted to Dr. Hagai Rottenberg of Tel Aviv University for 27. Harold, F. M. (1970) Adv. Microbiol. Physiol. 4,45-104. suggesting the use of acetate, and for helpful discussions. We are also 28. Harold, F. M., Altendorf, K. H. & Hirata, H. (1974) Ann. N.Y. indebted to Dr. Etana Padan of The Hebrew University for allowing Acad. Sci. 235, 149-160. us access to her data prior to publication. Finally, we thank Dr. Arnold 29. Kaback, H. R. & Milner, L. S. (1970) Proc. NatI. Acad. Sci. USA Liebman and The Isotope Synthesis Group of Hoffmann-La Roche for 66, 1008-1012. the preparation of [3H]TPMP+. S. R. is a postdoctoral fellow of the 30. Kaback, H. R., Reeves, J. P., Short, S. A. & Lombardi, F. J. (1974) Ministerio de Educacion y Ciencia of Spain. Arch. Biochem. Biophys. 160,215-222. Downloaded by guest on October 2, 2021