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Stoichiometry of H+ ejection coupled to electron transport through site 2 in ascites tumor mitochondria
ARCHIVES Vol. 205,
OF BIOCHEMISTRY No. 1, November,
Stoichiometry
BIOPHYSICS
210-216,
1980
of H+ Ejection Coupled to Electron Transport Site 2 in Ascites Tumor Mitochondria’
ANTONIO Department
AND
pp.
VILLALOB02
ALBERT
AND
L. LEHNINGER3
of Physiological Chemistry, The Johns Hopkins University School 725 North Wolfe Street, Baltimore, Maryland 21205 Received
April
through
of Medicine,
9, 1980
The stoichiometry of H+ ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and ASSO-D hepatoma cells was determined. Values close to 4.0 for the H+/%ejection ratio were found in both cell lines, with either Ca*+ or K+ (+valinomycin) as charge-compensating permeant cation. The 4 H+ ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)63to 2 Fe(CN)G”-, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and(c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H+ ejection occurred, thus eliminating possible overestimation of the H+/%ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H+/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the H+/Ze- stoichiometry for site 2 in normal rat liver mitochondria.
Earlier reports from this laboratory have provided evidence that the average number of H+ translocated into the medium per pair of electrons passing through each of the three energy-conserving sites of the respiratory chain (i.e., the H+/site ratio) of rat liver mitochondria is close to 4.0 (l-9). This information has come from stoichiometric studies of sites 2 + 3, as represented by electron flow from succinate to oxygen (24), of sites 1 + 2 + 3, represented by electron flow from NADH to oxygen (5), of site 3 alone, represented by electron flow from cytochrome (6, 7), of site 2 alone, as represented by electron flow from succinate to ferricyanide (8), and of sites 1 + 2 (9). Simi1 This work was supported by a grant from the National Cancer Institute (CA25360) and a Public Health Service International Research Fellowship to A. V. (1 F05 TW02585). 2 Present address: Laboratoire D’Enzymologie, Universite de Louvain, 1348 Louvain-La-Neuve, Belgium. a To whom reprint requests should be sent. 4 J. Benavides and A. L. Lehninger, unpublished observations. 0003-9861/80/130210-07$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
lar observations have been made independently by Azzone et al. (10, 11). Moreover, mitochondria isolated from rat heart (12), mouse Ehrlich ascites tumor, and rat AS30D cells (13), and rat brain,4 also have yielded average H+/site ejection ratios close to 4 for sites 2 + 3 (succinate * 02) and sites 1 + 2 + 3 (NADH + 0,). In this paper we report the results of stoichiometric studies of H+ ejection and permeant cation uptake coupled to electron flow through energy-conserving site 2 of the respiratory chain of mitochondria isolated from two malignant cell lines: the mouse Ehrlich ascites tumor and the rat ASSO-D ascites tumor, derived from a hepatoma. These experiments, in which succinate was used as electron donor and ferricyanide as electron acceptor, yielded observed H+/2eejection ratios very close to 4.0 and cation+1 2+- uptake ratios close to 2.0, in exact agreement with data on site 2 reported earlier on normal rat liver mitochondria (8). These observations not only provide new data on the stoichiometry of respiration-coupled H+ transport in mitochondria from malignant
H+ EJECTION
IN SITE 2 OF TUMOR
cells, but also add to the increasing evidence that the H+/site ratio is close to 4 in mitochondria from animal tissues generally. EXPERIMENTAL
DETAILS
The line of Ehrlich ascites tumor cells was originally supplied by Dr. E. L. Coe, Department of Biochemistry, Northwestern University Medical School, Chicago, Illinois. The AS30-D rat hepatoma in ascites form was obtained from Dr. E. F. Walborg, Department of Biochemistry, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. Inoculation of 0.1-0.2 ml ascitic fluid from donor mice bearing the AS30-D hepatoma was carried out in groups of 20 young adult male Swiss albino mice (Buckberg Labs, Tomkins Cove, N. Y.) and groups of 5 or 6 young adult male albino rats from Sprague-Dawley, Inc., Madison, Wisconsin, respectively. The tumor cells were harvested 1 week after inoculation. After sacrifice of the animals the ascitic fluid was collected and filtered twice through a double layer of gauze to remove clumped cells. The peritoneal cavity was washed with a cold solution of 150 mM NaCl, 5 mM KCl, and 20 mM Tris-chloride (pH 7.4) and the washings added to the filtered ascitic fluid. The cells were recovered by centrifugation at SOg for 4 min and washed three times with a total of about 240 ml of the above medium. The cells were washed once more with cold 0.25 M sucrose and collected by centrifugation at 6209 for 2 min. Such cells were used directly to prepare tumor mitochondria as has been previously described (13). Alternatively, the last washing of the ascites cells was performed with a medium (designated STE) of 0.25 M sucrose, 1 mM Tris-EGTA,” 5 mM Tris-chloride (pH 7.4), followed by centrifugation at 3000g for 3 min. Washing of the cells in the STE medium gave higher yields of mitochondria as well as slightly higher acceptor control ratios. The cells from either procedure were diluted to a total volume of 60 ml in the STE medium and preincubated for 8 min at 0°C with 1 mg Nagarse (Enzyme Development Corp., New York, N. Y.) with occasional gentle stirring. The cells were then washed three times with a total volume of 80 ml of ‘70 mM sucrose, 210 mM mannitol, 2.1 M K-HEPES, pH 7.4, and 0.1% fatty acid-free bovine serum albumin (designated H-medium) and centrifuged at 30009 for 3 min. The pellet was suspended in approximately 200 ml of H-medium and 40-ml batches were homogenized, using a tightly fitting i Abbreviations used: EGTA, ethylene glycol bi@aminoethylether)N,N,N’,N’-tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; STE medium, 0.25 M sucrose, 1 mM Tris-EGTA, 5 mM Tris-chloride; H-medium, 70 mM sucrose, 210 mM mannitol, 2.1 M K-Hepes, pH 7.4, 0.1% fatty acid-free bovine serum albumin.
MITOCHONDRIA
211
Teflon pestle rotated at 500 rpm. Five or six up-anddown strokes were used for each batch. Centrifugation of the homogenized cell suspension was performed three times at 480s for 5 min in order to remove the nuclear fraction and unbroken cells completely and to obtain as large a volume of the supernatant suspension as possible. The mitochondrial fraction was then collected by centrifugation at 12,000g for 10 min and the pellet resuspended in H-medium. Two more centrifugations were performed at 14,500g 10 min in order to wash the mitochondrial fraction, which was finally resuspended in a minimum volume of H-medium. A typical preparation contained a final protein concentration (14) of about 80-100 mg protein/ml, with a total yield of mitochondrial protein of about 50-100 mg. The acceptor control ratio of these preparations ranged from 2.5 to 4.0 using succinate as substrate and ADP as acceptor. The reactions were carried out in thermostatted glass cells of 2- to 3-ml capacity at the temperatures given in the legends. A combination pH electrode (Thomas 4094 L25) and, when used, a Ca*+-selective electrode (Radiometer, F2112 Calcium Selectrode) were inserted into the cells through independent openings. The electrode outputs were amplified through Beckman Expandomatic SS2 pH meters and fed into a dual-channel Sargent-Welch DSRG recorder. Internal standards of HCl and CaCl,, comparable in amounts to the changes being measured, were added to calibrate the pH and Ca2+ electrodes, respectively. Other experimental conditions of the incubations are given in the legends. RESULTS
The Hi12e- Ejection Ratio and Cationl2eUptake Ratios with Ca2+ as ChargeCompensating Cation
The first series of experiments was carried out on mitochondria isolated from Ehrlich ascites tumor cells. Mitochondria were preincubated aerobically in the buffered medium with rotenone to prevent electron flow from endogenous NAD(P)-linked substrate(s), oligomycin to prevent H+ movements associated with ATP synthesis or hydrolysis, and N-ethymaleimide to prevent underestimation of the H+/2e- ejection ratio due to rapid H+ reuptake with phosphate on the H+/H,PO; symport carrier (1, 3, 6, 15). After the short (1-3 min) aerobic preincubation to deplete possible endogenous flavinlinked substrate(s), neutralized KCN was added to block electron flow through cyto-
212
VILLALOBO
AND LEHNINGER
chrome oxidase. Succinate was then added, followed 30 s later by Ca*+. After the electrode traces became stable a known amount of ferricyanide (>99% purity) was added as electron acceptor and the ensuing H+ ejection and Ca*+ uptake recorded simultaneously from the same vessel. Since these experiments were of the pulse type, the total amount of H+ ejected (in ng-ion H+) obtained from the trace was divided by one-half the amount of ferricyanide added (in nmol) to give the number of H+ ejected for each 2eflowing from succinate to ferricyanide via FIG. 1. The H+/%- ejection and Ca*+/2.e- uptake mitochondrial cytochrome c, i.e., the H+/&ratios with Ca*+ as charge-compensating cation. The ejection ratio. test system (3.4 ml, 24.5%) contained sucrose (124 A typical experiment is shown in Fig. 1. mM), KC1 (60 mM), K-HEPES (3 mM, pH ‘7.l), rotenone Upon addition of 200 nmol of ferricyanide (1 nmol mg-I), oligomycin (0.5 pg mg-I), N-ethylmaleivery rapid ejection of H+ took place, fol- mide (40 nmol mg-I), Ehrlich tumor mitochondria (20 lowed by a slow back-decay of H+ to half the mg protein), and CaCl, (100 nmol mg-I). KCN (4.0 mM) extent of the total H+ ejected. The total was added after a short aerobic preincubation, followed amount of H+ ejected was extrapolated by succinate (0.55 mM). After the H+ and Ca2+ traces from the ejection and back-decay curves as became stable a pulse of 200 nmol of ferricyanide was shown, giving an Hf/2e- ejection ratio of added and H+ ejection and Ca*+ uptake recorded simultaneously. Where indicated, antimycin A (0.2 nmol 400 + l/2(200> = 4.0. Without the correcmg-‘) or FCCP (2 pM) were added. Also shown are tion for H+ back-decay, the H+/2e- ejection traces from an experiment in which succinate was ratio taken from the observed H+ peak (Fig. omitted from the system. 1) was 3.75; thus the correction for backdecay is only about +6%. Figure 1 also shows that when succinate Table I summarizes data collected from was omitted no H+ ejection or Ca*+ uptake many such experiments on Ehrlich ascites H+/2e- ejection ratios took place, indicating that no coupled elec- tumor mitochondria; tron flow from endogenous substrates oc- close to 4.0 and Ca*+/&- uptake ratios of curred. The rotenone block thus prevented approximately 1.0 were consistently observed, Similar experiments carried out on interfering electron flow from possible endogenous NAD(P)-linked substrates. AS30-D hepatoma mitochondria under esMoreover, it is also seen that antimycin A sentially the same conditions also gave valinhibited electron flow from succinate to ues of the H+/2t- ejection ratio very close to ferricyanide over 97% at the time of the 4.0 (Table I). In experiments such as that in peak H+ ejection, demonstrating that no Fig. 1 a succession of ferricyanide pulses significant reduction of ferricyanide takes could be added to the same system, all giving place at the expense of electron donors on nearly identical H+/2e- ratios close to 4.0. In the substrate side of the antimycin block such experiments the ferricyanide pulses were added only after the back-decay of H+ under the conditions used. In the presence of the H+-conducting uncoupler FCCP (Fig. from the preceeding pulse to the new baseline (H+/&- = 2) was complete. 1) a net of 1.99 H+ was formed per 2e- transferred, very close to the expected value 2.0 (8). The 2 H+ released in the presence of The H+lZe- Ejection Ratio with K+ FCCP ultimately arise from the methylene (+Valinom&in) as Chargecarbons of succinate by the action of succiCompensating Cation nate dehydrogenase; net electrogenic translocation of H+ from the matrix to the meExperiments were also carried out in dium is abolished in the presence of FCCP. which Ca*+ was replaced with 60 InM K+
H+ EJECTION
213
IN SITE 2 OF TUMOR MITOCHONDRIA TABLE
I
THE H+/%- EJECTION RATIO AND THE CATION/%- UPTAKE RATIO IN SITE 2 EXPERIMENTS”
Tumor Ehrlich
AS30-D hepatoma
Permeant cation Ca2+ (11) Caz+ (3) K+ ( + valinomycin) (5) K+ (+valinomycin) (2) None (4) Ca*+ (2) Ca2+ (1)
Other conditions No NEM No rotenone FCCP No NEM
H+/2eEjection ratio
Ca2+/% Uptake ratio
4.03 f 0.09 3.11 3.98 + 0.02 3.85 1.97 i 0.03
1.02 k 0.09 0.97
4.00 2.90
a The test system (3.0-3.5 ml at 25°C) contained sucrose (125 mM), KC1 (60 mM), K-HEPES (3 mM, pH 7.1), rotenone (2 to 4 nmol mg-I), KCN (l-4 mM), oligomycin (0.5 pg mg-I), CaCl, where indicated (100-600 ng-ion mg-I), valinomycin where indicated (50-200 ng mg-I), N-ethylmaleimide (40 nmol mg-*), succinate (0.4 to 0.5 mM), and mitochondria (5-20 mg protein). Ferricyanide was added last at 5 to 10 nmokmg protein. Other conditions are specified. Figures in brackets indicate the number of experiments carried out; standard deviations are given where appropriate.
(+ valinomycin) as charge-compensating permeant cation. Under these conditions K+ moves into the matrix to maintain bulk electroneutrality. A typical set of traces is presented in Fig. 2. Upon addition of a pulse of 100 nmol of ferricyanide a total of 203 ng-ion H+ were ejected, equivalent to an H+/2eejection ratio of 203150 = 4.06. A similar experiment in which FCCP replaced valinomycin resulted in a net H+/%- ejection ratio of 103/50 = 2.06, in agreement with the expected value 2.0 and with the data in Fig. 1. Figure 2 also shows that in the absence of succinate no H+ ejection took place. When antimycin A was included in the system, to block electron flow through site 2, only a very slow H+ ejection took place in the presence of FCCP, again indicating that endogenous substrates on the reducing side of the antimycin block do not react with ferricyanide at a significant rate under the conditions employed. The H+/%- ejection ratio for the span succinate to ferricyanide was also measured in a system not containing rotenone, after the mitochondria were first depleted of endogenous substrates. Under these conditions several experiments gave an H+/2eejection ratio of 3.85 + 0.03, essentially the same as when rotenone was present (H+/&= 3.94 2 0.05; see Table I).
Table I summarizes the average H+/2eejection ratios of a number of experiments carried out under a variety of conditions, in the presence of either Ca2+ or K+ (+valinomycin) as permeant cations. The average H+/2e- ejection ratio was very close to 4.0. The Ca2+/2e- uptake ratio, as indicated above, was very close to 1.0 in a number of experiments, equivalent to uptake of two compensating positive charges per 2e-. Table I also shows the H+/%- ejection ratios in experiments in which N-ethylmaleimide was omitted from the system, conditions under which underestimation of the H+/2eejection ratio was expected (1,3,6, 15). The H+/&- ejection ratio under these conditions was reduced to slightly over 3, due to the rapid reuptake of H+ with phosphate in response to the matrix alkalinity generated by electron flow. Nevertheless, the Ca2+/2euptake ratio was unchanged in these conditions, as expected, since reentry of H+ by H+/phosphatesymport is electroneutral (4). As was shown above in Figs. 1 and 2, addition of FCCP to the complete system resulted in the appearance of only 2 H+ per 2~~. In other experiments in which neither Ca2+ nor valinomycin was present, and EGTA was included to bind Ca2+ from endogenous sources, the presence of FCCP
214
VILLALOBO
AND LEHNINGER
again yielded an observed H+/2e- ejection ratio of 1.97 & 0.03, very close to the theoretical value of 2.0. Table I also shows some data for mitochondria from the AS30-D tumor, which gave results almost identical to those in the Ehrlich mitochondria. Further stoichiometric data on the AS30-D and mitochondria from other tumor cell livers are being collected. DISCUSSION
The H+/2e- ejection ratios close to 4.0 reported here for the segment succinate + ferricyanide in mitochondria from the Ehrlich ascites and AS30-D tumors are in complete agreement with our earlier report showing that these mitochondria yield average H+/site ejection ratios close to 4 for the spans succinate + 0, and NADH + 02, which encompass energy-conserving sites 2 + 3 and 1 + 2 + 3, respectively (13). The H+/2e- ratios reported here also agree with earlier data from this laboratory on site 2 in rat liver mitochondria (8), and with data on site 2 in intact mitochondria reported from other laboratories (10, 11, 16-20). The experiments reported here also remove a criticism advanced by Mitchell and Moyle (21) on the use of N-ethylmaleimide to inhibit H+ reuptake on the H+/phosphatesymporter. They have proposed that this reagent increases the observed H+ ejection, not because of its well-known capacity to inhibit interfering reuptake of H+ on the phosphate carrier, as has been demonstrated (1, 3, 6, 15, 19), but because it inhibits succinate dehydrogenase and at the same time evokes flow through sites 1 and 2 from endogenous pyridine-linked substrates, despite the presence of rotenone. As is shown here, this claim is invalid, since in the absence of succinate no significant endogenous H+ ejection occurred. Moreover, the succinate -+ ferricyanide span yielded an H+/%- ejection ratio of 4 whether or not rotenone was present. Although there is general agreement that the observed H+/2e- ejection ratio for site 2 as reflected by the span succinate + ferricyanide is 4.0 (7, 10, 16, B-20), there has
Ferrwonide
(100)
plus FCCP
H+/&- =+=
2.06
FIG. 2. The H+/2e- ejectionratio with K+ (+valinomycin) as charge-compensating cation. The test system (2.5 ml at 25°C) contained the same sucrose-KCI-KHEPES medium as in Fig. 1, rotenone (2 nmol mg-I), N-ethylmaleimide (40 nmol rng-I), oligomycin (1 pg mg-I), valinomycin (200 ng mg-I), and Ehrlich tumor mitochondria (20 mg protein). After a short aerobic preincubation period KCN (2 mM) was added, followed by succinate (0.55 mM). A pulse of 100 nmol ferricyanide was then added to start the reaction and H+ ejection was recorded. Where indicated, antimycin A (0.4 nmol mg-‘) and FCCP (1.0 PM) were added. A trace is also shown in which no succinate was added to the system.
been some disagreement as to the meaning of this value. Mitchell has postulated a mechanism for H+ ejection coupled to sites 2 + 3, the Q cycle (22, 23), which proposes that 2 of the 4 H+ ejected as 2~ flow from succinate to ferricyanide represent the action of an outward-directed H+-transporting arm corresponding to the inward-directed electron-transporting arm furnished by cytochrome oxidase, which he regards as intrinsically unable to transport protons. However, the necessity for this proposal has been removed by recent demonstrations in several laboratories that H+ ejection is in fact coupled to electron flow through cytochrome oxidase (6, 7, 9, 11, 18-20, 25-27). There are additional uncertainties regarding the specific mechanisms involved in the ejection of 4 H+ as 2~ pass from succinate to ferricyanide. First, the precise sequence of the electron carriers in this complex segment of the respiratory chain is still not known. Second, another problem is posed by the observation previously re-
H+ EJECTION
IN SITE
2 OF TUMOR
ported from this laboratory (6) that the H+/O ratios for the oxidation of the site 2 substrates succinate and a-glycerol phosphate in rat liver mitochondria are identical at close to 8. Since succinate dehydrogenase is located on the M side of the inner membrane and glycerol phosphate dehydrogenase on the C side, it is uncertain to which side the 2 H+ removed from these two substrates by their respective dehydrogenases are delivered and whether they contribute in both cases to the observed 8 H+ ejected. This question is being studied directly by comparison of the H+/2e- ejection ratios for glycerol phosphate and succinate as the substrates with ferricyanide or cytochrome c as electron acceptor. Also to be considered is the probability that the intrinsic H+LW ratio for site 2 is at least 3 and probably 4 when the electron donor is endogenous NADH since the observed H+/%- ratio for the segment NADH + ferricyanide can significantly exceed 7 (9, lo).’ Further investigation is also required to identify the specific electron transport steps in site 2 that yield 4 H+ per %- transported. Recent experiments have indicated that during succinate oxidation by ferricyanide, 2 of the 4 H+ ejected appear during electron flow prior to the point of antimycin A inhibition when N,N,N’,N’-tetramethyl-pphenylenediamine is used to bypass the antimycin block (9, lo).’ Presumably the other 2 H+ are ejected during electron flow through the b-c, complex. The H+/site data reported here, as well as those reported earlier (13), are also germane to further analysis of the profound inhibition of phosphorylating electron transport caused by matrix Ca2+ in mitochondria from these tumor cells (28). Since the H+/site ratio of electron transport in the tumor mitochondria appears to be identical with that in normal mitochondria, some other aspect of oxidative phosphorylation appears to be the locus of the inhibitory action of matrix Ca*+. B A. Villalobo, A. Alexandre, manuscript in preparation. ’ A. Alexandre, F. Galiazzo, J. Biol. Chem., In press.
and A. L. Lehninger, and A. L. Lehninger,
215
MITOCHONDRIA ACKNOWLEDGMENTS
The authors of Irene Wood
acknowledge and Jocelyn
the technical King.
assistance
REFERENCES 1. BRAND, M. D., REYNAFARJE, B., AND LEHNINGER, A. L. (1976) Proc. Nat. Acad. Sci. USA 73,437-441. 2. BRAND, M. D., CHEN, C.-H., AND LEHNINGER, A. L. (1976) J. Biol. Chem. 251, 968-974. 3. REYNAFARJE, B., BRAND, M. D., AND LEHNINGER, A. L. (1976) J. Biol. Chem. 251, 7442-7451. 4. REYNAFARJE, B., AND LEHNINGER, A. L. (1977) Biochem. Biophys. Res. Commun. 77, 12731279. 5. REYNAFARJE, B., AND LEHNINGER, A. L. (1978) J. Biol. Chem. 253, 6331-6334. 6. LEHNINGER, A. L., REYNAFARJE, B., AND ALEXANDRE, A. (1977) in Structure and Function of Energy-Transducing Membranes (vanDam, K. and vanGelder, B. V., eds.) pp. 95-106, Elsevier/North Holland, Amsterdam. 7. ALEXANDRE, A., REYNAFARJE, B., AND LEHNINGER, A. L. (1978)Proc. Nat. Acad. Sci. USA 75, 5296-5300. 8. ALEXANDRE, A., AND LEHNINGER, A. L. (1979) J. Biol. Chem. 254, 11555-11560. 9. LEHNINGER, A. L., REYNAFARJE, B., ALEXANDRE, A., AND VILLALOBO, A. (1979) in Membrane Bioenergetics (Lee, C. P., Schatz, G., and Emster, L., eds.), pp. 393-404, Addison-Wesley, Reading, Mass. 10. POZZAN, T., MICONI, V., DIVIRGILIO, F., AND AZZONE, G. F. (1979) J. Biol. Chem. 254, 10200-10205. 11. AZZONE, G. F., POZZAN, T., AND DIVIRGILIO, F. (1979) J. Biol. Chem. 254, 10206-10212. 12. VERCESI, A., REYNAFARJE, B., AND LEHNINGER, A. L. (1978) J. Biol. Chem. 253, 6379-6385. 13. VILLALOBO, A., AND LEHNINGER, A. L. (1979) J. Biol. Chem. 254,4352-4358. 14. MURPHY, J. B., AND KIES, M. W. (1960) Biochim. Biophys. Aeta 45, 382-384. 15. BRAND, M. D., REYNAFARJE, B., AND LEHNINGER, A. L. (1976) J. Biol. Chem. 251, 5670-5679. 16. MITCHELL, P., AND MOYLE, J. (1967) in Biochemistry of Mitochondria @later, E. D., Kaniuga, A., and Wojtczak, L., eds.), pp. 53-74, Academic Press and Polish Sci. Publ., London and Warsaw. 17. MOYLE, J., AND MITCHELL, P. (1978) FEBS Lett. 88, 268-272.
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AND
18. WIKSTR~M, M., AND KRAB, K. (1978) in Energy Conservation in Biological Membranes (Schafer, G., tid Klingerberg, M., eds.), pp. 128- 139, Springer-Verlag, Berlin. 19. WIKSTR~M, M., AND KRAB, K. (1979) Biochim. Biophys. Acta 549, 177-222. 20. BRAND, M., HARPER, W. G., NICHOLLS, D. G., AND INGLEDEW, W. J. (1978) FEBS Lett. 95, 125-129. 21. MOYLE, J., AND MITCHELL, P. (1978) FEBS Lett. 90,361-365. 22. MITCHELL, P. (1975) FEBS Lett. 59, 137-139. 23. MITCHELL, P. (1976) J. Theor. Biol. 62,327-367. 24. PAPA, S., LORIJSSO, M., GUERRIERI, F., Izzo, G., AND CAPUANO, F. (1978) in The Proton and
LEHNINGER
25.
26. 27. 28.
Calcium Pumps (Azzone, G. F., Avron, M., Metcslfe, J. C., Quagliariello, E., and Siliprandi, N., eds.), pp. 227-238, Elisevier/NorthHolland, Amsterdam. LEHNINGER, A. L., REYNAFARJE, B., AND ALEXANDRE, A. (1978) in Frontiers of Biological Energetics (Dutton, P. L., Leigh, J. S., and Scarpa, A., eds.), pp. 384-393, Academic Press, New York. SIGEL, E., AND CARAFOLI, E. (1978) Eur. J. Biothem. 89, 119-123. CASEY, R. P., CHAPPELL, J. B., AND AZZI, A. (1979) Biochem. J. 182, 149-156. VILLALOBO, A., AND LEHNINGER, A. L. (1980), J. Biol. Chem. 255, 2457-2464.
OF BIOCHEMISTRY No. 1, November,
Stoichiometry
BIOPHYSICS
210-216,
1980
of H+ Ejection Coupled to Electron Transport Site 2 in Ascites Tumor Mitochondria’
ANTONIO Department
AND
pp.
VILLALOB02
ALBERT
AND
L. LEHNINGER3
of Physiological Chemistry, The Johns Hopkins University School 725 North Wolfe Street, Baltimore, Maryland 21205 Received
April
through
of Medicine,
9, 1980
The stoichiometry of H+ ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and ASSO-D hepatoma cells was determined. Values close to 4.0 for the H+/%ejection ratio were found in both cell lines, with either Ca*+ or K+ (+valinomycin) as charge-compensating permeant cation. The 4 H+ ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)63to 2 Fe(CN)G”-, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and(c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H+ ejection occurred, thus eliminating possible overestimation of the H+/%ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H+/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the H+/Ze- stoichiometry for site 2 in normal rat liver mitochondria.
Earlier reports from this laboratory have provided evidence that the average number of H+ translocated into the medium per pair of electrons passing through each of the three energy-conserving sites of the respiratory chain (i.e., the H+/site ratio) of rat liver mitochondria is close to 4.0 (l-9). This information has come from stoichiometric studies of sites 2 + 3, as represented by electron flow from succinate to oxygen (24), of sites 1 + 2 + 3, represented by electron flow from NADH to oxygen (5), of site 3 alone, represented by electron flow from cytochrome (6, 7), of site 2 alone, as represented by electron flow from succinate to ferricyanide (8), and of sites 1 + 2 (9). Simi1 This work was supported by a grant from the National Cancer Institute (CA25360) and a Public Health Service International Research Fellowship to A. V. (1 F05 TW02585). 2 Present address: Laboratoire D’Enzymologie, Universite de Louvain, 1348 Louvain-La-Neuve, Belgium. a To whom reprint requests should be sent. 4 J. Benavides and A. L. Lehninger, unpublished observations. 0003-9861/80/130210-07$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
210
lar observations have been made independently by Azzone et al. (10, 11). Moreover, mitochondria isolated from rat heart (12), mouse Ehrlich ascites tumor, and rat AS30D cells (13), and rat brain,4 also have yielded average H+/site ejection ratios close to 4 for sites 2 + 3 (succinate * 02) and sites 1 + 2 + 3 (NADH + 0,). In this paper we report the results of stoichiometric studies of H+ ejection and permeant cation uptake coupled to electron flow through energy-conserving site 2 of the respiratory chain of mitochondria isolated from two malignant cell lines: the mouse Ehrlich ascites tumor and the rat ASSO-D ascites tumor, derived from a hepatoma. These experiments, in which succinate was used as electron donor and ferricyanide as electron acceptor, yielded observed H+/2eejection ratios very close to 4.0 and cation+1 2+- uptake ratios close to 2.0, in exact agreement with data on site 2 reported earlier on normal rat liver mitochondria (8). These observations not only provide new data on the stoichiometry of respiration-coupled H+ transport in mitochondria from malignant
H+ EJECTION
IN SITE 2 OF TUMOR
cells, but also add to the increasing evidence that the H+/site ratio is close to 4 in mitochondria from animal tissues generally. EXPERIMENTAL
DETAILS
The line of Ehrlich ascites tumor cells was originally supplied by Dr. E. L. Coe, Department of Biochemistry, Northwestern University Medical School, Chicago, Illinois. The AS30-D rat hepatoma in ascites form was obtained from Dr. E. F. Walborg, Department of Biochemistry, The University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. Inoculation of 0.1-0.2 ml ascitic fluid from donor mice bearing the AS30-D hepatoma was carried out in groups of 20 young adult male Swiss albino mice (Buckberg Labs, Tomkins Cove, N. Y.) and groups of 5 or 6 young adult male albino rats from Sprague-Dawley, Inc., Madison, Wisconsin, respectively. The tumor cells were harvested 1 week after inoculation. After sacrifice of the animals the ascitic fluid was collected and filtered twice through a double layer of gauze to remove clumped cells. The peritoneal cavity was washed with a cold solution of 150 mM NaCl, 5 mM KCl, and 20 mM Tris-chloride (pH 7.4) and the washings added to the filtered ascitic fluid. The cells were recovered by centrifugation at SOg for 4 min and washed three times with a total of about 240 ml of the above medium. The cells were washed once more with cold 0.25 M sucrose and collected by centrifugation at 6209 for 2 min. Such cells were used directly to prepare tumor mitochondria as has been previously described (13). Alternatively, the last washing of the ascites cells was performed with a medium (designated STE) of 0.25 M sucrose, 1 mM Tris-EGTA,” 5 mM Tris-chloride (pH 7.4), followed by centrifugation at 3000g for 3 min. Washing of the cells in the STE medium gave higher yields of mitochondria as well as slightly higher acceptor control ratios. The cells from either procedure were diluted to a total volume of 60 ml in the STE medium and preincubated for 8 min at 0°C with 1 mg Nagarse (Enzyme Development Corp., New York, N. Y.) with occasional gentle stirring. The cells were then washed three times with a total volume of 80 ml of ‘70 mM sucrose, 210 mM mannitol, 2.1 M K-HEPES, pH 7.4, and 0.1% fatty acid-free bovine serum albumin (designated H-medium) and centrifuged at 30009 for 3 min. The pellet was suspended in approximately 200 ml of H-medium and 40-ml batches were homogenized, using a tightly fitting i Abbreviations used: EGTA, ethylene glycol bi@aminoethylether)N,N,N’,N’-tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; STE medium, 0.25 M sucrose, 1 mM Tris-EGTA, 5 mM Tris-chloride; H-medium, 70 mM sucrose, 210 mM mannitol, 2.1 M K-Hepes, pH 7.4, 0.1% fatty acid-free bovine serum albumin.
MITOCHONDRIA
211
Teflon pestle rotated at 500 rpm. Five or six up-anddown strokes were used for each batch. Centrifugation of the homogenized cell suspension was performed three times at 480s for 5 min in order to remove the nuclear fraction and unbroken cells completely and to obtain as large a volume of the supernatant suspension as possible. The mitochondrial fraction was then collected by centrifugation at 12,000g for 10 min and the pellet resuspended in H-medium. Two more centrifugations were performed at 14,500g 10 min in order to wash the mitochondrial fraction, which was finally resuspended in a minimum volume of H-medium. A typical preparation contained a final protein concentration (14) of about 80-100 mg protein/ml, with a total yield of mitochondrial protein of about 50-100 mg. The acceptor control ratio of these preparations ranged from 2.5 to 4.0 using succinate as substrate and ADP as acceptor. The reactions were carried out in thermostatted glass cells of 2- to 3-ml capacity at the temperatures given in the legends. A combination pH electrode (Thomas 4094 L25) and, when used, a Ca*+-selective electrode (Radiometer, F2112 Calcium Selectrode) were inserted into the cells through independent openings. The electrode outputs were amplified through Beckman Expandomatic SS2 pH meters and fed into a dual-channel Sargent-Welch DSRG recorder. Internal standards of HCl and CaCl,, comparable in amounts to the changes being measured, were added to calibrate the pH and Ca2+ electrodes, respectively. Other experimental conditions of the incubations are given in the legends. RESULTS
The Hi12e- Ejection Ratio and Cationl2eUptake Ratios with Ca2+ as ChargeCompensating Cation
The first series of experiments was carried out on mitochondria isolated from Ehrlich ascites tumor cells. Mitochondria were preincubated aerobically in the buffered medium with rotenone to prevent electron flow from endogenous NAD(P)-linked substrate(s), oligomycin to prevent H+ movements associated with ATP synthesis or hydrolysis, and N-ethymaleimide to prevent underestimation of the H+/2e- ejection ratio due to rapid H+ reuptake with phosphate on the H+/H,PO; symport carrier (1, 3, 6, 15). After the short (1-3 min) aerobic preincubation to deplete possible endogenous flavinlinked substrate(s), neutralized KCN was added to block electron flow through cyto-
212
VILLALOBO
AND LEHNINGER
chrome oxidase. Succinate was then added, followed 30 s later by Ca*+. After the electrode traces became stable a known amount of ferricyanide (>99% purity) was added as electron acceptor and the ensuing H+ ejection and Ca*+ uptake recorded simultaneously from the same vessel. Since these experiments were of the pulse type, the total amount of H+ ejected (in ng-ion H+) obtained from the trace was divided by one-half the amount of ferricyanide added (in nmol) to give the number of H+ ejected for each 2eflowing from succinate to ferricyanide via FIG. 1. The H+/%- ejection and Ca*+/2.e- uptake mitochondrial cytochrome c, i.e., the H+/&ratios with Ca*+ as charge-compensating cation. The ejection ratio. test system (3.4 ml, 24.5%) contained sucrose (124 A typical experiment is shown in Fig. 1. mM), KC1 (60 mM), K-HEPES (3 mM, pH ‘7.l), rotenone Upon addition of 200 nmol of ferricyanide (1 nmol mg-I), oligomycin (0.5 pg mg-I), N-ethylmaleivery rapid ejection of H+ took place, fol- mide (40 nmol mg-I), Ehrlich tumor mitochondria (20 lowed by a slow back-decay of H+ to half the mg protein), and CaCl, (100 nmol mg-I). KCN (4.0 mM) extent of the total H+ ejected. The total was added after a short aerobic preincubation, followed amount of H+ ejected was extrapolated by succinate (0.55 mM). After the H+ and Ca2+ traces from the ejection and back-decay curves as became stable a pulse of 200 nmol of ferricyanide was shown, giving an Hf/2e- ejection ratio of added and H+ ejection and Ca*+ uptake recorded simultaneously. Where indicated, antimycin A (0.2 nmol 400 + l/2(200> = 4.0. Without the correcmg-‘) or FCCP (2 pM) were added. Also shown are tion for H+ back-decay, the H+/2e- ejection traces from an experiment in which succinate was ratio taken from the observed H+ peak (Fig. omitted from the system. 1) was 3.75; thus the correction for backdecay is only about +6%. Figure 1 also shows that when succinate Table I summarizes data collected from was omitted no H+ ejection or Ca*+ uptake many such experiments on Ehrlich ascites H+/2e- ejection ratios took place, indicating that no coupled elec- tumor mitochondria; tron flow from endogenous substrates oc- close to 4.0 and Ca*+/&- uptake ratios of curred. The rotenone block thus prevented approximately 1.0 were consistently observed, Similar experiments carried out on interfering electron flow from possible endogenous NAD(P)-linked substrates. AS30-D hepatoma mitochondria under esMoreover, it is also seen that antimycin A sentially the same conditions also gave valinhibited electron flow from succinate to ues of the H+/2t- ejection ratio very close to ferricyanide over 97% at the time of the 4.0 (Table I). In experiments such as that in peak H+ ejection, demonstrating that no Fig. 1 a succession of ferricyanide pulses significant reduction of ferricyanide takes could be added to the same system, all giving place at the expense of electron donors on nearly identical H+/2e- ratios close to 4.0. In the substrate side of the antimycin block such experiments the ferricyanide pulses were added only after the back-decay of H+ under the conditions used. In the presence of the H+-conducting uncoupler FCCP (Fig. from the preceeding pulse to the new baseline (H+/&- = 2) was complete. 1) a net of 1.99 H+ was formed per 2e- transferred, very close to the expected value 2.0 (8). The 2 H+ released in the presence of The H+lZe- Ejection Ratio with K+ FCCP ultimately arise from the methylene (+Valinom&in) as Chargecarbons of succinate by the action of succiCompensating Cation nate dehydrogenase; net electrogenic translocation of H+ from the matrix to the meExperiments were also carried out in dium is abolished in the presence of FCCP. which Ca*+ was replaced with 60 InM K+
H+ EJECTION
213
IN SITE 2 OF TUMOR MITOCHONDRIA TABLE
I
THE H+/%- EJECTION RATIO AND THE CATION/%- UPTAKE RATIO IN SITE 2 EXPERIMENTS”
Tumor Ehrlich
AS30-D hepatoma
Permeant cation Ca2+ (11) Caz+ (3) K+ ( + valinomycin) (5) K+ (+valinomycin) (2) None (4) Ca*+ (2) Ca2+ (1)
Other conditions No NEM No rotenone FCCP No NEM
H+/2eEjection ratio
Ca2+/% Uptake ratio
4.03 f 0.09 3.11 3.98 + 0.02 3.85 1.97 i 0.03
1.02 k 0.09 0.97
4.00 2.90
a The test system (3.0-3.5 ml at 25°C) contained sucrose (125 mM), KC1 (60 mM), K-HEPES (3 mM, pH 7.1), rotenone (2 to 4 nmol mg-I), KCN (l-4 mM), oligomycin (0.5 pg mg-I), CaCl, where indicated (100-600 ng-ion mg-I), valinomycin where indicated (50-200 ng mg-I), N-ethylmaleimide (40 nmol mg-*), succinate (0.4 to 0.5 mM), and mitochondria (5-20 mg protein). Ferricyanide was added last at 5 to 10 nmokmg protein. Other conditions are specified. Figures in brackets indicate the number of experiments carried out; standard deviations are given where appropriate.
(+ valinomycin) as charge-compensating permeant cation. Under these conditions K+ moves into the matrix to maintain bulk electroneutrality. A typical set of traces is presented in Fig. 2. Upon addition of a pulse of 100 nmol of ferricyanide a total of 203 ng-ion H+ were ejected, equivalent to an H+/2eejection ratio of 203150 = 4.06. A similar experiment in which FCCP replaced valinomycin resulted in a net H+/%- ejection ratio of 103/50 = 2.06, in agreement with the expected value 2.0 and with the data in Fig. 1. Figure 2 also shows that in the absence of succinate no H+ ejection took place. When antimycin A was included in the system, to block electron flow through site 2, only a very slow H+ ejection took place in the presence of FCCP, again indicating that endogenous substrates on the reducing side of the antimycin block do not react with ferricyanide at a significant rate under the conditions employed. The H+/%- ejection ratio for the span succinate to ferricyanide was also measured in a system not containing rotenone, after the mitochondria were first depleted of endogenous substrates. Under these conditions several experiments gave an H+/2eejection ratio of 3.85 + 0.03, essentially the same as when rotenone was present (H+/&= 3.94 2 0.05; see Table I).
Table I summarizes the average H+/2eejection ratios of a number of experiments carried out under a variety of conditions, in the presence of either Ca2+ or K+ (+valinomycin) as permeant cations. The average H+/2e- ejection ratio was very close to 4.0. The Ca2+/2e- uptake ratio, as indicated above, was very close to 1.0 in a number of experiments, equivalent to uptake of two compensating positive charges per 2e-. Table I also shows the H+/%- ejection ratios in experiments in which N-ethylmaleimide was omitted from the system, conditions under which underestimation of the H+/2eejection ratio was expected (1,3,6, 15). The H+/&- ejection ratio under these conditions was reduced to slightly over 3, due to the rapid reuptake of H+ with phosphate in response to the matrix alkalinity generated by electron flow. Nevertheless, the Ca2+/2euptake ratio was unchanged in these conditions, as expected, since reentry of H+ by H+/phosphatesymport is electroneutral (4). As was shown above in Figs. 1 and 2, addition of FCCP to the complete system resulted in the appearance of only 2 H+ per 2~~. In other experiments in which neither Ca2+ nor valinomycin was present, and EGTA was included to bind Ca2+ from endogenous sources, the presence of FCCP
214
VILLALOBO
AND LEHNINGER
again yielded an observed H+/2e- ejection ratio of 1.97 & 0.03, very close to the theoretical value of 2.0. Table I also shows some data for mitochondria from the AS30-D tumor, which gave results almost identical to those in the Ehrlich mitochondria. Further stoichiometric data on the AS30-D and mitochondria from other tumor cell livers are being collected. DISCUSSION
The H+/2e- ejection ratios close to 4.0 reported here for the segment succinate + ferricyanide in mitochondria from the Ehrlich ascites and AS30-D tumors are in complete agreement with our earlier report showing that these mitochondria yield average H+/site ejection ratios close to 4 for the spans succinate + 0, and NADH + 02, which encompass energy-conserving sites 2 + 3 and 1 + 2 + 3, respectively (13). The H+/2e- ratios reported here also agree with earlier data from this laboratory on site 2 in rat liver mitochondria (8), and with data on site 2 in intact mitochondria reported from other laboratories (10, 11, 16-20). The experiments reported here also remove a criticism advanced by Mitchell and Moyle (21) on the use of N-ethylmaleimide to inhibit H+ reuptake on the H+/phosphatesymporter. They have proposed that this reagent increases the observed H+ ejection, not because of its well-known capacity to inhibit interfering reuptake of H+ on the phosphate carrier, as has been demonstrated (1, 3, 6, 15, 19), but because it inhibits succinate dehydrogenase and at the same time evokes flow through sites 1 and 2 from endogenous pyridine-linked substrates, despite the presence of rotenone. As is shown here, this claim is invalid, since in the absence of succinate no significant endogenous H+ ejection occurred. Moreover, the succinate -+ ferricyanide span yielded an H+/%- ejection ratio of 4 whether or not rotenone was present. Although there is general agreement that the observed H+/2e- ejection ratio for site 2 as reflected by the span succinate + ferricyanide is 4.0 (7, 10, 16, B-20), there has
Ferrwonide
(100)
plus FCCP
H+/&- =+=
2.06
FIG. 2. The H+/2e- ejectionratio with K+ (+valinomycin) as charge-compensating cation. The test system (2.5 ml at 25°C) contained the same sucrose-KCI-KHEPES medium as in Fig. 1, rotenone (2 nmol mg-I), N-ethylmaleimide (40 nmol rng-I), oligomycin (1 pg mg-I), valinomycin (200 ng mg-I), and Ehrlich tumor mitochondria (20 mg protein). After a short aerobic preincubation period KCN (2 mM) was added, followed by succinate (0.55 mM). A pulse of 100 nmol ferricyanide was then added to start the reaction and H+ ejection was recorded. Where indicated, antimycin A (0.4 nmol mg-‘) and FCCP (1.0 PM) were added. A trace is also shown in which no succinate was added to the system.
been some disagreement as to the meaning of this value. Mitchell has postulated a mechanism for H+ ejection coupled to sites 2 + 3, the Q cycle (22, 23), which proposes that 2 of the 4 H+ ejected as 2~ flow from succinate to ferricyanide represent the action of an outward-directed H+-transporting arm corresponding to the inward-directed electron-transporting arm furnished by cytochrome oxidase, which he regards as intrinsically unable to transport protons. However, the necessity for this proposal has been removed by recent demonstrations in several laboratories that H+ ejection is in fact coupled to electron flow through cytochrome oxidase (6, 7, 9, 11, 18-20, 25-27). There are additional uncertainties regarding the specific mechanisms involved in the ejection of 4 H+ as 2~ pass from succinate to ferricyanide. First, the precise sequence of the electron carriers in this complex segment of the respiratory chain is still not known. Second, another problem is posed by the observation previously re-
H+ EJECTION
IN SITE
2 OF TUMOR
ported from this laboratory (6) that the H+/O ratios for the oxidation of the site 2 substrates succinate and a-glycerol phosphate in rat liver mitochondria are identical at close to 8. Since succinate dehydrogenase is located on the M side of the inner membrane and glycerol phosphate dehydrogenase on the C side, it is uncertain to which side the 2 H+ removed from these two substrates by their respective dehydrogenases are delivered and whether they contribute in both cases to the observed 8 H+ ejected. This question is being studied directly by comparison of the H+/2e- ejection ratios for glycerol phosphate and succinate as the substrates with ferricyanide or cytochrome c as electron acceptor. Also to be considered is the probability that the intrinsic H+LW ratio for site 2 is at least 3 and probably 4 when the electron donor is endogenous NADH since the observed H+/%- ratio for the segment NADH + ferricyanide can significantly exceed 7 (9, lo).’ Further investigation is also required to identify the specific electron transport steps in site 2 that yield 4 H+ per %- transported. Recent experiments have indicated that during succinate oxidation by ferricyanide, 2 of the 4 H+ ejected appear during electron flow prior to the point of antimycin A inhibition when N,N,N’,N’-tetramethyl-pphenylenediamine is used to bypass the antimycin block (9, lo).’ Presumably the other 2 H+ are ejected during electron flow through the b-c, complex. The H+/site data reported here, as well as those reported earlier (13), are also germane to further analysis of the profound inhibition of phosphorylating electron transport caused by matrix Ca2+ in mitochondria from these tumor cells (28). Since the H+/site ratio of electron transport in the tumor mitochondria appears to be identical with that in normal mitochondria, some other aspect of oxidative phosphorylation appears to be the locus of the inhibitory action of matrix Ca*+. B A. Villalobo, A. Alexandre, manuscript in preparation. ’ A. Alexandre, F. Galiazzo, J. Biol. Chem., In press.
and A. L. Lehninger, and A. L. Lehninger,
215
MITOCHONDRIA ACKNOWLEDGMENTS
The authors of Irene Wood
acknowledge and Jocelyn
the technical King.
assistance
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AND
18. WIKSTR~M, M., AND KRAB, K. (1978) in Energy Conservation in Biological Membranes (Schafer, G., tid Klingerberg, M., eds.), pp. 128- 139, Springer-Verlag, Berlin. 19. WIKSTR~M, M., AND KRAB, K. (1979) Biochim. Biophys. Acta 549, 177-222. 20. BRAND, M., HARPER, W. G., NICHOLLS, D. G., AND INGLEDEW, W. J. (1978) FEBS Lett. 95, 125-129. 21. MOYLE, J., AND MITCHELL, P. (1978) FEBS Lett. 90,361-365. 22. MITCHELL, P. (1975) FEBS Lett. 59, 137-139. 23. MITCHELL, P. (1976) J. Theor. Biol. 62,327-367. 24. PAPA, S., LORIJSSO, M., GUERRIERI, F., Izzo, G., AND CAPUANO, F. (1978) in The Proton and
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Calcium Pumps (Azzone, G. F., Avron, M., Metcslfe, J. C., Quagliariello, E., and Siliprandi, N., eds.), pp. 227-238, Elisevier/NorthHolland, Amsterdam. LEHNINGER, A. L., REYNAFARJE, B., AND ALEXANDRE, A. (1978) in Frontiers of Biological Energetics (Dutton, P. L., Leigh, J. S., and Scarpa, A., eds.), pp. 384-393, Academic Press, New York. SIGEL, E., AND CARAFOLI, E. (1978) Eur. J. Biothem. 89, 119-123. CASEY, R. P., CHAPPELL, J. B., AND AZZI, A. (1979) Biochem. J. 182, 149-156. VILLALOBO, A., AND LEHNINGER, A. L. (1980), J. Biol. Chem. 255, 2457-2464.