THE JOURNAL OF Bm.ocm~~ CHEMISTRY Vol. 251, No. 23, Issue of December 10, pp. 7442-1451, 1976

Printed m U.S.A.

Evaluation of the H+/Site Ratio of Mitochondrial Electron Transport from Rate Measurements*

(Received for publication, April 26, 1976, and in revised form, August 2, 1976)

BALTAZAR REYNAFARJE, MARTIN D. BRAND, AND ALBERT L. LEHNINGER

From the Department of Physiological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N- ethylmaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca*+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO,-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by cY-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate move- ments are eliminated, may be close to 4.0.

Mitochondria supplemented with an oxidizable substrate and oxygen are able to translocate protons from the matrix to the suspending medium (for reviews l-3). The chemiosmotic theory of oxidative phosphorylation (l-4) proposes that the passage of a pair of electrons from NADH to oxygen will cause the ejection of a fixed number of protons at each of the three energy-conserving sites of the respiratory chain (hereafter called the H+/site ratio), thus generating an electrochemical gradient of H+ which may subsequently be used to drive the transport of ions or the synthesis of ATP. Mitchell and Moyle have determined, in oxygen-pulse experiments, that 2 protons are ejected per energy-conserving site (5-7). This ratio has been confirmed by a number of other investigators using es- sentially the same conditions but employing pulses of oxidants other than oxygen or pulses of reductants in the presence of oxygen (8-10). Similar results were also found in mitochon- drial membrane vesicles which are inverted and thus take up H+ instead of ejecting them into the medium (11-13).

There is increasing evidence, however, that the value of 2.0 for the mitochondrial H+/site ratio is an underestimate. Ther-

* This work was supported by Grant BMS-75-21923 from the Na- tional Science Foundation, Grant GM05919 from the National Insti- tutes of Health, and Contract NOl-CP-45610 from the National Cancer Institute.

modynamic comparisons of the electrochemical H+ gradient generated by electron transport with the phosphate potential which it can sustain indicate that the H+ gradient is insuffl- cient to account for phosphorylation of ADP unless the H+/site ratio is greater than 2.0 (14-16). Measurements made on pho- tosynthetic phosphorylation also indicate that the H+/ATP ratio is greater than 2.0; values up to 4.0 have been reported (17). In earlier work from this laboratory it has been shown that from 1.7 to 2 Ca2+ ions are accumulated by mitochondria per 2e- per site (18); since the inward movement of Ca*+ is by electrophoretic uniport (19-21), 3.5 to 4 positive charges are transported inward per 2e- per site. After the ambiguities in proton movements inherent when phosphate is the counteran- ion for Ca*+ uptake were eliminated by replacing phosphate with a weak monocarboxylic acid, such as 3-hydroxybutyrate, the H+/site ratio of Ca2+-stimulated electron flow was found to be 3.5 to 4.0 (22). We have also re-examined the H+/site ratio in oxygen-pulse experiments of the type originally described by Mitchell and Moyle (5-7). We have found the H+/site ratio of 2.0 observed under their conditions to be seriously underes- timated, owing to the rapid, simultaneous uptake of phos- phate from the medium during the course of H+ ejection in- duced by addition of an oxygen pulse (23). Uptake of each phosphate ion (H,PO,J into the matrix requires simultaneous

7442

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Mitochondrial H+lSite Ratio 7443

loss of an OH- from the matrix (or its equivalent, entry of an H+ from the medium), thus causing the Hi/site ratio to be underestimated. In the absence of phosphate or when phos- phate movements across the mitochondrial membrane were blocked by N-ethylmaleimide, an inhibitor of the phosphate- hydroxide exchange carrier (24, 25), the observed H+/site ra- tios with the oxygen-pulse method were close to 3.0 (23).

In this paper we describe a third approach to the evaluation of the H+/site ratio of mitochondrial electron transport. In this method the initial rates of both oxygen uptake and H+ ejection during a period of electron flow induced by the addition of succinate to rotenone-inhibited, energy-depleted mitochondria were measured. This approach involves no extrapolation pro- cedure to estimate Hi ejection, such as is required by the oxygen-pulse method, which measures the total amount of H+ ejected. To prevent the generation of a transmembrane poten- tial either K+ + valinomycin or Ca’+ were added as mobile, permeant cation. The results described here show that in the absence ofN-ethylmaleimide the rate of H+ ejection divided by the rate of oxygen consumption (hereafter called the H+/O rate quotient) was near 4.0 with succinate as substrate, correspond- ing to a H+/site ratio of 2.0. However, whenN-ethylmaleimide was added to inhibit movements of phosphate in response to the H+ gradient generated, the observed H+/O rate quotient for succinate oxidation approached 8 under a variety of experi- mental conditions, corresponding to a H.+/site ratio of close to 4.0. By the three methods we have employed, we estimate the H+/site ratio of mitochondrial electron transport to be at least 3.0 and it may be as high as 4.0. Preliminary accounts of this work have been published (26, 27).

EXPERIMENTAL PROCEDURES

Rat liver mitochondria were isolated in 0.25 M sucrose following the procedure of Schneider (28). After washing three times the mitochondria were resuspended in 0.25 rn~ sucrose at a stock con- centration of 50 mg of protein/ml. Protein was determined according to the method of Murphy and Kies (29) and inorganic phosphate following the method of Chen et al. (30). All reagents were of analyti- cal grade.

Reactions were carried out in a thermostated glass cell of 2-ml capacity. A Clark oxygen electrode (Yellow Springs Instrument Co.1 and a combination pa-glass electrode (Thomas, 4094 L15, connected to a Beckman Expandomatic SS2 pH meter) were placed in the cell through independent openings. The outputs from the electrodes were registered using a dual-channel Sargent-Welch DSRG recorder, at a speed of 5 or 10 inches/min.

Experiments were initiated by adding 0.1 ml of the stock suspen- sion of mitochondria (5 mg of protein) to 1.9 ml of stirred medium (120 rnM LiCl, 10 rnM KCl, 3 mM Hepes,’ pH 7.1, and 4 PM rotenone) at 25”. Any endogenous two-site substrates or.endogenous ATP were depleted by incubation for 2 min. Addition of oligomycin to prevent hydrolysis of any ATP remaining had no effect on the results ob- tained. After this period valinomycin was added at a concentration of 100 ng/mg of protein. After an additional minute, succinate (usually 1 mM) was added and changes in oxygen and H’ concentrations in the medium were recorded. When Ca2+ was used as mobile cation valinomycin was omitted. Known amounts of HCl, or NaOH, or both, were added as internal standards to calibrate the electrode response in all experiments.

The phosphate-depleted mitochondria were prepared as follows: 1 ml of stock suspension of mitochondria (50 mg of protein) were added to 19 ml of medium (120 mM LiCl, 10 rnM KCl, 3 mM Hepes, pH 7.1) at 25”, with or without 4 PM rotenone as indicated. After 1 to 2 min of incubation mitochondria were collected by centrifugation; the super- natant fluid was saved for phosphate determinations and the pellet was resuspended in fresh sucrose at a concentration of 50 mg of protein/ml. This washing procedure was repeated a number of times

1 The abbreviations used are: Hepes, 4-(2hydroxyethyl)-l-pipera- zineethanesulfonic acid; EGTA, ethylene glycol bis(p-aminoethyl ether) N,N,N’,N’-tetraacetic acid.

until no phosphate was detected in the supernatant. When o-glycerol phosphate was used as respiratory substrate the

rats were treated with a subcutaneous injection of 50 pg of thyrox- ine/100 g of body weight/day for 1 week prior to preparation of the liver mitochondria.

The solubility of oxygen in the medium was assumed to be 475 ng atoms/ml (903 ng atoms in 1.9 ml) at 25” and 760 mm Hg.

RESULTS

Recording of Rates of Oxygen Consumption and H+ Ejec- tion - The initial rates of oxygen consumption and H+ ejection were recorded from experiments in which rat liver mitochon- dria were first preincubated aerobically for some minutes in the presence of rotenone to inhibit the endogenous respiration, which is very largely NAD-linked; a mobile permeant cation, either K+ + valinomycin or Ca2+ was also present. During this preincubation period in the absence of significant electron flow, the intramitochondrial ATP is substantially depleted (31) and the mitochondria are in the de-energized state. To initiate electron flow to oxygen, a pulse of 1.0 mM succinate is added and both oxygen consumption and H+ ejection recorded at a fast chart speed (5 to 10 inches/min). As is seen in Fig. 1, short lag periods of about 1.2 s for glass electrode and about 2.4 s for the Clark oxygen electrode (1.8 s for a vibrating platinum electrode) are seen in the recorder traces. In the subsequent period of from 6 to 9 s the rates of both oxygen consumption and H+ ejection are linear and constant; after this steady state period the rate of H+ ejection falls off as significant back- leakage of H+ sets in; a slight acceleration of oxygen consump- tion accompanies back-leakage of H+, as is expected (32). The initial rates are obtained from the slopes of the linear portions of the traces (Fig. 1).

The H+/O rate quotient (nanogram ions of H+ ejected per min per mg of protein/nanogram atoms of oxygen consumed per min per mg) for the experiment in Fig. la is 4.2. Since two energy-conserving sites are involved in electron flow from succinate to oxygen the H+/site ratio is 4.2/2 = 2.1, very close to the H+/site ratio of 2.0 found in oxygen-pulse experiments when no precautions are taken to eliminate movements of inorganic phosphate across the mitochondrial inner mem- brane (23).

The experiment in Fig. la was carried out in a 120 mM lithium chloride medium containing 10 mM KC1 plus 100 ng of valinomycin/mg of protein. Nearly identical results were ob-

0

H’

(b)

Su$clnate

FIG. 1. Initial rates of oxygen consumption and H+ ejection. For details see “Experimental Procedures.” In a and c, mitochondria (5 mg of protein) were added to a system containing 120 rnM LiCl, 10 mM KCl, 3 mM Hepes, pH 7.2, and 4 PM rotenone. In b, 130 mM KC1 replaced the LiCl/KCl combination. After 2 min incubation at 25” valinomycin (100 ng/mg of protein) was added to a and b, and CaZ+ (40 ng ions/mg of protein) to c. One minute later, 1.0 mM succinate was introduced into the system and the rates recorded.

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7444 Mitochondrial H+lSite Ratio

served when the medium was 120 mM NaCl or 240 mM sucrose instead of 120 mM LiCl. When 130 mM KC1 was employed as the medium the rate of back-leakage of ejected H+ was much greater than in a 120 mM LiCl, 10 mM KC1 medium (Fig. lb), due to more rapid swelling of the mitochondria, as expected (32). Nevertheless, it was still possible to obtain good measure- ments of the H+/O rate quotient as is indicated by the observed value of H+/site = 2.0 (Fig. lb).

Fig. lc shows an experiment identical with that in Fig. la, but in which K+ + valinomycin was replaced by Ca2+ as mobile cation. Again, the H+/O rate quotient yielded a H+/site ratio of 2.0, in agreement with the results when K+ + valino- mycin served as mobile cation system.

In most of the following experiments K+ + valinomycin was employed as the mobile cation to exchange with the ejected H+, thus preventing development of a transmembrane poten- tial (14). Some additional experiments with Ca2+ as mobile cation are also shown.

Effect of K+ Concentration on H+lO Rate Quotient- Fig. 2 shows the effect of Kt concentration on the initial rates of H+ ejection, oxygen consumption, and the H+/site ratio obtained from the rate measurements. In the absence of added K+ there was virtually no net H+ ejection, but the usual State 4 respira- tion occurred, in which the rate of H+ ejection is nearly exactly counterbalanced by the rate of H+ influx. As the concentration of K+ was increased, the net rate of H+ ejection increased, as did the rate of oxygen consumption, due to the more rapid charge compensation at higher K+ concentrations. The H+/site ratio attained a constant value of 2.0 at about 6 mM KCl, with no significant change up to 130 mM KCl.

That both H+ ejection and oxygen consumption were reflec- tions of a common process dependent upon K+ (+ valinomycin) is shown in the double reciprocal plots of l/u uersus l/[K+l for the stimulated oxygen consumption and for the H+ ejection (Fig. 3); the observed K, for K+ was 7.6 mM for both processes, with an H+/site ratio of 2.1, calculated from the V,,, values.

Effect of Succinate Concentration- It was also important to demonstrate that the H+ appearing in the medium and the extra oxygen consumed are entirely the consequence of the oxidation of the added electron donor succinate. Fig. 4 shows double reciprocal plots of l/u versus l/[succinatel for both oxygen consumption and H+ ejection. As is seen, K, for succi- nate was 0.20 mM for H+ ejection and 0.19 mM for oxygen consumption. The nearly identical K,, values show that both

IA0 IlO I10 lb0 9’0 b mM LiCl

KCl,mM

FIG. 2. Effect of K+ concentration on the Hi/site ratio. For details see Fig. la. The concentration of K+ in the medium was changed as indicated. The osmolarity was kept constant by adding compensat- ing amounts of LiCl. The system contained 100 ng of valinomycinlmg of protein.

processes are wholly reflections of electron transport from succinate to oxygen. In the absence of added succinate the rate of oxygen consumption was negligible, i.e., less than 2% of the rate with only 0.1 mM succinate. Similarly, the rate of net H+ ejection in the absence of succinate was insignificant, indicat- ing that rotenone inhibition of endogenous oxygen consump- tion was sufficiently complete so that the H+ ejection measure- ments in the presence of added succinate were not compro- mised by irrelevant H+ movements. As is shown below, the presence of N-ethylmaleimide does not significantly alter the K, for succinate with respect to either H+ ejection or oxygen consumption.

Effect of Phosphate and of Inhibition of Phosphate Trans- port-It was earlier shown that inorganic phosphate in the suspending medium, whether added or arising from the efIlux of endogenous phosphate from the mitochondria, lowers the observed H+/site ratio in oxygen-pulse experiments (23,261, as a consequence of the entry of phosphate into the mitochondria in antiport with matrix OH- (or symport with medium H+). The effects of added phosphate and of N-ethylmaleimide, an inhibitor of the phosphate-hydroxide exchange carrier (24,251, on the rates of H+ ejection and oxygen consumption were examined. Fig. 5 shows recorder traces of a control experiment (Fig. 5~) and of experiments in which phosphate (Fig. 5b) and N-ethylmaleimide (Fig. 5~) were added to the medium. In the control experiment (cf. also Fig. la) the extrapolated H+/site ratio was 2.1. In Fig. 56 two important effects are noted on addition of 0.5 mM phosphate to the medium. First, the pres- ence of phosphate resulted in a lag period of about 2.4 s before

‘/ ‘/ - To * %’ I

Vmox= 55 ng-aioms/mm/mg ;y an zm /OZ

&+I, M-’

FIG. 3. Dependence of the rates of oxygen consumption and H+ ejection on K+ concentration. Double reciprocal plots were con- structed with data obtained in experiments similar to those in Fig. 2.

Km=0 19 mM

I/[SUCCINATE] . mM-’

FIG. 4. Effect of succinate concentration on the rates of H+ ejec- tion and oxygen consumption. For details see Fig. la. Succinate concentration was changed as indicated in the double reciprocal plot.

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Mitochondrial H+/Site Ratio 7445

net H+ ejection occurred. The second effect is a large decrease in the rate of the subsequent H+ ejection, with a significant increase in the rate of oxygen consumption, which are presum- ably the result of the very rapid entry of phosphate from the medium into the mitochondria following the induction of elec- tron flow. The phosphate-hydroxide transmembrane exchange is known to be extremely fast in rat liver mitochondria, about 205 nmol/min/mg of protein at 0” (25) and certainly more than 5-fold higher at 25”: this is much higher than the rate of H’ ejection coupled to electron flow from 0.5 mM succinate (-160 ng ionslminlmg at 25”, cf, Fig. 5~). As a consequence, the phosphate-hydroxide exchange, which is favored by the high external phosphate concentration, consumes all the net matrix OH- generated (or H+ ejected), accounting for the observed lag, until a steady state between H+ efflux coupled to electron transport and phosphate influx via the carrier is established. In this steady state the rate of net H+ ejection is only about a third of the normal control rate in the absence of added phos- phate. From the H+/O rate quotient in the presence of added phosphate the H+/site ratio is seen to be very low, 0.48, in contrast to the control value of 2.0.

Fig. 5c shows the effect of adding N-ethylmaleimide in addition to phosphate to the medium. It is seen that N-ethyl- maleimide not only abolishes the long lag in H+ ejection given by added phosphate, but also greatly increases the rate of net H+ ejection to a value much higher than the control rate in the absence of additions. The H+/O rate quotient of 8.18 gives an H+/site ratio of 4.09. These results with the rate quotient method mirror closely the effects of phosphate and N-ethyl- maleimide on the H+/site ratio determined by oxygen pulse measurements (23). The H+/site ratio in the rate method de- scribed here is much more sensitive to phosphate, due to the partial correction for phosphate movements possible with the oxygen-pulse method (6). These observations also suggest that the H+/site ratios of about 2.0 yielded by the experiments shown in Fig. 1 are underestimated, due to the efflux of endogenous phosphate from the mitochondria during the preincubation and its rapid reuptake after addition of succi- nate.

Effect of N-Ethylmaleimide Concentration on H+ISite Ra- tio - Fig. 6 shows the effect ofN-ethylmaleimide concentration on the rates of H+ ejection, oxygen consumption, and the,H+/O rate quotient in the absence of added phosphate. N-Ethylmal- eimide causes only a slight inhibition of oxygen consumption,

H%lk =2.1 H&k =0.48 I

H;/slte = 4 09

FIG. 5. Effect of phosphate and N-ethylmaleimide on the H+/site ratio. Conditions were as in Fig. lo, except that in b 0.5 mM phos- phate, and in c, 2.0 IIIM phosphate plus 30 nmol ofN-ethylmaleimidel mg of protein were present in the system.

but a very large increase in the rate of H+ ejection, which attains a maximum at about 20 nmols of N-ethylmaleimidel mg of mitochondrial protein, about the same amount required for maximal inhibition of the phosphate-hydroxide exchange carrier (24, 25). Above 20 nmol of N-ethylmaleimidelmg of protein, the observed H+/O rate quotient was constant at about 7.7, equivalent to a H+/site ratio of 3.9.

Fig. 7 shows the effect of varying the concentration of succi- nate on the rates of H+ ejection and oxygen consumption, and on the H+/site ratio in the absence and presence of N-ethyl- maleimide. In the absence of N-ethylmaleimide the H+/site ratio was approximately constant up to 2.0 mM succinate (as in Figs. la and 5~). In the presence of N-ethylmaleimide the H+/ site ratio was approximately constant at 3.75 in the range 0.2 to 2.0 mM succinate. At the concentration used (30 nmol/mg of protein) N-ethylmaleimide accelerated the net rate of H+ ejec- tion at all concentrations of succinate, the result of inhibition of the phosphate-hydroxide exchange, and inhibited up to 35% the rate of oxygen consumption, presumably by inhibiting succinate dehydrogenase, and, indirectly, succinate entry by

0 20 40 60 SO NEM. nmoleshg protem

FIG. 6. Effect of N-ethylmaleimide on the H+/site ratio. The sys- tem was identical with that in Fig. lo, with N-ethylmaleimide present at the concentrations indicated. NEM, N-ethylmaleimide.

a,

*s I

NO NEM

,2 I,

t* . .-

. l No NEM

SUCCINATE tmM) SUCCINATE tmM)

FIG. 7. Effect of succinate on the H+/site ratio in the presence of N-ethylmaleimide. Conditions were as in Fig. 4. Experiments were carried out both in the presence and in the absence of N-ethylmal- eimide (30 nmol/mg of protein). NEM, N-ethylmaleimide.

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‘7446 Mitochondrial H+lSite Ratio

succinate-phosphate exchange. Nevertheless, the H+/site ratio remained constant at a value close to 4.0 at all succinate concentrations tested. It will also be noted from Fig. 7 that the concentrations of succinate yielding half-maximal rates of H+ ejection and oxygen consumption in the presence of N-ethyl- maleimide are essentially identical at about 0.2 mM, in agree- ment with the results of Fig. 4. Thus succinate is essentially the sole source of the electron flow to oxygen in experiments with N-ethylmaleimide present.

Effect of Phosphate Concentration on H+ISite Ratio- Ex- periments in Figs. la and 5a have shown that the H+/site ratio is close to 2.0 when no N-ethylmaleimide is added to the system. Fig. 8 shows that the effect of added phosphate in lowering the H+/site ratio is nearly maximal at 0.1 mM added phosphate, with a half-maximal effect occurring at less than 50 PM added phosphate. The H+/site ratio was lowered from 2.05 to 0.75 by phosphate in this particular experiment; in others (see, for example, Fig. 56) the H+/site ratio was lowered to less than 0.5 compared to the control H+/site ratio of 2.0. Actually, however, the concentration of phosphate required to decrease the H+/site ratio significantly is much less than the data of Fig. 8 suggest (see Fig. 111, since it has been found that considerable endogenous phosphate undergoes eMux from rat liver mitochondria on incubation under anaerobic conditions or when electron flow is inhibited. In fact, substantial efflux of mitochondrial phosphate occurs in the stock suspension of mitochondria kept at 0” (50 mg of mitochondrial protein/ml of 0.25 M sucrose), or at the moment of suspension in the reaction medium. When a O.l-ml aliquot of the stock mitochondrial suspension is added to a system of total volume 2.0 it yields an extramitochondrial concentration of about 40 PM phosphate within the time span required for sampling (less than 12 s); thiL> concentration of phosphate is already sufficient to de- crease the H+/site ratio from its maximum value of 4.0 to about 2.0 (Fig. lib).

To show that medium phosphate is in fact taken up by the mitochondria in response to the initiation of electron flow, the phosphate concentration in the suspending medium was fol- lowed before and after the addition of succinate to mitochon- dria supplemented with K+ + valinomycin in the presence of rotenone (Fig. 9). No phosphate was added to the medium; the phosphate present was that contributed by etIlux from the mitochondria during (anaerobic) storage of the stock suspen- sion, the short (2 min) initial aerobic incubation period, and the additional emux following de-energization due to addition

Plus NEM .-a-

- 0 05 IO 15 P, (mhl)

FIG. 8. The H+/site ratio as a function of phosphate concentra- tion For details see Fig. la. Phosphate was added as indicated with and without N-ethylmaleimide (30 nmol/mg of protein). NEM, N- ethylmaleimide.

of rotenone. It is seen that the subsequent addition of succi- nate produced immediate lowering of the phosphate concen- tration. This appeared to proceed at a rate consonant with the time scale of the induced oxygen consumption, but could not be measured rapidly enough by manual sampling to attempt quantitative correlations between the rates of phosphate up- take, oxygen consumption, and H+ ejection. Nevertheless, the amount of phosphate taken up in this experiment following succinate addition, about 7 nmol/mg of protein, corresponds to uptake of about 53 ng ions of H+ in the 2.0-ml system (with 5 mg of protein), assuming net influx of 1.5 H+ with each phos- phate. (1 H+ by phosphate-hydroxide antiport and about 0.5 H+ by secondary ionizations). The reuptake of mitochondrial phosphate in the experiment of Fig. 9 is thus equivalent to at least 53 ng ions of H+ taken up during the initial 6 to 10 s steady state period. The traces in Fig. 5a show that this is close to the deficit in total H+ ejection observed when no N- ethylmaleimide is added. Thus the observed uptake of medium phosphate is sufficiently large in magnitude to account for the large deficit in H+ ejection from the maximal H+/site ratio of 4.0, as observed in Fig. 5c.

Effect of Depletion of Endogenous Phosphate on H+ISite

Ratio - Fig. 10 shows that successive washes of the mitochon- dria to deplete them of phosphate caused the observed H+/site ratio to increase to a maximum value of 4 (Fig. l&z). Addition of N-ethylmaleimide to the reaction medium elevated the H+/ site ratio to 4 when the mitochondria were incompletely de- pleted of endogenous phosphate (Fig. 10~). When washed mito- chondria were assayed in the supernatant from the first wash (containing about 40 PM phosphate released from the endoge- nous pool) the H+/site ratio was reduced to values close to 2 (Fig. lob). This effect was mimicked by addition of 40 pM

phosphate (Fig. Ilb). The addition of exogenous phosphate (0.5 mM) to washed mitochondria in the absence of N-ethyl- maleimide decrease the ratio to 0.8 (Fig. 1oC); however, 0.5 mM phosphate was far in excess of that required to lower the ratio from 4 to 2. Fig. 11 shows the effect of small amounts of phosphate on the rates of H+ ejection and oxygen uptake and the H+/O ratio. From 20 to 40 PM phosphate was enough to

OL I I I I I 0 I 2 3 4 5 6

MINUTES

FIG. 9. Appearance of phosphate in the medium during the course of a typical experiment. For details see Fig. la. At the indicated times samples were taken and centrifuged for 2 min in an Eppendorf 3200 centrifuge. Phosphate was determined in the supernatant. Open circles indicate experiments carried out using Ca2+ (80 ng ions/ mg of protein) as permeant cation instead of the K+-valinomycin system (solid circles).

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Mitochondrial H+lSite Ratio 7447

It xp’uspi x (d)

I

-- 0 2 3 NUMBER OF WASHES

FIG. 10. Effect of depletion of endogenous phosphate on the H+/ site ratio. Mitochondria were incubated in the medium (120 rnM LiCl, 10 rnM KCl, 3 rnM Hepes, pH 7.2, and 4 PM rotenone) for 2 min at 25”. After centrifugation the pellet was resuspended in 0.25 M sucrose for further washes, or assays, or both, in the LiCl medium. The supernatant was saved for phosphate analysis and to be used as medium (containing the released Pi) in experiments shown in Curue b. In Curve a washed mitochondria were assayed in fresh medium; in Curve c N-ethylmaleimide (30 nmol/mg of protein), and in Curve d phosphate (0.5 mM) were present in the medium.

a

0 28 100 200 300 400 500 [Pi] (PM)

FIG. 11. Effect of added phosphate on the H+/site ratio of washed mitochondria. Twice washed mitochondria were assayed in fresh medium containing the indicated amounts of phosphate, and in the presence or absence of N-ethylmaleimide (30 nmol/mg of protein). NEM, N-ethylmaleimide.

reduce the H+/site ratio from 4, observed in phosphate-de- pleted or N-ethylmaleimide-treated mitochondria to the value of 2 characteristic of fresh mitochondria.

Ca*+ as Mobile Cation- Nearly all the experiments de- scribed by Figs. 1 through 11 employed valinomycin + K+ as the mobile cation to exchange with the ejected Hi. In similar experiments essentially identical results were observed when Ca2+ was employed as the mobile cation in place of K+ + valinomycin; in this case no ionophore is required since the mitochondrial membrane contains a Ca2+ carrier system (18). To avoid repetition, the collected data from only one important experiment with Ca2+ will be given. Fig. 12 shows the H+/site ratios obtained from controls and from experiments with added phosphate and N-ethylmaleimide, as a function of

Plus NEM o

I I I I I 0 40 80 120 160

CO’+ ADDED (ng-atoms/mg)

FIG. 12. The H+/site ratio with Ca2+ as mobile cation. The basic system was the same as in Fig. la, except that valinomycin was omitted and Ca2+ was added instead of K+ at the indicated concentra- tions. The effects of phosphate (0.5 mM) and N-ethylmaleimide (30 nmol/mg of protein) are shown in the lower and upper curves, respectively. NEM, N-ethylmaleimide.

added Ca*+ concentration. In the control experiments (neither phosphate nor N-ethylmaleimide added), the H+/site ratios were nearly constant in the range 2.5 to 2.85 over the range 100 to 400 KM added Ca*+. When phosphate was added (supple- menting that already present in the medium as a result of eMux from the mitochondria) the H+/site ratios were de- pressed to a nearly constant value of 1.5 attained at Ca*+ levels above 150 pM. However, whenN-ethylmaleimide (30 nmol/mg of protein) was present, the H+/site ratio was greatly increased and became constant at about 3.8 in the range 50 to 400 PM

Ca2+, in full agreement with the maximal H+/site ratios of about 4.0 observed in the presence of K+ + valinomycin as the mobile cation system.

Two other points might be made. It has already been estab- lished that endogenous mitochondrial Ca2+ undergoes efflux into the medium under anaerobic conditions to yield as much as 5 to 10 nmol/mg of protein in the suspending medium (33). This amount of Ca2+ is already sufficient to exchange with ejected H+ within the usual dimensions of the classical oxygen- pulse experiment, in which it is unnecessary to add extra Ca*+ to measure the H+/site ratio (6). However, in the rate experi- ments as carried out in this investigation, the endogenous Ca2+ present in the suspending medium is not sufficient to exchange with all the H+ ejected over the usual 6 to 9 s intervals of succinate-induced electron flow, since Ca2+ is accu- mulated in the mitochondria stoichiometrically with electron transport. For this reason extra Ca*+ needs to be added to the medium to observe maximum rates of H+ ejection.

It is also to be noted that the H+/site ratio in this set of experiments is higher than 2.0 in the absence of added phos- phate and N-ethylmaleimide, whereas the experiments with K+ + valinomycin in this paper show lower values close to 2.0. However, this is not caused by an intrinsic difference in the mobile cations per se, but is a reflection of the fact that the amount of available phosphate prior to the addition of succi- nate is less in Ca2+ than in the K+-valinomycin experiments (see Fig. 9). since more phosphate leaks from mitochondria in the absence of Ca2+ than in its presence. The degree of under- estimation of the H+/site ratio in the absence of N-ethylmal- eimide is thus a function of the phosphate concentration in the medium. As shown in Fig. 12, the observed H+/site ratio may vary from 0.5 up to nearly 3 depending on the concentration of phosphate available for reuptake. Only in the presence of N- ethylmaleimide to block phosphate transport are consistent stoichiometric values obtained and they are in all cases close

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7448 Mitochondrial H+lSite Ratio

to 4.0. Fig. 13 shows that the K,,, (Ca2+) values for H+ ejection and 0, uptake are identical when small amounts of Ca2+ are added in the absence of Pi.

Effect of Temperature on H+ISite Ratio- Fig. 14 shows that when experiments are carried out at temperatures below 20”, in the absence of added phosphate and N-ethylmaleimide, the H+/site ratio tends to increase above the usual value of 2 observed at -25”. Addition of phosphate (0.5 mM) decreases the ratio to about 0.6 and N-ethylmaleimide increases it to about 4 at all temperatures, whether or not phosphate is present. The rate of phosphate influx is evidently not suffi- ciently rapid at 5” to reduce the Hi/site ratio to 2. In these experiments correction must be made for the largely nonenzy- matic oxygen consumption by the electrode system before the addition of succinate, which may constitute up to 20% of the low rate of oxygen consumption in the presence of succinate at 5”.

Effect ofpH of Medium - Fig. 15 shows the results of a series of experiments on the effect of medium pH on the rates of H+ ejection, oxygen consumption, and the H+/site ratio, in the absence and presence of N-ethylmaleimide. In the absence of N-ethylmaleimide the rates of H+ ejection and oxygen con- sumption are essentially constant between pH 6.0 and 7.2, but fall sharply as the pH is increased from pH 7.2 to pH 8.0; nevertheless, the H+/site ratio remains constant at about 2.0

over this pH range. In the presence of N-ethylmaleimide the rates of oxygen consumption and H+ ejection decrease with increasing pH, but the H+/site ratio is constant near 4.0. The H+/site ratio in the presence or absence ofN-ethylmaleimide is thus essentially independent of the pH of the medium over a range much wider than likely to be encountered in the biologi- cal function of mitochondria. In the same manner the amount of mitochondrial protein did not affect the H+/site ratio, al- though the rates of oxygen consumption and H+ ejection were strictly proportional to protein concentration.

Effect of Znhibitors of Mitochondrial Transport-Table I shows that the inhibition of transport of tricarboxylates (by benzene 1,2,3-tricarboxylate, BTC), Ca2+ (by EGTA), adenine nucleotides (by atractyloside), or HCOBm (by sodium acetazo- lamide, Diamox), had no effect on the H+/site ratio. Similarly, no effect was observed when Ca2+ was added in the presence of

400 1 I I , I SO Plus NEM

300 t

H+ l -. 60

- “v,J ? Vmnr=42 2 na-atoms/mln/ma

FIG. 13. The kinetics of oxygen uptake and H+ ejection in the CaZ+ system. The medium was the same as the control in Fig. 12 (i.e. no N-ethylmaleimide).

I Plus NEM

4 -l--e-* . . 0 .-

0 0

0 5 IO 15 20 25 TEMPERATURE (“C)

FIG. 14. Effect of temperature on the H+/site ratio. Experiments were performed as described in Fig. 1. Open circles indicate experi- ments in which Ca2+ was used as mobile cation (Fig. 12) and closed circles those in which the K+-valinomycin system was employed. NEM, N-ethylmaleimide.

100

I

tit l

-*-+A

2.0 I9 19 23 H*/siie ratios

0' I I '0 6.0 7.0 so

PH

FIG. 15. Effect of pH on the H+/site ratio. For details see Fig. 1. The system contained 100 ng of valinomycin/mg of protein. The pH of the succinate was previously carefully adjusted to be the same as the medium. NEM, N-ethylmaleimide.

TABLE I

Effect of various compounds on H+lsite ratio

The reaction medium was that described under “Experimental Procedures” for the K+-valinomycin system. Mitochondria were added to a medium containing N-ethylmaleimide (30 nmol/mg of protein) plus the compounds listed in the table. Figures represent mean values of at least three different experiments.

Compound Concentration H+/site

Control Benzene 1, 2, 3-tricar-

boxylate (BTC) BTC + citrate Citrate Ca2+ EGTA Oligomycin Atractyloside Diamox (sodium ace-

tazolamide)

1.0 rnM

1.0 rnM + 5.0 rnM 3.93 5.0 rnM 4.01 40 ng atoms/mg 3.89 0.5 rnM 4.00 650 ng/mg 3.98 2 nmol/mg 4.02 100 KM 4.03

3.98 3.86

n-Butylmalonate 5.0 rnM -a

a n-Butylmalonate prevented oxygen uptake and proton ejection when succinate was added.

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Mitochondrial Hi/Site Ratio 7449

valinomycin or when oligomycin was added to inhibit ATPase. As predicted n-butylmalonate prevented oxygen uptake and proton ejection when succinate was added, due to inhibition of the dicarboxylate carrier.

a-Glycerol Phosphate as Substrate-In order to eliminate possible H+ movements associated with succinate transport H+/site determinations were carried out with a-glycerol phos- phate as respiratory substrate, using mitochondria from rats pretreated with thyroxine in order to induce high levels of mitochondrial glycerol phosphate dehydrogenase (34). a-Glyc- erol phosphate dehydrogenase is located on the outer surface of the inner membrane; thus o-glycerol phosphate does not need to enter the matrix compartment to undergo oxidation (35). The a-glycerol phosphate was rapidly oxidized, accompanied by H+ ejection. In the absence ofN-ethylmaleimide the H+/site ratio was close to 2.0; in the presence ofN-ethylmaleimide the ratio increased to 3.8, demonstrating that the results obtained in this study were not dependent upon the use of succinate, but could be duplicated with another two-site substrate.

Mitochondria from Other Sources - H+/site ratios of about 2 and 4 were obtained in the absence and presence of N-ethyl- maleimide, respectively, when mitochondria isolated from rat heart, rat kidney, and mouse liver were used.

DISCUSSION

The experimental method employed in this paper represents the third approach taken in this laboratory for evaluation of the H+/site ratio of mitochondrial electron transport. Our earlier papers (23, 26) have employed (a) the classical oxygen- pulse method first developed by Mitchell and Moyle (5, 6) and (b) measurement of the respiration-dependent uptake of Ca2+ together with a proton-carrying weak acid as source of coun- ter-anions (22). When appropriate precautions were taken to eliminate the acid-base exchanges across the inner mitochon- drial membrane accompanying movement of phosphate via the phosphate-hydroxide antiporter, the three methods yielded results in rather good agreement. The Ca2+ uptake method gave H+/site ratios from 3.5 to 4.0 (22), the H+/O rate ratio method described here yielded H+/site ratios very close to 4.0, whereas the oxygen pulse method gave H+/site ratios of 3.0 (23). These may be contrasted with the value of 2.0 re- ported by Mitchell and Moyle (6) and subsequently observed by others using similar conditions (8-13). However, the lower values reported by the latter groups were obtained under conditions in which movements of phosphate were not consid- ered or inadequately eliminated.

The interference caused by phosphate movements stems from the fact that freshly prepared rat liver mitochondria quickly lose their endogenous phosphate to the suspending medium, particularly when they are anaerobic or respiration is inhibited, as shown here and by earlier investigators (23,26, 361. Presumably the loss of phosphate occurs via the phos- phate-hydroxide antiporter, which is highly active even at 0 (25). When electron flow is initiated in energy-depleted anaer- obic (or respiration-inhibited) mitochondria in the presence of a mobile cation such as K+ (+ valinomycin) or Ca2+ there is a rapid ejection of H+, generating a transmembrane H+ gra- dient. But as this process takes place, the phosphate earlier lost to the medium from the mitochondria is quickly reaccu- mulated in antiport with matrix OH- (or symport with H+), resulting in net loss of H+ from the medium. Such inward movement of phosphate is so fast that the full extent of H+ ejection from a given pulse of oxygen is never realized (231, unless phosphate transport is inhibited. The net result is that

the amount of H+ ejected per given amount of electron flow is underestimated. These considerations, first developed in our re-evaluation of the oxygen-pulse method (23, 261, also apply to the measurements of the H+/O rate quotient described in this paper. In the absence of a means of preventing phosphate movements the H+/O rate quotient was relatively low, about 4.0 for succinate oxidation (equivalent to a H+/site ratio of 2.01, but increased to nearly 8.0, corresponding to a H+/site ratio of 4.0, when N-ethylmaleimide was added to inhibit phosphate transport.

It is important to consider the nature of the errors that may be inherent in the H+/O rate quotient measurements described here. One question is to what extent endogenous three-site substrates contribute to oxygen consumption and H+ ejection. It appears very unlikely that release of H+ occurring at the expense of endogenous NADP- or NAD-linked substrates in the presence of rotenone contributes significantly (although there is a small residue of rotenone-insensitive electron flow), since there was no significant net ejection of H+ in the absence of succinate over time intervals up to 30 s or more (Figs. 1 and 5). Moreover, the base-line oxygen consumption in the pres- ence of rotenone amounts to less than 5% of the rate of oxygen consumption after addition of succinate at 25”. A second ques- tion may be raised regarding the accuracy of assessing by graphical means the initial rates of both H+ ejection and oxygen consumption in the 6 to 9 s interval after succinate addition in which they are essentially linear. In a typical experiment, however, calculation of the Hi/O rate quotient using the steepest or the shallowest slopes that can reasonably be drawn causes a variation of only 2% in the quotient ob- tained. Measurement of the initial rates of both H+ ejection and oxygen consumption recorded in the same way as em- ployed here have been used by Mitchell in another context (14); he has also evaluated H+/ATP ratios from such rate measurements (37).

Another possible source of error might be proton movements coupled to entry of succinate. In these experiments succinate entry into the matrix to allow reaction at the active site of succinate dehydrogenase must precede and may be concomi- tant with the period in which H+ movements are monitored. Exchange of added succinate with endogenous citrate on the tricarboxylate carrier, which catalyzes succinate’-/H-citrate2- antiport (38), would lead to overestimation of the H+/site ratio, due to liberation into the medium of the H+ transported with citrate. On the other hand, exchange of succinate with endoge- nous phosphate would lead to underestimation of the H+/site ratio, because the exchange of succinate- with matrix HPOd2- via the dicarboxylate carrier (24, 39) will be followed by the partial reprotonation of the HP042- in the medium to form a mixture of H,PO,- and HPOd2-. This reaction will consume H+ from the medium. Reuptake of the phosphate on the phos- phate-hydroxide antiporter will cause additional H+ disap- pearance from the medium, in the absence of N-ethylmaleim- ide. However, the data in Table I show that abolition of citrate movements via the tricarboxylate carrier by addition of ben- zene 1,2,3-tricarboxylate (40) does not affect the observed Hi/ site ratio in the presence of N-ethylmaleimide, neither does removal of endogenous phosphate by washing. The only me- tabolite of the matrix available for exchange with incoming succinate under these conditions is therefore malate, which is also the product of succinate oxidation. Since succinate-malate exchange does not involve movements of H+ (39), it must be concluded that the entry of succinate produces no systematic error in measuring the H+/site ratio. Furthermore, the use of

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7450 Mitochondrial H+lSite Ratio

a-glycerol phosphate as substrate, which is oxidized in the outer surface of the inner membrane and thus does not require transport into the matrix, gave results that were identical to those with succinate.

Pressman and colleagues (41, 42) have reported K+/site ratios of 3 to 4, as has Azzone’s group (43, 44). In these experiments valinomycin was added last to energized mito- chondria in the presence of K+. Their results cannot be re- garded as reliable indicators of the H+/site ratio since, under their conditions, an initial superstoichiometric uptake of KC will be observed in response to the pre-existing membrane potential (45) and ATP stores (31), leading to overestimation of the K+/site ratio. The agreement between their results and ours may therefore be partly fortuitous.

The three different methods we have employed to estimate the H+/site ratio are subject to different types of errors. The oxygen-pulse method requires a substantial correction to allow for the decay of the H+ gradient, which already begins during H+ ejection, so that the peak value of the H+ ejected must be extrapolated by assuming that the rate of the H+ decay in its early phase is a simple exponential (6). The magnitude of this correction, which is added to the observed peak, is about 20%. It appears possible, however, that an additional upward cor- rection may be required in oxygen-pulse experiments, since the maximum H+/site ratio we observed is 3.0 with the oxygen pulse approach, even in the presence of N-ethylmaleimide to inhibit phosphate movements (23), compared to up to 4.0 for the two other approaches. Alternatively, the results yielded by the other two methods (i.e. H+/site = 3.5 to 4.0) may require downward correction to 3.0. We cannot at present distinguish between these two possibilities.

Estimation of the H+/site ratio based upon measurements of energized Ca2+ and counteranion uptake (22) are subject to errors of a qualitatively different type. In this procedure it is necessary to correct for the presence of Ca2+ and anion in the adhering extramitochondrial water phase in the pellet by use of radioactive sucrose to determine extra-matrix water space. Another type of interference is caused by a certain amount of nonenergized binding of Ca*+ and anion to the mitochondrial membranes, which is corrected for by a graphical procedure (22). The most important correction, however, involves the question of whether only the extra oxygen uptake stimulated by Ca*+ or the total oxygen uptake is to be used for the calculation of the H+/O ratio. Using the extra oxygen uptake the H+/site ratio was found to be almost exactly 4.0, but when the total oxygen uptake was used the H+/site ratio was about 3.5. Older experiments from this laboratory (46), as well as more recent work by Stucki and Ineichen (47) are consistent with the view that the H+/site ratio derived from Ca”+ uptake measurements should correct to somewhere between these values.

Since the three methods we have employed involved differ- ent kinds of measurements and different kinds of errors, it is particularly significant that they yield H+/site ratios agreeing closely, in the range 3.0 to 4.0, when phosphate movements are absent or are prevented. We have also eliminated possible interference from H+ movements coupled to nearly all the relevant transport processes promoted by rat liver mitochon- dria, i.e. transport of Pi, ADP, ATP, tricarboxylates, and HCOB- (CO,) by use of specific inhibitors. That we may have overestimated the H+/site ratio by not recognizing yet another and unknown complicating factor is of course possible; if SO,

such an overestimation would also be inherent in the experi- ments of Mitchell and Moyle (5-7) and others (8-12). To sum-

marize, we conclude that we have identified and eliminated a source of serious underestimation of the H+/site ratio not fully appreciated by Mitchell and Moyle (5, 6) and others who have reported values of about 2.0 (8-12). The evidence reported here and in our preceding papers indicate that the true value of the H+/site ratio is at least 3.0 and may be as high as 4.0.

Acknowledgments-We thank Irene Wood, Carl Mahle, and Garvon Givan for technical assistance.

1.

2.

3.

Mitchell, P. (1966) Chemiosmotic Coupling in Onidatiue and Photosynthetic Phosphorylation, Glynn Research Ltd., Bodmin

Mitchell. P. (1968) Chemiosmotic Counlina and Energy Trans- duction, Glynn Research Ltd., Bodkin -

-”

Greville. G. D. (1969) in Current Tonics in Bioenerpetics (Sanadi,

4. 5. 6. 7. 8.

D. R., ed) Vol. 3, pp. l-78, Academic Press, New York Mitchell, P. (1961) Nature 191, 144-148 Mitchell, P., and Moyle, J. (1965) Nature 208, 147-151 Mitchell, P., and Moyle, J. (1967) Biochem. J. 105, 1147-1162 Moyle, J., and Mitchell, P. (1973) &o&em. J. 132, 571-585 Downie, J. A., and Garland, P. B. (1973) Biochem. J. 134, 1045-

1049 9.

10.

11.

12.

13.

14. 15. 16. 17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

Lawford, H. G., and Garland, P. B. (1972)Biochem. J. 130,1029- 1044

Lawford, H. G., and Garland, P. B. (1973) &o&em. J. 136, lll- 720

Hinkle, P. C., and Horstman, L. L. (1971) J. Biol. Chem. 246, 6024-6028

Papa, S., Guerrieri, F., and Lorusso, M. (1974) Biochim. Bio- phys. A& 357, 181-192

Ragan, C. I., and Hinkle, P. C. (1975) J. Biol. Chem. 250, 8472- 8476

Mitchell, P., and Moyle, J. (1969) Eur. J. Biochem. 7, 471-484 Nicholls, D. G. (1974) Eur. J. Biochem. 50. 305-315 Rottenberg, H. (1975) J. Bioenerg. 7, 61-74 Witt, H. T. (1975) inBioenergetics of-Photosynthesis (Govindjee,

ed) pp. 493-554, Academic Press, New York Lehninger, A. L., Carafoli, E., and Rossi, C. S. (1967) Adu.

Enzymol. 29, 259-320 Selwyn, M. J., Dawson, A. P., and Dunnett, S. J. (197O)FEBS

Lett. 10, l-5 Lehninger, A. L. (1974) Proc. Natl. Acad. Sci. U.S. A. 71, 1520-

1524 Rottenberg, H., and Scarpa, A. (1974)Biochemistry 13,4811-4817 Brand, M. D., Chen, C-H., and Lehninger, A. L. (1976) J. Biol.

Chem. 251, 968-974 Brand, M. D., Reynafarje, B., and Lehninger, A. L. (1976) J.

Biol. Chem. 251, 5670-5679 Johnson, R. N., and Chappell, J. B. (1973) Biochem. J. 134,769-

774 Coty, W. A., and Pedersen, P. L. (1974) J. Biol. Chem. 249,2593-

2598

29.

Brand, M. D., Reynafarje, B., and Lehninger, A. L. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 437-441

Reynafarje, B., Brand, M. D., and Lehninger, A. L. (1976) Fed. Proc. 35, 1601

Schneider, W. C. (1957) inManometric Techniques (Umbreit, W. W., Burris, R., and Staufer, J. F., eds) p. 188, Burgess Pub- lishing Co., Minneapolis, Minn.

Murphy, J. B., and Kies, M. W. (1960) Biochim. Biophys. Acta 45, 382-384

30. Chen, P. S., Jr., Toribara, T. Y., and Warner, H. (1956)AnaZ. Chem. 28, 1756-1758

31. Brand, M. D., and Lehninger, A. L. (1975) J. Biol. Chem. 250, 7958-7960

32. Nicholls, D. G. (1974) Eur. J. Biochem. 49, 573-583 33. Thomas, R. C., Manger, J. R., and Harris, E. J. (1969) Eur. J.

Biochem. 11, 413-418 34. Lee. Y.-P.. and Lardv. H. A. (1965) J. Biol. Chem. 240.1427-1436 35. Klingenbeig, M. (l<lb) Eur. J. Biochem. 13, 247-252 36. Taeer. J. M., Veldsema-Currie, R. D., and Slater, E. C. (1966)

Nat&e 212; 376-379 37. 38

Moyle, J., and Mitchell, P. (1973) FEBS Lett. 30, 317-320 McGivan, J. D., and Klingenberg, M. (1971)Eur. J. Biochem. 20,

392-399

REFERENCES

by guest on February 3, 2018http://w

ww

.jbc.org/D

ownloaded from

Mitochondrial H+lSite Ratio 7451

39. Chappell, J. B., and Haarhoff, K. N. (1967) in Biochemistry of chemistry 5, 2326-2335 Mitochondria (Slater, E. C., Kaniuga, Z., and Wojtczak, L., 43. Rossi, E., and Azzone, G. F. (1969)Eur. J. Biochem. 7,418-426 eds) pp. 75-91, Academic Press, New York 44. Azzone, G. F., and Massari, S. (1971) Eur. J. Biochem. 19,97-107

40. Robinson. B. H.. Williams. G. R.. Haluerin. M. L.. and Leznoff, 45. Mitchell. P. (19721 FEBS Svmv. 28. 353-370 C. C. (i972) J: Membrane Biol.’ 7, 391-401 46. Drahota; Z., Carafoli, E.,” Rossi, C. S., Gamble, R. L., and

41. Pressman. B. C. (1965) Proc. N&l. Acad. Sci. U. S. A. 53, 1076- Lehninger. A. L. (1965) J. Biol. Chem. 240, 2712-2720 1083 47. Stucki, JYW., and Ineichen, E. A. (1974) Eur. J. Biochem. 48,

42. Cockrell, R. S., Harris, E. J., and Pressman, B. C. (1966) Bio- 365-375

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.jbc.org/D

ownloaded from

B Reynafarje, M D Brand and A L Lehningermeasurements.

Evaluation of the H+/site ratio of mitochondrial electron transport from rate

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