re-evaluation of the h+/site ratio of mitochondrial electron

THE JOURNAL OF BIOL~CICAL CHEMISTRY Vol. 251, No. 18, Issue of September 25, pp. 5670-5679, 1976

Printed in U.S.A.

Re-evaluation of the H+/Site Ratio of Mitochondrial Electron Transport with the Oxygen Pulse Technique*

(Received for publication, March 4, 1976)

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

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

The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105,1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments.

1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate.

2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Ad- dition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/ site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0.

3. Lowering the temperature to 5”, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide.

The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca’+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site

ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during an-

aerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/ site ratio for the primary H+ ejection process during electron transport.

Mitochondrial electron transport is accompanied by translocation of H+ ions from the matrix to the suspending medium (for reviews see Refs. l-3). The chemiosmotic theory of oxidative phosphorylation (l-4) proposes that these protons are translocated by the components of the respiratory chain, vectorially arranged in loops in the inner membrane, and that the electrochemical H+ gradient so generated is subsequently used to drive the proton-translocating ATPase in the direction

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

j: Present address, Department of Biochemistry, University of Cam- bridge, Cambridge CB2 l&W, United Kingdom.

of ATP synthesis. In this theory passage of a pair of electrons through each of the three energy-conserving sites in the respiratory chain between NADH and oxygen is linked to ejection of a fixed number of protons, hereafter called the H+/site ratio.

In his original formulation of the chemiosmotic hypothesis Mitchell (4) suggested that the H+/site ratio was 1.0. However “oxygen pulse” experiments carried out by Mitchell and Moyle to measure the quantity of H+ ejected during utilization of a known amount of oxygen subsequently gave H+/site values of 2.0 (5-9). This latter value has been supported by a number of other experiments following essentially the same procedures but using pulses of oxidants other than oxygen, by using pulses of reductants rather than oxidants (7, 10, ll), or by using

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Stoichiometry of Mitochondrial Proton Ejection 5671

submitochondrial particles, which are inverted and thus take up H+ rather than ejecting it (12, 13). The number of H+ ejected per molecule of ATP hydrolyzed by the mitochondrial ATPase (the H+/ATP ratio), has been measured by very simi-

lar procedures, also giving a value of 2.0 (2, 5, 14-16). Recently three separate lines of evidence have suggested

that the value of 2.0 for the mitochondrial H+/site ratio may be

an underestimate. (a) Simultaneous measurements of the magnitude of the electrochemical gradient of H+ and the phosphate potential (a function of the ratio [ATP]/[ADP]. [pi]) which it

can sustain indicate that the observed gradient falls some 50 to

80 mV short of accounting for the phosphate potential if the

H+/site and H+/ATP ratios are taken to be 2.0, but agrees

fairly well using higher values of 3 or 4 (17-19). (5) Many obser-

vations that 2 Ca2+ ions are accumulated per 2e- per site (20),

and more recent evidence that Ca’+ transport into mitochon-

dria is by electrophoretic uniport (21-23), indicate that 4 positive charges are transported inward per 2e- per site. More-

over, we have recently shown that the H+/site ratio is between

3.5 and 4.0 during uptake of Ca2+ with weak acids as source of

counteranions, rather than phosphate (24, 25). This stoichiometry is distinct from the superstoichiometric H+ ejection due to

hydrolysis of endogenous ATP seen during Ca2+ uptake (26,

27). (c) Measurements made on photosynthetic phosphorylation also indicate that the H+/ATP ratio may be greater than

2.0; values between 3.0 and 4.0 have been reported (see Ref.

28).

In oxygen pulse experiments as devised by Mitchell and

Moyle (5-11) compensating inward movement of cations such

as Ca*+ or KC in the presence of valinomycin or compensating

outward movements of anions such as thiocyanate (6, 7, 29-31) are required in order to prevent the development of a trans- membrane potential, while allowing respiration-driven ejection of a sufficient quantity of H+ to be measured with a glass

electrode. Providing these movements of Ca2+ or K+ are not

directly linked to H+ movement (for example, by an obligatory

cation/H+ antiport carrier) they should not affect the magni-

tude of the observed H+/site ratio. On the other hand, respiration-coupled inward movement of phosphate, which

traverses the membrane via a specific HzPO,-/OH- antiporter

(see Ref. 32), or of weak acids, which can carry protons, will

reduce the amount of H+ ejected during electron transport and may lead to underestimated H+/site ratios. Mitchell and

Moyle (6) recognized this possibility, but considered that such movements would be completely corrected for by calculation of the amounts of H+ ejected by means of an extrapolation

procedure based on the exponential decay of the acidity generated by the oxygen pulse, particularly if the experiment was carried out at 5”, when phosphate transport was assumed

to be greatly reduced. In this paper we describe a re-examination of the classical

oxygen pulse method of measuring H+/site ratios. We show that the H+/site ratios observed in this method are underestimated due to re-uptake of endogenous phosphate released from

the mitochondria during the anaerobic preincubation; more-

over, the corrections applied by Mitchell and Moyle (6) do not sufficiently compensate for the movements of phosphate. Prevention of phosphate transport by specific inhibition of the

phosphate transport system of the inner membrane, or by

depleting the mitochondria of endogenous phosphate, causes

the observed H+/site ratio to rise from 2.0 to 3.0. Preliminary

accounts of these findings have been published (25, 33).

EXPERIMENTAL PROCEDURE

Liver mitochondria were isolated from male Charles River rats (retired breeders) (Charles River Breeding Laboratories, Wilmington, Mass.). After 3 washes in 250 mM sucrose they were resuspended in sucrose (50 mg of protein per ml) and stored at 4” until use. The acceptor control ratio was generally about 6.0 with succinate as substrate. Protein was determined according to Murphy and Kies (34) and phosphate according to Gomori (35). N-Ethylmaleimide and mersalyl were obtained from Sigma Chemical Co., St. Louis. A fresh solution of N-ethylmaleimide was prepared for use each day. Other reagents were of analytical grade.

Oxygen pulse experiments were carried out in a 2-ml glass cell with magnetic stirring within a water jacket maintained at 28”. A Clark oxygen electrode (Yellow Springs Instrument Co.) was inserted through the side of the chamber and a combination pH-sensitive glass electrode (Thomas, 4094 L15, connected to a Beckman Expandomatic SS2 pH meter) through the top. The cell had a restricted opening over which was directed a stream of oxygen-free nitrogen. The output from the electrodes was fed into a dual-channel recorder (Sargent-Welch DSRG); the response time of the whole system to injected HCl was less than 1 s.

Experiments were carried out basically as described by Mitchell and Moyle (6). The medium (120 mM KCl, 3 mM Hepes,’ pH 7.1, at 28”), flushed with oxygen-free nitrogen to render it anaerobic, was pumped into the cell. Mitochondria (200 ~1 of stock suspension, containing 10 mg of protein) were added to yield a total volume of 2.0 ml and were allowed to reduce the small amount of oxygen present with the use of endogenous substrates. On anaerobiosis, which was attained within 1 to 2 min, the substrate and either valinomycin or Ca*+ were added. When the pH trace became steady, after about 10 min, the pH was readjusted to 7.10 and after further equilibration oxygen was added as a Z-n1 pulse (equivalent to 11.3 ng atoms of oxygen) of air-saturated KCl/Hepes medium at 28”, which was assumed to contain 453 ng atom of oxygen per ml (36). The recorder response was calibrated in each experiment by injection of standard 0.10 M HCl. Some experiments were carried out with preincubation periods up to and exceeding 20 min (cf. Ref. 6) with identical H+/site ratios. A number of oxygen pulses were performed with each incubation; in many experiments N-ethylmaleimide or other inhibitors were added only after two preceding oxygen pulses had established the inhibitor-free H+/site values. Similar results to those reported here were obtained when nitrogen was replaced by argon or when Hepes was replaced by glycylglycine.

Extrapolation of the rate of decay of acidity in the medium following an oxygen pulse to give H+/O and thus H+/site ratios was carried out as described by Mitchell and Moyle (6), except that log AH+ was plotted as a function of time. The point on this line from which the amount of H+ ejected was calculated was based on the uncoupled rate of oxygen consumption in control aerobic experiments with all conditions and additions identical with those of the experiment being controlled; the rate of oxygen consumption was thus corrected for the slight inhibition caused by N-ethylmaleimide when succinate or glutamate plus malate were used as substrates. N-Ethylmaleimide caused substantial inhibition of uncoupled 3.hydroxybutyrate oxidation; in this case the correction for N-ethylmaleimide inhibition was taken to be of the same relative magnitude as that for succinate. H+/site ratios were calculated from the observed H+/O ratios by assuming two coupling sites for succinate and three sites for 3-hydroxybutyrate and glutamate plus malate.

The phosphate-depleted mitochondria used in the experiments reported in Fig. 9 and Table IV were prepared as follows. Mitochon- dria (10 mg of protein) were incubated in 2.0 ml of anaerobic medium (120 mM KCl, 3 mM Hepes, pH 7.1, 28”) until residual oxygen was consumed (about 1 min). The suspension was transferred anaerobically to centrifuge tubes which were then sealed and incubated for 20 min. The mitochondria were collected by a 10s centrifugation in an Eppendorf 3200 centrifuge. The supernatant was discarded (except where stated), and the pellet was resuspended in 2.0 ml of fresh anaerobic medium by gentle mixing in a Vortex.

‘The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; EGTA, ethylene glycol bis(P-amino- ethyl ether) NJ’-tetraacetic acid.


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5672 Stoichiometry of Mitochondrial Proton Ejection

RESULTS

Measurement of H+lSite Ratio-When a small quantity of

air-saturated medium was added to an anaerobic mitochon-

drial suspension with succinate present as a source of reducing

equivalents there was a rapid acidification of the medium followed by a relatively slow decay back to the initial pH (Fig.

1). The decay was exponential (although frequently biphasic)

and’was extrapolated back to a point calculated to be midway

through the oxygen pulse (6). In all cases the H+ decay curve

ultimately returned to the original base-line pH. Calculation of

the H+/site ratio under these conditions gave a value of 2.04

with either added Ca2+ (Fig. 1A) or K+ in the presence of

valinomycin (Fig. 1B) as permeant cation. A similar value was

I 1 I I 30sec 30sec

I I I 1 30sec 30sec

FIG. 1. H’ ejection by mitochondria pulsed with oxygen. For details see under “Experimental Procedure.” The system during the anaerobic preincubation contained 109 mM KCl, 23 mM sucrose, 2.7 mM Hepes, 0.5 mM succinate, 5 pM rotenone, 5.0 mg of mitochondrial protein per ml, and either 17 ng ions of Ca2+ per mg of protein (A, C) or 100 ng of valinomycin per mg of protein (B, D). N-Ethylmaleimide was present at 40 nmol per mg of protein in C and D. The temperature was 28” and the pH was 7.10. H+ ejection was initiated by addition of 25 ~1 of air-saturated medium (120 mM KCl, 3 mM Hepes, pH 7.10) at the times indicated by the arrows. Extrapolated H+/site values were: A, 2.04; B, 2.04; C, 2.95; D, 3.06. The dashed lines represent the magnitude of the extrapolation.

obtained without either added Ca2+ or valinomycin; under

these conditions endogenous Ca’+ lost from the mitochondria

during the anaerobic preincubation (29-31) was able to col-

lapse the membrane potential. This type of experiment con-

sistently yielded values of the H+/site ratio of close to 2.0, thus

confirming the reports of Mitchell and Moyle (5-7). Effect of N-Ethylmaleimide and Other Inhibitors of Phos-

phate Transport on H’lSite Ratio-In marked contrast to the

results described above, the addition of N-ethylmaleimide at a concentration sufficient to inhibit the translocation of phos-

phate across the mitochondrial membrane via the phosphate/

hydroxide antiporter (37, 38) caused the extrapolated H+/site ratios to rise to about 3.0 (Fig. 1, C and D). This occurred

whether N-ethylmaleimide was added at the start of the

experiment or after a number of “normal” pH jumps had been

observed. Even without extrapolation the peak value of H+

ejected routinely gave H+/site ratios greater than 2.4, well

above the value of 2.0 obtained after extrapolation in the

absence of the inhibitor, demonstrating that values greater

than 2.0 were not dependent on the assumptions inherent in

the method of correction.

The results of a number of such experiments are presented in

Table I. The H+/site ratio with succinate as substrate was very

close to 2.0 in the absence and 3.0 in the presence of

N-ethylmaleimide, with either Ca2+ or K+ as the charge-com-

pensating cation. With 3-hydroxybutyrate or glutamate plus

malate as substrate similar values were obtained, showing that

the effect of N-ethylmaleimide was not site-specific. Table I

also shows the effect of mersalyl, another inhibitor of phos-

phate translocation, on the H+/site ratios; it was almost in-

distinguishable from N-ethylmaleimide in raising the ratio

from 2.0 to 3.0. Other experiments showed that p-chloromer-

curibenzene sulfonate also had this effect, although this inhib-

itor of phosphate transport also substantially increased the

decay rate.

It was important to demonstrate that the H+/site value of 3.0

obtained in the presence of inhibitors of phosphate transport was not dependent on a very specific, narrow range of experi-

mental conditions. Fig. 2 shows the effect of varying the

concentration of N-ethylmaleimide and mersalyl. The curves

are virtually identical with those seen by others (37-40) for

inhibition of phosphate transport in mitochondria. In our

experiments N-ethylmaleimide was routinely used at a con-

centration of 40 nmol per mg of protein, twice the amount

necessary for maximum effect; mersalyl was used at 10 nmol

per mg of protein, rather than at the higher concentrations at

TABLE I

H+/site ratios under different conditions

For details see Fig. 1. Succinate was present at 0.5 mM, 3-hydroxybutyrate at 0.25 mM, and glutamate and malate at 0.5 mM each. Rotenone was present only with succinate. N-Ethylmaleimide was added at a final concentration of 40 nmol per mg of protein, mersalyl at 10 nmol per mg of protein.

-

Substrate Mobile cation

No inhibitor added

H+/site * S.E.

+N-ethylmaleimide

-

+Mersalyl

Succinate K+” 2.02 * 0.02 (57)b 3.00 f 0.02 (52) 2.96 + 0.02 (6) Succinate Caz+c 2.06 i 0.03 (27) 3.01 + 0.03 (36) 3.06 i 0.11 (4)

3-Hydroxybutyrate K+ 1.95 * 0.04 (13) 2.93 * 0.07 (10) 2.57 i 0.07 (5) 3-Hydroxybutyrate Cal+ 1.72 + 0.06 (12) 2.89 i 0.05 (9) 2.72 i 0.07 (6) Glutamate plus malate K+ 1.81 * 0.09 (3) 2.95 * 0.02 (2) 2.71 + 0.01 (3)

a Indicates that 100 ng of valinomycin was added per mg of protein. b Figures in parentheses indicate the number of experiments carried out ‘Indicates that 17 ng ion of Ca2+ was added per mg of protein.


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2

T 2.5 l x

0 IO 20 40 100

NEM or Mersolyl (nmol / mg protein)

1 l

I

FIG. 2. Titration of the H+/site ratio by N-ethylmaleimide (N&W) and mersalyl. The experiments were carried out as described in Fig. 1, in the presence of 0.5 mM succinate and 100 ng of valinomycin per mg of protein. The NEM or mersalyl concentration was varied as indicated.

2 3 I

No NEM = 2. -------?--------.------d ---_--- _‘--, /-,

1.0 0

I 05

Succinate (mM)

FIG. 3. Effect of succinate concentration on the H+/site ratio. For details see Fig. 1. Valinomycin was present at 100 ng per mg of protein; succinate concentration was varied as indicated. NEM, N-ethylmaleimide.

which it inhibits dicarboxylate transport (40). Fig. 3 shows that

altering succinate concentration had no effect on the H+/site ratios obtained. Although the rate of decay of the H+ gradient increased with succinate concentration, the extrapolation procedure was able to compensate, in agreement with results obtained by Mitchell and Moyle (6). In the presence of antimycin A no H+ ejection was seen after pulsing with oxygen. Figs. 4 and 5 show that the amount of oxygen added or the amount of mitochondrial protein present did not affect the H+/site rat.io, unless the ratio of ng atoms of oxygen/mg of protein exceeded 1.8 to 2.0, at which point the H+/site ratio declined, presumably because the pH gradient set up across the mitochondrial membrane became too great (6). Other experiments demonstrated that the H+/site ratios of 2.0 and 3.0 in the absence and presence of N-ethylmaleimide, respec- tively, were unaffected by altering the concentration of rotenone (with succinate as substrate) from 2.5 to 20 FM, or the amount of Ca*+ added (in the presence or absence of valinomy-

/ No NEM

2.0 _ aI

--~ _____ 2-O ____ - ---.---- l -2 --.--.--- 5--\ .

C . . ‘. . .

L-3 ‘;\

“.

I

l.O-

0 I I I I 05 1.0 1.5 2.0

Oxygen Added (ng-atoms/mg protein)

FIG. 4. Effect of amount of oxygen added on the H+/site ratio. For details see Fig. 1. Succinate was present at 0.5 mM, valinomycin at 100 ng per mg of protein. The quantity of oxygen added was varied by injecting different volumes of air-saturated medium. NEM, N-ethylmaleimide.

,I- 2.5 5 7.5 IO 12.5

Mitochondria (mg protein/ml)

FIG. 5. Effect of mitochondrial protein concentration on the H+/site ratio. For details see Fig. 1. The systems contained 0.5 mM succinate and 100 ng of valinomycin per mg of protein; the volume of mitochondrial suspension added was varied. The total volume was maintained at 2.0 ml by appropriate reduction in the volume of medium added; no correction was made for the dilution of the KC1 medium by sucrose added with the mitochondria. The same quantity of oxygen (i.e. 25 ~1 of air-saturated medium) was added at each protein concentration used. NEM, N-ethylmaleimide.

TABLE II

Effect of pH on H+lsite ratios

For details see Fig. 1. The test systems contained 0.5 mM succinate and 100 ng of valinomycin per mg of protein; the initial pH was adjusted to the values given.

PH H+/site

Control Plus N-ethylmaleimide

6.5 2.09 3.05 7.1 2.02 3.00 7.5 2.10 2.90

KC1 by LiCl or addition of up to 2 mM NaCl did not alter the H+/site ratios. Table II shows that variation of the initial pH of the test system over the range 6.5 to 7.5 did not affect the H+/ site ratio. Results similar to those reported here (i.e. H+/site

tin) from 0 to 30 nmol/mg of protein. Similarly, replacement. of ratios of 2.0, rising to 3.0 in the presence of N-ethylmaleimide)


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were obtained with mitochondria isolated from rat heart or from Ehrlich ascites tumor cells, or using a-glycerol phosphate as substrate with liver mitochondria from thyroxine-treated rats.

Effect of Permeant Weak Acids on N’lSite Ratio-The fact that the curves showing the effect of the concentration of N-ethylmaleimide and mersalyl on the increase in the H+/site ratio (Fig. 2) were very similar to those for inhibition of phosphate transport (37-40) strongly indicated that re-uptake of endogenous phosphate lost during the anaerobic preincubation was lowering the H+/site ratio in the absence of the inhibitors. It was therefore of interest to investigate the effects of added phosphate on the H+/site ratio in the presence and absence of N-ethylmaleimide (Fig. 6). In general agreement with Mitchell and Moyle (6) it was found that phosphate markedly reduced the observed H+/site ratio at 28” from about 2.0 to about 1.0 at 2 IIIM phosphate, a 50% reduction. However, N-ethylmaleimide was strongly protective against phosphate; with N-ethylmaleimide present 2 mM phosphate altered the H+/site ratio by only about 17%, reducing it from 3.0 to 2.5. This again supports the interpretation that the action of N-ethylmaleimide was by inhibition of phosphate transport.

0 .Z 2 *... I

!

‘--.- ‘-.J ----__ /No NEM

1.0

l ------- -----.~~~~~~~~~~ ~

01 0

I I I 0.5 I .o 1.5 2.0

Phosphote (mM1

FIG. 6. Effect of added phosphate on the H+/site ratio. For details see Fig. 1. The test systems contained 0.5 mM succinate and 100 ng of valinomycin per mg of protein. Potassium phosphate (pH 7.1) was added as indicated. NEM, N-ethylmaleimide.

II

I.01 I I 0 0.5 1.0

3-Hydroxybutyrote or Propionate ImM)

FIG. 7. Effect of 3-hydroxybutyrate and propionate on the H+/site ratio. For details see Fig. 1. The systems contained 0.5 mM succinate and 100 ng of valinomycin per mg of protein. Sodium 3-hydroxybutyrate (0, 0) or potassium propionate (A, A) were added as shown. NEM, N-ethylmaleimide.

Further evidence is presented in Fig. 7, where it is shown that low concentrations of 3-hydroxybutyrate and propionate, which cross the membrane in protonated form as free acids, decreased the H+/site ratio from 3.0 to near 2.0 in the presence of N-ethylmaleimide, as would be expected because of their ability to carry a proton back into the matrix (see Ref. 22). In the absence of the inhibitor these weak acids did not affect the H+/site ratio of 2.0, because the extrapolation procedure was able to correct for the increased decay rate of the H+ gradient.

This decrease in the observed H+/site ratio by weak acid anions in the presence of N-ethylmaleimide indicated that the value of 3.0 might itself be an underestimate if there were other endogenous acids in mitochondria which could move out during the anaerobic preincubation and move in again during the oxygen pulse, carrying protons with them. For this reason a number of compounds known to inhibit effective translocation of various acids and ions across the mitochondrial membrane were tested for their effect on the H+/site ratio. From the results in Table III it is seen that inhibition of the movements of the major tricarboxylic acid cycle intermediates by inhibitors of the dicarboxylate and tricarboxylate carriers did not increase the H+/site ratio and neither did removal of Ca’+ by EGTA (in the presence of valinomycin) nor inhibition of the mitochondrial ATPase by oligomycin. It is thus unlikely that movements of dicarboxylates or tricarboxylates were sufficient to affect the value of the H+/site ratio measured by this, technique. Inhibition of dicarboxylate transport did not inhibit H+ ejection with succinate as substrate, presumably because sufficient succinate was able to enter the mitochondria during the preincubation even in the presence of the inhibitor. Data in Table III also show that inhibitors of the transport of pyruvate (a-cyano-4-hydroxycinnamate), adenine nucleotides (atractyloside), bicarbonate (Diamox) (41), and free fatty acids (bovine serum albumin), failed to influence the H+/site ratio.

Efflux of Phosphate and H+/Site Ratios of Phosphate-depleted Mitochondria-By direct analysis of the suspending medium from mitochondria preincubated anaerobically, it was found that rapid efflux of endogenous phosphate occurs, as originally noted by Tager et al. (42). Fig. 8 shows that the concentration of inorganic phosphate in the anaerhoic incuba-

TABLE III Inhibitors which did not affect H+/site ratio in absence orpresence of

N-ethylmaleimide

For details see Fig. 1. The test systems contained 0.5 mM succinate and 100 ng of valinomycin per mg of protein.

Compound Final

concentration Mode of action

oc-Cyano-4-hydroxycinnamate

Phenylsuccinate Benzene-1,2,3-

tricarboxylate Atractyloside

LiCl EGTA Bovine serum

albumin Diamox (sodium

acetazolamide) Oligomycin

0.5 mM Inhibits pyruvate transport

2.5 mM Inhibits dicarboxylate transport 0.5 mM Inhibits tricarboxylate transport

2 nmol per mg Inhibits adenine nucleotide protein transport

110 mM Inhibits Na+/H+ antiport (?) 1mM Sequesters Ca*+ 2 mg per ml Sequesters fatty acids

0.1 mM

1.0 pg per mg protein

Inhibits net accumulation of HCO,- derived from medium

Inhibits mitochondrial ATPase


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I I 0 IO 20

Time of onaerobic incubation (mid

0

FIG. 8. Appearance of endogenous phosphate in the extramitochondrial medium during the anaerobic preincubation. Mitochondria (5 mg of protein per ml) were incubated in the anaerobic medium (120 mM KCl, 3 mM Hepes, pH 7.1, 28”) until residual oxygen was consumed (about 1 min). The suspension was then transferred under anaerobic conditions to centrifuge tubes which were sealed, and incubated for the times stated. The mitochondria were collected by a 2-min centrifugation in an Eppendorf 3200 centrifuge, and the supernatant fraction was assayed for inorganic phosphate after precipitation of remaining protein with 150 mM HClO,. Open circles, 0, show effect of continuous bubbling of air through the suspension after 20 min of anaerobic preincubation.

tion medium rose to 100 pM within 10 min. The subsequent slower increase in extramitochondrial phosphate concentration was attributed to hydrolysis of organic phosphate, which is known to occur under anaerobic conditions (42). Fig. 8 also shows that aeration of the suspension after 20 min of anaerobiosis resulted in a very rapid uptake of the extramitochondrial phosphate, about 10 nmol of phosphate/mg of protein being removed from the medium within 10 s. The rate of phosphate uptake under these conditions, in excess of 1 nmol per mg per s, is obviously sufficient to cause lowering of the H+/site ratio in oxygen pulse experiments; the phosphate movement required to reduce the ratio from 3.0 to 2.0 in our experiments is calculated to be about 1.5 to 2.0 nmol per mg within 2 to 3 s.

The anaerobic efflux of phosphate observed in these experiments suggested another experimental approach. Mitochon- dria were depleted of endogenous phosphate by incubating them anaerobically for 20 min at 28” and then collecting them by centrifugation under anaerobic conditions, followed by resuspension in phosphate-free anaerobic medium. The washed mitochondria obtained in this way were then tested in oxygen pulse experiments exactly as before. The results are shown in Fig. 9. Phosphate-depleted mitochondria gave H+/ site ratios approaching 3.0 even in the absence of N-ethylmaleimide or other inhibitors of phosphate transport (Fig. 9A). Such mitochondria were now insensitive to N-ethylmaleimide (Fig. 9B), which produced no change in the H+/site ratio. In a control experiment mitochondria were taken through the same procedure except that the resuspension was in the original suspending medium rather than in a fresh phosphate-free

ApB(\ +-ion 02 t 02

NEM

I

I min I FIG. 9. H+ ejection by phosphate-depleted mitochondria pulsed

with oxygen. The mitochondria were washed anaerobically as described under “Experimental Procedure” and then treated as described in Fig. 1. 17 ng ions of Ca2+ per mg of protein was added in all cases; 40 nmol of N-ethylmaleimide (NEW per mg of protein and 90 pM potassium phosphate were present where indicated. Extrapolated H+/site ratios are given in parentheses. A, control (2.96); B, plus NEM (2.94); C, mitochondria resuspended not in fresh medium but in the original supernatant (1.95); D, as C but plus NEM (2.67); E, control (2.88); F, plus phosphate (2.01); G, plus phosphate plus NEM (2.86). Each horizontal set represents a single experiment.

medium. These mitochondria gave an H+/site ratio of about 2.0 (Fig. 9C), which was increased to about 2.7 by N-ethylmaleimide (Fig. 9D). That phosphate was the responsible factor present in the original supernatant but absent from the fresh medium was shown by adding phosphate to washed, phosphate-depleted mitochondria. The H+/site ratio was reduced from 2.9 (Fig. 9E) to 2.0 (Fig. SF’) by addition of 90 pM

phosphate; addition of N-ethylmaleimide was now able to raise the H+/site ratio to 2.9 (Fig. 9G). Thus it is shown that energized re-uptake of the endogenous phosphate leaking from the mitochondria during the anaerobic preincubation is responsible for the low H+/site ratio of near 2.0 observed in the absence of N-ethylmaleimide.

The phosphate data and other results are summarized in Table IV. It will be noted that washed mitochondria usually gave increased H+/site ratios on addition of either Ca*+ or valinomycin, indicating a shortage of endogenous mobile cations, whereas unwashed mitochondria contained sufficient endogenous Ca’+, which, after leaking into the medium anaerobically, could support H+ ejection during an oxygen pulse. Measurements of efflux of Ca2+ during the anaerobic preincubation gave results similar to those seen previously (30, 31). Washed mitochondria gave H+/site ratios near 3.0 with either Ca*+ or K+ as permeant cation, and with either succinate or 3-hydroxybutyrate as substrate. In all such experiments N-ethylmaleimide had little or no effect unless phosphate was first added to the medium and thus depressed the H+/site ratio.

Oxidation of Endogenous Substrates during Oxygen FUse-


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The experiments in this section were carried out in order to (A4) showing that the mitochondria contained no utilizable eliminate a possible trivial explanation of our results, namely, substrate. Subsequent addition of N-ethylmaleimide did not that N-ethylmaleimide acted not only on phosphate transport, allow the endogenous 3-site substrates to be used, since there but also in some way allowed or promoted utilization of was still no response to an oxygen pulse (A7). Addition of endogenous 3-site substrates even in the presence of rotenone. succinate to these substrate-limited mitochondria allowed the The increased H+/O ratios caused by N-ethylmaleimide in the oxygen which had accumulated in the medium from the presence of succinate would thus be due to an increase in the previous pulses to be reduced, with concomitant H+ ejection number of sites involved in electron transport rather than to an (A8). Following this, normal oxygen pulse traces were seen, increase in the H+/site ratio. Our method of disproving this with the expected H+/site ratios of near 3.0 (A9). Fig. 10B explanation was to deplete the pool of endogenous substrate shows a similar experiment demonstrating that after depletion which could be used during an oxygen pulse and then to of endogenous substrate (BO to B8) and addition of succinate measure the H+/site ratio in the presence of N-ethylmaleimide (RIO). H+/site ratios of 2.0 in the absence (&I) and 3.0 in under conditions where added succinate was the only available the presence (B13) of N-ethylmaleimide could be demon- substrate. strated. Thus the effects of N-ethylmaleimide were not due to

Fig. 1OA shows an experiment in which endogenous 2-site promotion of the oxidation of endogenous 3-site substrates, substrates were depleted by repeated addition of small pulses since these effects could be demonstrated with succinate as of oxygen (A0 to A4). After a number of such pulses (usually substrate under conditions where utilization of endogenous between 4 and 10) further addition of oxygen was without effect substrates was not occurring.

In any case, observations recorded in Fig. 10 (A0 and BO) also

TABLE IV indicate that the endogenous substrate available in fresh

H+lsite ratios in phosphate-depleted mitochondria unpulsed mitochondria in the presence of rotenone was 2-site.

The experiments were carried out as described in Fig. 9 except that Other experiments demonstrated that this endogenous sub-

0.25 mM 3.hydroxybutyrate replaced succinate plus rotenone in strate gave H+/site ratios of 3.0 when N-ethylmaleimide was

Experiment 3, and 100 ng of valinomycin per mg of protein replaced present. In the absence of rotenone, prolonged pulsing with

Ca*+ in Experiments 2 and 5. In Experiments 4 and 5 the mitochondria oxygen did not deplete the endogenous pool of NAD-linked

were resuspended not in fresh medium but in the original supernatant. substrates. Such pulses gave the expected H+/site ratios of 2.0

The figures represent mean values of a number of experiments. in the absence and near 3.0 in the presence of N-ethylmalei-

Hi/site mide, assuming 3 sites.

Effect of Low Temperatures on H+lSite Ratio-Fig. 11 Plus 40

Experi- nmol of shows that when the temperature at which the experiments

merit Substrate Cation NO

Plus ca*+ Plus 90 N-ethyl- were carried out was decreased from 28” to 5-lo”, the ob-

additions or valinomycin WP, maleimide

per mg of served H+/site ratios rose to 3.0 even in the absence of N-

protein ethylmaleimide although in the presence of the inhibitor the ratios were unaffected. This occurred with 0.5 mM succinate

1 Succinate Caa+ 2.70 2.71 2.17 and, to a variable extent, with 2 mM 3-hydroxybutyrate, as 2 Succinate K+ 2.47 2.68 2.64 3 3-Hydroxy- Caa+

substrate. Our interpretation of this effect is that phosphate 2.76 2.72 2.86

butyrate transport is sufficiently slow at lower temperatures to allow the

4 Succinate Ca*+ 1.99 1.96 2.59 extrapolation procedure to compensate for the phosphate-in-

5 Succinate K’ 1.81 1.92 2.76 duced decay of the H+ gradient. At 5” the calculated time for

6 Succinate Cal+ 2.84 2.85 2.08 2.83 influx of sufficient phosphate to reduce the ratio from 3.0 to 2.0 is 10 to 15 s, compared with 1 to 2 s at 28” (see “Discus-

FIG. 10. Contribution of endogenous substrate to the reduction of oxygen. The experiments were carried out as described for Fig. 1, with rotenone and valinomycin present, except that succinate was not added until shown. A and B were separate experiments. Selected curves are presented from a sequence of pulses lasting up to 90 min; the figures by each curve indicate the number of preceding oxygen pulses. Extrapolated H+/site values were: AO, 2.08; A3, 1.17; A9, 2.95; BO, 2.08; B7, 1.10; BIZ, 2.00; 813, 2.80. iVEA4, N-ethylmaleimide.

A

P A3 A4 -lb

t t t A7tA* t Ag t

02 02 02 t

02 succinate 02

NEM

B

T jOng-ion

“i 80 il,r.. YbF

t t 02 02

J2 EK) t e” J2 B’3 ot, succinate

t NEM


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. +x

20.

I I 1 I I

5 IO 15 20 25 30 Temperature PC)

FIG. 11. Effect of temperature on the H+/site ratio. The experiment was carried out as described in Fig. 1 in the presence of 0.5 mM

succinate and 100 ng of valinomycin per mg of protein. NEM, N-ethylmaleimide.

sion”). The reason for the discrepancy between our observations and those of Mitchell and Moyle (6), who reported no increase in the H+/site ratio at 5” with 3-hydroxybutyrate as substrate, is unclear. In any case, many experiments at 5 to 10” with succinate in the absence of N-ethylmaleimide have consis- tently yielded H+/site ratios of 3.0.

DISCUSSION

The results presented here show that the observed H+/site ratio in oxygen pulse experiments may be increased from 2.0 to 3.0 by three different procedures, (a) addition of N-ethylmaleimide or other inhibitors of phosphate/hydroxide antiport, (b) depletion of the endogenous phosphate pool of the mitochondria, or (c) reduction of the rate of phosphate transport by de- creasing the temperature. The common element in these procedures is restriction of the movement of phosphate across the mitochondrial membrane during an oxygen pulse. In addition we have shown that sufficient endogenous phosphate leaks out of the mitochondria during the anaerobic preincubation preceding an oxygen pulse to give an extramitochondrial phosphate concentration greater than 100 pM (Fig. 8); on aeration a substantial proportion of this is very rapidly reabsorbed by the mitochondria.

The evidence that the effect of N-ethylmaleimide is due to its inhibition of phosphate transport rather than to some undefined effect such as the membrane damage seen at high concentrations of sulfhydryl reagents (e.g. Ref. 43) may be summarized as follows.

1. The titration curves of the increase in the H+/site ratio caused by N-ethylmaleimide and mersalyl (Fig. 2) are virtually identical with the titration curves for inhibition of phosphate transport by these inhibitors reported by others (37-40). Thus, the maximal effect of N-ethylmaleimide in raising the H+/site ratio is produced by concentrations that produce maximal inhibition of the phosphate/hydroxide antiporter.

2. A number of other inhibitors of mitochondrial transport processes which do not prevent phosphate movements did not increase the H+/site ratio (Table III).

3. N-ethylmaleimide protected the H+/site ratio against the deleterious (i.e. lowering) effect of added phosphate (Fig. 6).

4. In the presence of N-ethylmaleimide the addition of permeant weak acids other than phosphate reduced the H+/site ratio from 3.0 to near 2.0 (Fig. 7).

5. Washing the mitochondria to reduce the level of endoge-

nous phosphate raised the H+/site ratio from 2.0 to near 3.0; no further increase was then given by N-ethylmaleimide (Table IV).

6. Addition of 90 pM phosphate to phosphate-depleted mitochondria reduced the H+/site ratio from near 3.0 to 2.0; subsequent addition of N-ethylmaleimide reversed this effect of added phosphate and raised the H+/site ratio to near 3.0 (Table IV).

These results provide strong evidence that movements of endogenous phosphate are sufficient in magnitude to reduce the value of the H+/site ratio from 3.0 to 2.0 in the absence of N-ethylmaleimide in our experiments and those of previous workers (5-13). We propose that this occurs as follows. During the anaerobic preincubation phosphate leaks out of the mitochondria via the phosphate/hydroxide antiporter (see Ref. 32) and equilibrates with the suspending medium. The amount of inorganic phosphate may be augmented by hydrolysis of organic phosphates of the mitochondria (42). On addition of oxygen (or of reductant in variations of the pulse method) there is rapid H+ ejection coupled to electron transport, very quickly followed by cation uptake (or anion efflux) down the potential gradient so formed. Simultaneously there is rapid movement of the phosphate from the suspending medium to the matrix, down the pH gradient being set up; this occurs by phosphate/ hydroxide antiport (or phosphate/proton symport). Phosphate uptake is therefore accompanied by net H+ uptake, which reduces the extent and rate of acidification of the medium caused by electron transport and thus leads to a low observed H+/site ratio. Movements of phosphate on the phosphate/ dicarboxylate antiporter are not directly proton-linked (see Ref. 44) and occur more slowly (38), and are thus unimportant in this context. After consumption of the oxygen the pH gradient across the membrane decays exponentially to zero and the phosphate and cation once again leak out.

An alternative explanation of the results is that intramito- chondrial phosphate is necessary for synthesis of ATP from endogenous ADP during an oxygen pulse. Such a synthesis might decrease the net H+ ejection and thus cause an underestimation of the H+/site ratio in the absence of N-ethylmaleimide. This explanation seems unlikely since excess oligomycin does not affect the H+/site ratio, with or without N-ethylmaleimide present (see Table III). Moreover, it is well known that mobile cation uptake (which occurs during the oxygen pulse) takes precedence over ADP phosphorylation in its demand on respiratory energy (45).

Uptake of phosphate is extremely rapid at 28”, particularly in comparison with that of other mitochondrial anions. From the kinetic constants given by McGivan and Klingenberg (32) or by Coty and Pedersen (38) for phosphate/hydroxide antiport, or from the minimum value of phosphate reuptake obtained in Fig. 8, we have estimated the time required in our experiments for sufficient phosphate to move inward to reduce the H+/site ratio from 3.0 to 2.0. This was found to be between 1 and 2 s, although this value is only approximate due to the uncertainties involved in extrapolating to 28” from data obtained at much lower temperatures. Nevertheless, this pe- riod is substantially less than the time taken by the mitochondria to utilize all the oxygen added; thus it is probable that in the absence of N-ethylmaleimide the theoretical amount of H+ ejection is not attained even at times very soon after the oxygen is added. The fact that techniques designed to monitor very rapid changes, such as continuous or stopped flow measurements, also yield H+/site ratios no greater than 2.0 in the


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absence of N-ethylmaleimide (13, 46) supports this interpretation. The observation of Penniston (47) that the H+/site ratios

are elevated during the first 300 ms of oxygenation may be ex-

plained by partial inhibition of phosphate transport by the

fluorescein mercuric acetate (a sulfhydryl reagent) present in his experiments.

It is interesting that Mitchell (48) has also observed an

increase in the H+/O ratio (for endogenous substrate) in the

presence of N-ethylmaleimide. He attributed this to a relative

increase in NADP-linked (i.e. 4.site) oxidation of the endoge-

nous substrate in the presence of the inhibitor. It is evident

from the. results described in Fig. 10 that this explanation

cannot be applied to our experiments, since under conditions

where oxidation of endogenous substrates was demonstrated to

be zero, N-ethylmaleimide raised the H+/site ratio to 3.0 with added succinate as the only source of electrons for the

respiratory chain. Some earlier reports have suggested that more than two

positive charges are moved per Ze- per site during mitochon-

drial cation transport. In these experiments Pressman (49, 50)

and Azzone and colleagues (51,52) measured the number of K+

ions accumulated by respiration-energized mitochondria after

addition of valinomycin and calculated a K+/site ratio of 3.0 to

4.0, or higher. However, as Mitchell (48) has pointed out, some

K+ uptake will be associated with the pre-existing electrochemical gradients across the inner membrane. In addition,

hydrolysis of endogenous ATP may support cation transport

(27). These results are thus unreliable indicators of the H+/site

ratio.

It could be suggested that our measurements in the presence

of N-ethylmaleimide, or with washed mitochondria, or at low

temperatures, give overestimates of the H+/site ratio due to

stimulation of H+ ejection by some hypothetical process

dependent upon electron transport but not an intrinsic element in it. The more obvious explanations for such an effect have

been eliminated in this study, and the major effect of N-ethylmaleimide has been shown to be at the level of the phosphate

carrier. Moreover, estimation of the H+/site ratio by measure-

ment of respiration-energized uptake of large quantities of

Ca’+ and 3-hydroxybutyrate (24, 25) yields values between 3.5

and 4.0 without the necessity of adding N-ethylmaleimide. In

addition the calculation of H+/site ratios from measurements

of the rate of H+ ejection and oxygen consumption following

addition of reductant to a de-energized mitochondrial suspen-

sion also gives values up to 4.0 (25, 53). The three different

methods we have used to estimate the H+/site ratio are subject

to different experimental errors; thus the fact that they all

yield ratios between 3.0 and 4.0 is highly significant.

The values of up to 4.0 for the H+/site ratio obtained by our

other methods (24, 25, 53) raise the question of whether the

value of 3.0 obtained by the oxygen pulse technique in this

paper is a valid reflection of the true H+/site ratio, or whether

it is also an underestimate. Although we cannot answer this

question at the present time the following points may be

raised. In the oxygen pulse experiments the measured changes

in H+ distribution are small (about 5 to 10 ng ion/mg) in

comparison to the H+ ejection occurring in experiments where

large amounts of Ca*+ are accumulated (about 400 ng ions/mg)

(24, 25) or where excess substrate is added to an aerobic system

(about 10 to 20 ng ions/mg) (25,53). Any rapid but small

re-uptake of protons by some uncharacterized pathway would

therefore reduce the H+/site ratio more in oxygen pulse experiments than in the other experimental methods. To

reduce this possibility we have attempted to eliminate move-

ments of all endogenous acids either by direct inhibition (Table

III), or by the anaerobic washing procedure.- In neither case

were H+/site ratios of greater than 3.0 observed. Na+/H+

antiport seems an unlikely cause of rapid proton backflow due to the low rate of this exchange, and also since added Na+ does

not reduce the H+/site ratio, nor does Li+ have any effect even

at the high concentrations at which it may inhibit Na+/H+

exchange (54). Possible exchanges of Ca2+ with H+ were

prevented by addition of EGTA. We have not been able to

eliminate the possibility that a transient increase in H+

permeability or a change in the degree of protonation of some

specific membrane protein occurs on addition of oxygen,

although the lack of dependence on amount of oxygen added or

protein present (Figs. 4 and 5) argues against such effects.

In a recent report van Dam and colleagues (55) have

provided independent support for the results presented here. They found that addition of mersalyl decreased the rate of

decay of the H+ gradient after an oxygen pulse and suggested

that this might be due to inhibition of succinate or phosphate

transport. In addition they observed an increased H+/site

ratio at 3” compared with 32”.

The H+/site ratio has been measured in submitochondrial

particles by means of the oxygen pulse technique and has been

found to be close to 2.0 (12, 46). Since these particles would not

be expected to contain large amounts of endogenous phos-

phate, on the basis of our results with intact mitochondria we

might predict an H+/site ratio of 3.0. We do not know the

reason for this discrepancy, but can suggest that it may be due

to the presence of small (but sufficient) amounts of endogenous

phosphate or other weak acids within the particles. Alterna- tively, either a small proportion of right-side-out vesicles or

some “leakiness” to H+ might cause these lower values.

Similar considerations might apply to the recent determina- tions of the H+/site ratio in reconstituted systems (56, 57).

In conclusion, we have shown in intact mitochondria that

prevention of re-uptake of endogenous phosphate during oxygen pulse experiments results in an increase of the observed

H+/site ratio from 2.0 to 3.0. The mechanism by which 3 (or

more) H+ are ejected per site remains to be defined. However,

it is apparent that existing models of electron transport based

on a stoichiometry of 2.0 H+ per site, such as the “loop”

mechanism of the chemiosmotic hypothesis, may have to be

revised to account for these experimental findings.

Acknowledgments-We thank Irene Wood and Garvon

Givan for technical assistance.

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M D Brand, B Reynafarje and A L Lehningeroxygen pulse technique.

Re-evaluation of the H+/site ratio of mitochondrial electron transport with the

1976, 251:5670-5679.J. Biol. Chem.

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