Proton electrochemical gradient and phosphate potential in mitochondria

307

Biochimica et Biophysica Acta, 501 (1978) 307--316 © Elsevier/North-Holland Biomedical Press

BBA 47438

PROTON ELECTROCHEMICAL GRADIENT AND PHOSPHATE POTENTIAL IN MITOCHONDRIA

G.F. AZZONE, T. POZZAN and S. MASSARI With the technical assistance of Mr. L. PREGNOLATO

C.N.R. Unit for the Study of Physiology of Mitochondria and Institute of General Pathology, University of Padua, Padua (Italy) (Received April 1st, 1977) (Revised manuscript received August 31st, 1977)

Summary The paper reports an analysis of the relationship between A~H the proton electrochemical potential difference, and AGp, the phosphate potential. Depression of A~H and AGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b)nigericin (+ valinomycin); (c)KCI (+ valinomycin); and (d)rotenone. The uncoupler depresses A~H more than nigericin (+ valinomycin), KC1 (+ valinomycin) and rotenone at equivalent AGp. The AGp/A~H ratio is about 3 at high values of A~ H. When AGp and A~H are depressed by nigericin {+ valinomycin) the AGp/A/~ H ratio remains constant. When AGp and A/~H are depressed by uncouplers, the AGp/A~ H ratio increases hyperbolically tending to infinity while A~ H tends to zero. The absence of constant proportionality between AGp and A~H indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.

Introduction

Lardy [1] and Chance and Williams [2,3] considered respiratory control to be of kinetic origin. Klingenberg [4,5] considered respiratory control as due to an increased rate of reversed electron transfer. It is now generally accepted that oxidation reduction reactions and ADP phosphorylation are near equilibrium i.e. mitochondrial respiration is dependent on AGp, the phosphate potential [6--10]. If A~H is the obligatory intermediate between electron transfer and ATP synthesis some sort of constant relationship should exist between A~H and AGp. Abbreviations: TPMP, t r i p h e n y l m e t h y l p h o s p h o n i u m ; FCCP, carbonyl cyanide p-trifluoromethyl-

phenylhydrazone.

308 In a previous paper [11] we s h o w e d that the relationship between rate of controlled respiration and A~H is not that expected from a simple thermodynamic equilibration. In the present paper we investigate the relationship between A~H and AGp in order to further ascertain the kinetic competence of A~ H as the sole and obligatory energy transducing intermediate. The approach is similar to that used in the preceding paper, namely determination of A~H on the ion distribution under conditions of steady fluxes. Decline of AGp and depression of A~H have been obtained by titrations against increasing concentrations of various ionophores. It appears that the effects of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), valinomycin, rotenone and nigericin + valinomycin are not equivalent. The capacity of uncouplers to cause depression of ~PH, not proportional to the decline of AGp, results in an increase of the AGp/A~H ratio. The observation is discussed in relation to the question of whether the proton circuits driving ATP synthesis involve microscopic events. Experimental Preparation of mitochondria and incubation medium and determination of A~ n were as described in the preceeding paper [11]. The matrix cation concentration [cation]m was calculated from Eqn. 1. Cp -- C s Y e

[cation]m =

Vi

(1)

where Cp and Cs are the amounts of cations in the pellet and in the supernatant, Ve and Vi are the extramatrix and the matrix water in the pellet. Corrections for the a m o u n t of cation bound, in the case of Ca 2* were also as described in the preceding paper [ 11 ]. The phosphate potential was determined essentially as described by Slater et al. [6]. Time of incubation was 7 min except in the case of rotenone where it was prolonged to 15 min. The length of the incubation period was selected after ascertaining that the ATP/ADP ratio had become independent of the time of incubation. The reaction was terminated with perchloric acid. After cooling and neutralization with triethanolamine/KOH, the samples were centrifuged and the supernatants were assayed for nucleotides. ATP was determined in the presence of hexokinase, glucose-6-phosphate dehydrogenase and NADP. The formation of NADPH was followed with an Eppendorf fluorometer. The amount of net ATP synthesis was obtained by correcting for the amount of ATP found in a sample, incubated under identical conditions, supplemented with 2 • 10-4 M FCCP and antimycin A. This latter ATP is presumably due to residual adenylate kinase activity. ADP was determined, also fluorimetrically, in the presence of pyruvate kinase, phosphoenolpyruvate, lactic dehydrogenase and NADH. The phosphate potential was calculated by employing a value of --7.9 kcal/mol for the standard free energy of hydrolysis of ATP [6,12,13]. The data reported in the figures are the averages of 12--16 independent experiments. Chemicals were o f the highest purity commercially available. [3H]Triphenylmethylphosphonium was kindly provided by Dr. R. Kabach.

• 309

Results and Discussion

The effects of FCCP, nigericin and valinomycin on AGp and A~ H Fig. 1 shows the effect of FCCP, rotenone and nigericin (+ valinomycin) on AGp. The values of AGp with FCCP and rotenone were determined either in the presence or in the absence of valinomycin. The difference in AGp values due to treatment with valinomycin was negligible. FCCP, rotenone and nigericin caused a decline in AGp which was proportional to the amount of the inhibition. AGp fell from values of 530 mV, in the absence of inhibitors, to values of 420 mV with 80 pmol nigericin • mg -L protein of 450 mV wifh 100 pmol rotenone • mg -1 protein, and of 419 mV with 200 pmol FCCP • mg -1 protein. The decline of AGp from values around 530--550 mV. in the absence of ionophores, to values of 420 mV at the high ionophore concentrations implies a decrease of the ATP concentration in the medium from about 190 to 15 pM. This latter ATP concentration can be determined enzymatically with sufficient accuracy to obtain reproducible results. AGp values below 410 mV were determined only occasionally since they correspond to ATP concentrations in the medium lower than 10 pM.

FCCP

4501

Rotenone

~9 ,q Nigericin

40C l

40 '

100

80

Nigericin

I

FCCP

I00

'

I 2~)0 200

Rotenone p rnol•mg -I protein Fig. I . E f f e c t o f FCCP and nigericin (+ v a l i n o m y c i n ) and r o t e n o n e on AGp. The m e d i u m c o n t a i n e d 0.2 M s u c r o s e , 10 rnM s u c c i n a t e - T r i s , 3 m M Pi-Tris, 10 m M Tris-C1, p H 7.4, 1 m M E D T A , 2 0 0 #M ADP, i pM r o t e n o n e and 4 nag m i t o c h o n d r i a l p r o t e i n . In the s a m p l e s t i t r a t e d w i t h nigericin 0 . 5 m M KCI and 0.2 #g v a l i n o m y c i n / m l w e r e also a d d e d . In t h e s a m p l e s t i t r a t e d w i t h r o t e n o n e , s u c c i n a t e w a s r e p l a c e d w i t h 1 m M f l - h y d r o x y b u t y r a t e . T i m e o f i n c u b a t i o n 7 m i n , w i t h r o t e n o n e 1 5 rain.

310 The effects of FCCP and nigericin on &PH as calculated on the K ÷ distribution are shown in Fig. 2. 200 pmol FCCP • mg -t protein depressed A~H to 45 mV, while 80 pmol nigericin • mg -t protein depressed A~H to 118 mV. In Fig. 2B the values of AGp are plotted against the values of A~H. Since the decline of AGp induced by nigericin was accompanied by a smaller depression of ASH, with respect to the depression induced by FCCP, the slope of the plot was much steeper in the case of nigericin than in the case of FCCP. In the preceding paper [11] it was found that the rates of respiration induced by ADP and ionophores were not accompanied by similar levels of A~ H. In the experiment of Fig. 2 a similar discrepancy is observed. Nigericin + valinomycin cause a decline of AGp with minor variation of Ap~: levels of AGp around 420 mV are obtained while A ~ H is still above 100 mV. However with FCCP, levels of AGp around 420 mV are reached when / ~ H falls below 50 mV. As discussed in the preceding paper [11] the calculation of A~ on the K ÷ distribution in the presence of nigericin is incorrect, since nigericin induces a K ÷ efflux and therefore leads K ÷ out of electrochemical equilibrium. This means, as discussed previously, that the values of / ~ calculated on the K ÷ distribution in the presence of nigericin are lower than the real A~. Therefore the discrepancy between the effects of FCCP and of nigericin + valinomycin is larger than apparent from the data of Fig. 2. To test this conclusion, decline of AGp and depression of A~ H have been obtained in the experiments of Figs. 3 and 4, in the absence of nigericin. In Fig. 3 the depression of ASH and AGp was obtained by increasing the a m o u n t of valinomycin at three KC1 concentrations, 0.3--0.1 and 1.5 mM. It is seen that the increase of valinomycin caused no decline of AGp at 0.3 mM KC1, except at the highest valinomycin concentration. On the other hand the decline was marked at 1.0 and 1.5 mM KC1. The decline of AGp was accompanied only by minor depression of A~ H. As a consequence the slope of the

200 500 Nigericin E ,-& ::1:

10( FCCP

Nigericin

4b

4513

~o

loin

/

/

8b

~Bo FCCP(pmol/mg protein)

~6o ~D

2b

,d/2H(mV)

Fig. 2. Effect of F C C P a n d nigericin (+ v a l i n o m y c i n ) on A~H and AGp. A~ H measured on K +. Experim e n t a l c o n d i t i o n s a s in Fig. 1. A~ H w a s m e a s u r e d a s d e s c r i b e d in Experimental.

311

•

~

e

"%

0.3 mM ,~1

0.3 mM

~

500

500

1.5mM I , \ z~

E

v

450

450

\in

i 400

400

go

Valinomycin(log pmol. rng-1protein)

loo

1~o

zl/~M(mV)

Fig. 3. E f f e c t o f v a l i n o m y c i n and KC1 o n AGp and A ~ H . ~ ' # H m e a s u r e d o n K +. E x p e r i m e n t a l c o n d i t i o n s as in Fig. 1 e x c e p t that t h e c o n c e n t r a t i o n s o f KCl and o f v a l i n o m y c i n w e r e as i n d i c a t e d in the figure.

plot AGp vs. A~a was very steep, low values of AGp being reached at still high values of A~ H. The experiments of Fig. 3 thus show that a marked decline of AGp in the presence of a minor change of A~H can also be obtained in the absence of nigericin. In Fig. 4 the depression of AGp and A ~ H in valinomycin treated mitochondria was obtained by increasing KC1 between 0.3 and 20 mM. It is seen that AGp fell from 525 to 400 mV. This is due to the fact that increase of [K*]0 leads to considerable swelling and membrane stretching. The plot of AGp vs. A~H shows

500i

~3L CD

45C

\

450

ok

/o

t Z

0 ~ 0

4oc T

~'~o lJO KCI (raM)

4oc

210

./

T

lC)0 zl/~H(mV)

2~)0

Fig. 4. E f f e c t o f KCI on A G p and A ~ H. A ~ H m e a s u r e d o n K +. T h e m e d i u m c o n t a i n e d 0 . 2 M s u c r o s e , 1 0 m M s u c c i n a t e - T r i s , 1 m M / ~ - h y d r o x y b u t y r a t e , 3 m M Pi-Tris, p H 7 . 4 , 1 m M E D T A , 2 0 0 p M A D P , 0 . 5 ~g v a l i n o m y c i n / m l a n d 4 m g p r o t e i n . T h e o s m o l a r i t y w a s k e p t c o n s t a n t by d e c r e a s i n g the s u c r o s e c o n c e n t r a f l a n n~r~11~l t n t h ~ ~ n r r ~ p

nf th~ ~1

~nnP~nf1"~t|~n

h~ts~n

~ ~n~1 9 N

n~1~ r T i m ~

nf in~llh~'inn

~

min

312 t h a t the decline of AGp occurred parallel to a depression of A~ a between 120 and 50 mV. A c o m m e n t is needed as to the mechanism involved in the depression of A~H in the experiments shown in Figs. 1--4. This is clear in the case of FCCP and o f nigericin + valinomycin: (a) electrical ent ry of H ÷ via FCCP and (b) electrical e n t r y by K ÷ via valinomycin followed by electroneutral efflux o f K + via nigericin. In the case of the depression induced by high KC1 concentrations, it is k n o wn th at increase of KC1 in the m edi um above 0.5 mM leads to extensive K ÷ uptake, matrix osmotic swelling and m e m b r a n e stretching. Increased H ÷ influx and K ÷ efflux then occur through the leaky m em brane with a marked stimulation o f the basal respiratory rate. In the case of the A ~ H depression induced by high valinomycin concentrations, m e m b r a n e damage is also likely although the molecular mechanism for this effect is n o t clear. In conclusion it appears that the slope of the plot AGp vs. A g H tends always to be steeper u n der conditions where the ef f ect of the ionophores is t hat of inducing a cycling o f b o th H ÷ and cations through the membrane. In a preceding paper [11] we c om par e d the values of A~H calculated on the K ÷ distribution with those calculated on the TPMP ÷ and Ca ~÷ distribution. Fig. 5 shows a plot of AGp vs. A~H as measured on either TPMP ÷ or Ca 2+ distribution. The values of A~H, based on Ca 2÷, were obtained by titrating the effect o f FCCP, of nigericin + valinomycin (cf. Fig. 2 ref. 11) and of r o t e n o n e in the presence o f 200 pM Ca 2÷ and 10 mM acetate. These conditions are differe n t from those used to measure AGp (involving no Ca 2÷ and 3 mM Pi). The implicit assumption is that the e x t e n t of depression o f A~H induced by the

TPMP

F

50C

450

CQ2 +

+

//

. 1~0

t~)

~

1~)

1~)

J ~ H (mV) Fig, 5. E f f e c t o f FCCP, nigericin (+ v a l i n o m y c i n ) a n d r o t e n o n e o n AGp a n d A~ H. A ~ H m e a s u r e d o n T P M P + a n d C a 2+. T h e m e d i u m c o n t a i n e d 0 . 2 M s u c r o s e , 1 0 m M s u c c i n a t e - T r i s , 3 m M Pi-Tris, 1 0 m M TrisCI° p H 7 . 4 , 1/~M r o t e n o n e , 1 m M E D T A , 2 0 0 p M A D P a n d 4 m g m i t o c h o n d r i a l p r o t e i n . In the s a m p l e s t i t r a t e d w i t h n i g e r i c i n 0 . 5 m M KC1 and 0 . 2 /~g v a l i n o m y c i n / m ! w e r e also a d d e d . In the s a m p l e s t l t r a t e d w i t h r o t e n o n e , s u c c i n a t e w a s r e p l a c e d w i t h ~ - h y d r o x y b u t y r a t e . TPMP + d i s t r i b u t i o n w a s m e a s u r e d in t h e p r e s e n c e o f 2 5 ~ M T P M P +. C a 2+ d i s t r i b u t i o n , in t h e p r e s e n c e o f various u n c o u p l e r and nigericin c o n c e n t r a t i o n s , w a s m e a s u r e d in a m e d i u m w e r e E D T A , Pi a n d A D P w e r e o m i t t e d and 2 0 0 # M C a 2 + + 1 0 m M acetate were added.

313 various inhibitors is similar under the two experimental conditions. Fig. 5 shows, in accordance with the data of Fig. 2, a marked difference in the relationship AGp vs. A~H between FCCP on one side and nigericin and rotenone on the other. The slope of the plot was much steeper in the case of nigericin (+ valinomycin) than in the case of FCCP. With TPMP ÷ as permeant cation the initial decline of AGp occurred in the presence of an apparent slight increase of A~H at the low nigericin concentrations. This is presumably due to the further uptake of TPMP ÷ following K ÷ release induced by nigericin. The fact that the values of A~H during the FCCP titration are lower with Ca 2÷ than with TPMP ÷ (cf. Fig. 2 of ref. 11) is presumably due to absence of correction for internal binding in the case of TPMP ÷. The binding plays a major role at low TPMP ÷ uptake. The lower values of ASH, during FCCP titration, with Ca 2÷ in respect to TPMP ÷, lead to a more marked difference between effects of FCCP and nigericin when based on Ca 2÷ in respect to TPMP+. The effect of rotenone was more like t h a t of nigericin (+ valinomycin) than that of FCCP, i.e. it affected AGp more than A~H. The AGplA~Hratio From the plots of Figs. 2 and 5 it is possible to calculate the AGp/A/~H ratios as a function of A~H. In the case of the effect of nigericin the AGp/A~H ratios are constant in the range of 3 [13--17]. However in the presence of FCCP the AGp/A~H ratio tended to increase with the depression of APH. In Fig. 6 are reported the various AGp/A~H ratios obtained at the various FCCP concentrations (cf. Figs. 2 and 5). Fig. 6 shows that the plot AGp/A~H is hyperbolical and that all ratios obtained with either K ÷, or TPMP ÷ or Ca 2÷ as permeant cation are lying on the same curve. Energy for ATP synthesis can be provided in the various transducing systems either by metabolic reactions (respiration or illumination) or by the difference of proton electrochemical potential. If /~t~H is the obligatory intermediate between metabolism and ATP synthesis, aerobic or illumination driven ATP

2O O

C a 2+

A

K+

•

TPMP +

16o

2~o

z/~ H ( m Y )

Fig. 6. Relationship b e t w e e n A G p / A ~ H ratio and A~H. The values were t a k e n from Figs. 2 and 5.

314 synthesis is equivalent to ion driven ATP synthesis. If ATP synthesis is driven by A~H the following relation holds: AGp/A'fiH = n, where n is the number of H + translocated per ATP. However if n is a finite number, whether 2 or 4, an osmotic threshold might be predicted, i.e. a value of A~H below which ATP synthesis is abolished [17]. The lower the n value, the higher is the osmotic threshold in mV. For AGp = 560 mV and n = 2 the osmotic threshold is 280 mV; for AGp = 560 and n = 4, t!,.e threshold is at 140 mV. Furthermore the lower AGp, the lower is the level of the osmotic threshold. The osmotic threshold concept predicts that ATP synthesis should be abolished when the AGp/A~H ratio is higher than 2 or 4. An osmotic threshold during ion driven ATP synthesis has been reported in intact mitochondri~ [18], submitochondrial particles [19], chloroplasts [20,21] and bacterial chromatophores [22]. Junge et al. found a minimal /X~ for ATP synthesis in chloroplasts [23]. In the case of metabolism-driven ATP synthesis the results reported vary [24]. Graber and Witt [25] found a threshold of 50 mV in chloroplasts at low ApH, and no threshold at high ApH. Melandri et al. [26] found in chromatophores a A/~H threshold at 120 mV when energy supply was limited by uncoupler; however when energy supply was limited by electron transfer the Aftn threshold was at 300 mV. Jackson et al. [27] found that following short flash excitation in chromatophores the extent of ADP + Pi induced decay of the carotenoid shift was a constant fraction, 10% of the total decay. This is not in accord with an osmotic threshold if a large part of/k~H exists as A~. Massari and Azzone [28] found lack of ADP induced stimulation of respiration below A~Hof 120 inV. In the present study [29,30] the decline of AGp induced by either valinomycin alone or nigericin (+ valinomycin) is accompanied by negligible changes of the AGp/A~H ratio. A constant AGp/A~H ratio of about 3 is observed at the various concentrations of valinomycin and of nigericin (+ valinomycin). On the other hand the decline of z~Gp induced by uncoupler is accompanied by a major increase in the ,SGp/,5~n ratio. The occurrence of relatively high values of AGp in the presence of low values of A~H gives rise to the hyperbolic relationship between AGp/A'fi H and A~ H. The ratio tends to infinity while A~ n tends to zero. It may be argued that the AGp/A'fiH ratio should have been calculated on the endogenous AGp {taking into account the concentration of nucleotides and Pi in the matrix) rather than on the exogenous AGp. However the effect of A/~H is that of increasing the exogenous in respect to the endogenous AGp. Therefore the decrease of A~ H should decrease rather than increase the ,SGp/,5"fiH ratio. Conclusion Whether A~ H is the obligatory high energy intermediate in energy transductions is controversial [17,31--35]. In the present study the relationship between Z~Gp and A~H is n o t constant but depends on the pathway for energy drain. While inophores inducing H+-cation cycling maintain a fixed AGp/A~H ratio, classical uncouplers inducing only electrical H ÷ entry, cause an increase of the AGp/A'fiH ratio at low A~H values. This is in accord with the view of a discrete interaction between the two energy transducing units catalyzing electron transfer and ATP synthesis. This is shown in the scheme of

315 In

Out Electron

Transport

A~H

A~H ~active .- ~trans_~_rt AT P ase

Fig. 7. R e l a t i o n s h i p b e t w e e n e n e r g y t r a n s d u c i n g u n i t s .

Fig. 7 where active transport is seen an intrinsic property of the energy transducing units. If energy is drained for ATP synthesis or for active transport at a rate faster than for equilibration with A~H, the stimulation of the respiration or the decline of AGp are more pronounced than the decline of A/~H. This may be the case with ADP or nigericin (+ valinomycin) or A23187 or valinomycin which drain energy, the former at the level of the ATP synthesis and the latter at the level of active transport, respectively. The opposite occurs with FCCP which causes primarily a decline of A~H. It is likely that as the primary event leading to charge separation in each energy transducing unit is molecular in nature, so also the proton circuits connecting electron transfer and ATP synthesis transducing units are operating over microscopic environments. If this is so, then the determination of A~H in the bulk phases may have little relation to the driving force at the catalytic site of the ATP synthetase. The titrations with FCCP provide information about the dimension of the microscopic event for energy conservation. A~H was depressed below 20 mV and AGp below 420 mV at FCCP concentrations of 200 pmol • mg -1 protein, i.e. 12.4 • 10 '3 molecules FCCP • mg -1 protein. It has been calculated that 1 mg protein of rat liver mitochondria contains 7 . 2 . 1 0 9 mitochondria and each mitochondrion contains 20 000 respiratory chains [36]. The concentrations for full uncoupling then correspond to about 20 000 FCCP molecules per mitochondrion and 1 FCCP molecule per respiratory chain. The argument used here to assess the dimension of the enerTy conserving unit in rat liver mitochondria is similar to that used for chloroplasts and chromatophores. In this case it has been calculated that uncoupling requires 1 molecule of gramicidin per thylakoid (approx. 10 s chlorophyll molecules) and 1 molecule of valinomycin per vesicle (approx. 5 • 103 bacteriochlorophyll molecules) for chloroplasts and bacterial chromatophores, respectively [37]. The limitations o f this argument have already been discussed [38]. Note added in p r o o f (Received November 7th, 1977) Mitchell has recently proposed [39] a miniaturized version of the chemiosmotic hypothesis where the proton circulation is comparatively localised. In our view the critical question is n o t the extent of localisation of the proton cir-

316

c u l a t i o n b u t rather w h e t h e r e n e r g y transducing m e m b r a n e s o p e r a t e as fuel cells or as m o l e c u l a r energy m a c h i n e s (see ref. 17). T h e n u m b e r o f c y c l e s o f e l e c t r o n transfer required t o increase t h e t h e r m o d y n a m i c p o t e n t i a l o f a single m i t o c h o n d r i o n at a level c o m p a t i b l e w i t h A T P s y n t h e s i s is 2 • 10 4 in t h e case o f a fuel cell and 1 in t h e case o f a m o l e c u l a r e n e r g y m a c h i n e . References 1 2 3 4

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