Etir.
J. Hiochcm. 62. 77-86 (1976)
Anion and Amine Uptake and Uncoupling in Submitochondrial Particles Giovanni Felice AZZONE. Heidi GUTWENIGER, Elena VIOLA, Ermanno STRINNA, Stefano MASSARI, and Raffaele COLONNA 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 Padova (Received September 5. 1975)
1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KC1 or NH,CI. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases. 2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion. 3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-I-napthalene sulphonate cause a pH-independent, 50 uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 pM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two phases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation. 4. Addition of amines to energized submitochondrial particles results in two types of effects: uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling. 5. While the uptake of amines is dependent on the presence of non-permeant anions, such as C1- , the uncoupling effect is independent of C1- . Furthermore the amine uncoupling is markedly enhanced by lipophlic anions. 6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.
When active transport takes place other energyAlthough the inlinked reactions are inhibited [l]. hibition is loosely indicated as “uncoupling” it has been realized that the uncoupling due to active transport is different in nature from the classical uncoupling. In fact the former is due to stoichiometric energy utilization for ion transport while the latter is due to catalytic energy dissipation. The former is accompanied by a stoichiometric cycle of energy utilization, denoted as state 4- state 3 -state 4 transition, while the latter is accompanied by a shift of the steady-state energy level. While the distinction between stoichiometric energy utilization, linked to ion uptake, and catalytic energy dissipation, due to damage or un-
coupler cycling, is clearly established in intact mitochondria, this is not the case in submitochondrial particles. Skulachev el al. [2] reported an uncoupling effect of lipophilic anions in submitochondrial particles and suggested it be equivalent to the energy utilization accompanying cation uptake in mitochondria. Christiansen et al. [3] observed uncoupling by lipophilic anions in submitochondrial particles. Montal et al. [4] reported that addition of nigericin + KCl or amines in the presence of lipophilic anions caused a release of respiratory control and attributed the effect to stoichiometric energy utilization. Other lipophilic anions, such as 8-anilino-1-naphthalene sulphonate, phenyl-
Anion and Amine Uncoupling
78
dicarbaundecaborane, bromthymol blue, and SCN- , are also accumulated in submitochondrial particles [5 - 81. Although it has been generally assumed that anion accumulation is equivalent to uncoupling, the correspondence between energy utilization and energy dissipation has never been analyzed. Also, it is not clear the extent to which anion uptake and uncoupling are dependent on the presence of amines or nigericin + KCl. A marked difference in sensitivity to nigericin KCI and amines has becn found between chloroplasts on one side and chromatophores and submitochondrial particles on the other, which has been tentatively attributed to a difference in anion permeability [9- 141. The uncoupling effect of amines in isolated chloroplasts is well established. Izawa showed that the uncoupling effect of amines in chloroplasts is accompanied by swelling [15]. Crofts [12] proposed a general mechanism for the uncoupling of the amines based on diffusion of NH3 across the membrane, down the pH gradient, and accumulation of NH; . The swelling in NH4Cl was attributed to uptake of NH4Cl in thc inner osmotic space. Kraayenhof [16] observed in chloroplasts an uncoupling effect of the lipophilic amine atebrine, which was accompanied by an uptake of the dye [17]. Schuldiner et al. [18] considered the uptake of the acridines similar to that of NH, and utilized the spectroscopic changes to determine the internal pH [19,20]. In the case of submitochondrial particles a large number of acridine and aminophenazine dyes has also been found to be accumulated after supply of energy [21-241. Recently however it has been reported [25] that addition of lipophilic anions cause release rather than uptake of acridine dyes. We will show below that in contrast with Montal et al. [4] and in accord with Christiansen et al. [3] the uncoupling effect of lipophilic anions is largely independent of the presence of nigericin KCI or of amines. Furthermore the uncoupling effect corresponds to a n energy utilization larger than required for the accumulation of the anions. In regard to the effect of amines in submitochondrial particles we will show that lipophilic amines act as uncouplers and the uncoupling effect increases parallel to the amine lipophilicity and also that, unlike the chloroplasts, the uncoupling effect is not related with the uptake of the amine. The view will be discussed that the uncoupling effect of anions and amines is catalytic rather than stoichiometric in nature and is due to charge cycling.
+
+
EXPERIMENTAL PROCEDURE Beef heart mitochondria1 were prepared according to the procedure previously described [13].Preparation of phosphorylating fragments was obtained by sonicating under nitrogen, at 0 "C,5 ml of beef heart mito-
chondria in 10 mi of the following medium: 0.25 M sucrosc, 5 mM ATP, 5 mM MgCI,, 10 mM MnCI,, 1 mM succinate and 10 mM Tris-CI pH 7.5. After sonication for 30 s the suspension was centrifuged at 7000 x g for 5 min. The pellet was resuspended in the same medium as before and again sonicated for 30 s. The suspension was again centrifuged for 5 min at 7000 x g. The supernatant of the two centrifugations were pooled and centrifuged at 105000 x g for 45 min. The particles obtained by this treatment were denominated as manganese submitochondrial particles and possessed a high rate of phosphorylative activity. However, the phosphorylative activity was rapidly lost and therefore all phosphorylation experiments were carried out within few hours after sonication. In the experiments of Table 2 the preparation of submitochondrial particles was carried out by sonication in 20 mM pyrophosphate [26]. The response of bromthymol blue was measured in a dual wavelength spectrophotometer as described previously [27 - 291. The response of 8-anilino-1-naphthalene sulphonate was measured in an Eppendorf fluorometer [ S ] . The rate of oxygen uptakc was measured polarographically with a Clark electrode. The phosphorylative activity was measured by adding 0.5-2 mg protein of sonic fragments to a standard incubation medium of 2.5 ml containing 40 mM P, pH 7.0, 1 or 10 mM MgCI2 l00pM AMP, 200pM ADP, 0.2 M sucrose, 2.5 mg hexokinase (Sigma type IV), 20 mM glucose and 1 mM NADH or 2 mM succinate. After 10 or 15 min the reaction was blocked with perchloric acid and the system analyzed for the presence of glucose 6-phosphate fluorimetrically [30]. In the experiments where NADH is the substrate since the amount of oxygen consumed corresponds to the amount of NADH added, it is possible to calculate the P/O ratio in the absence of direct oxygen measurements. The energy-linked transhydrogenase [311was measured in a medium containing 0.25 M sucrose, 10 mM Tris-C1, pH 7.2, 1 pg oligomycin, 50 pM NADP, 200 pM NADH, 500 yM oxidized glutathione, glutathione reductdse, 1 pM rotenone, 10 mM MgS04 and 1.6 mg submitochondrial particles. The reaction was initiated by the addition of 2 mM succinate and the disappearance of NADH was followed either fluorimetrically or in the dual wavelength spectrophotometer. The succinate-linked pyridine nucleotide reduction was measured in a medium containing 0.25 M sucrose. 10 mM Tris-CI or 10 mM N-2-hydroxyethylpiperazineN'-Zethane sulphate pH 7.0, 2 mM NaCN, 5 mM MgCI2, 5 mM succinate, 200pM NAD and 1.6mg SMP. The reaction was initiated by the addition of 2 mM ATP and the formation of NADH was followed either fluorimetrically or in a dual-wavelcngth spectrophotometer.
G. F. Azzone. H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna 2.0
1
I
With nigericin I
5
10
[KSCN] (mM)
Fig. 1. Effect of' SCN oti ATf .3jnthesis. The medium for ATP synthesis contained 0.2 M sucrose, 40 m M Pi pH 7.4, 10 mM MgSO,, 200 pM ADP, 0.05",, bovine serum albumin, 3 mM succinate, 1.5 mg manganese submitochondrial particles. The same experiment was carried out either in the absence or in the presence of 300 ng nigericinjmg protein. Incubation time was 15 min. Synthesis of ATP was determined as described in the Methods. Final volume 2.5 ml
RESULTS
+ KCI
In chloroplasts, ATP synthesis is uncoupled by nigericin KCl or amines [9,11,12]. The uncoupling effect has been interpreted as due to abolition of the ApH. In submitochondrial particles, Montal et al. [4] reported an enhancement of the uncoupling effect of lipophilic anions after addition of nigericin KCl or NH4Cl. This effect has not been confirmed in the present study. Fig. 1 shows that KSCN caused a marked inhibition of the ATP synthesis, About 50 "/o inhibition was obtained around 2 mM KSCN. However, the inhibition was practically unaffected by the addition of nigericin. Briller and Gromet-Elhanan [I41 to explain the lack of uncoupling of nigericin KCI and NH,Cl in bacterial chromatophores, assumed an impermeability of the membrane to anions. In the case of mitochondrial sonic fragments it has been suggested that the membrane be impermeable to C1- but permeable to NO; [4]. We have made also a comparison of the effects of various C1- and NO; concentrations in the presence of K ,K + nigericin, NH:, Na' ,choline' and Li'. There was an inhibition of phosphorylation at increasing anion concentrations, the effect being more marked with NO;. However, replacement of cations, which are assumed as uncapable of equilibrating across the membrane such as Li', K' or Na' or choline, with others which are assumed to equilibrate according to ApH such as K + in the presence of nigericin and NH: resulted only in a very slight increase of the uncoupling effect. The effect of nigericin + KC1 and of NH,CI are negligible at concentrations
+
+
+
+
+
where they cause a marked uncoupling in chloroplasts. It seems therefore that with either C1- or NO;, most of the inhibition of ATP synthesis in submitochondrial particles is due to the properties of the anion and it is independent of a ApH-driven equilibration of the cation across the membrane. By using different anions it was found that the inhibition of phosphorylation followed the Hofmeister series. Table 1 shows the effect of various anions on the succinate-linked pyridine nucleotide reduction. The rate of the energy-linked reaction was titrated against increasing concentration of K salts of various anions, and the concentration for 50 uncoupling is reported in the table. For comparison are also reported the concentrations for 50 uncoupling in the presence of 20 mM NH4Cl and 1 pM nigericin. It is seen that the uncoupling effect increased in the order C1- < Br< NO, < I- < SCN- < tetraphenylboron-, which is an order of increasing lipophilicity. However as in the case of the inhibition of phosphorylation either the presence of NH4Cl or of nigericin increased only slightly the uncoupling effect of the anions. Again, uncoupling seems to be due to the properties of the anions and not to that of the cations. Similar results were obtained when the anion effect was tested on the energylinked transhydrogenase. +
I
Eflcct of NH,CI and Nigericin
19
Stoichiometry oj'Anion Effect
In intact mitochondria addition of permeant cations in limited amounts results in a stoichiometric cycle of H + ion extrusion, stimulation of respiration, oxidation of respiratory carriers ere. However permeant cations do not affect the steady-state energy level of mitochondria after accumulation [l]. Fig. 2 shows the effect of increasing amounts of tetraphenylboron to sonic fragments. In Fig. 2A the particles were energized with succinate, which induced the usual enhancement of 8-anilino-I-naphthalene sulphonate fluorescence. After reaching the steady state, addition of tetraphenylboron caused a decrease of fluorescence which was proportional to the amount of tetraphenylboron. However, in no case there was a return to the initial level of fluorescence (cf [32]). Thus the effect of tetraphenylboron was not cyclic, and the presence of tetraphenylboron affected the energy level in the steady state. When the energy level of submitochondrial particles was monitored by bromthymol blue [7] similar results were obtained. Furthermore no cyclic effect was observed when the energy level was monitored through determination of the respiratory rate. In Fig. 2B the energy level of submitochondrial particles was followed through the rate of the succinate-linked pyridine nucleotide reduction. Addition of ATP initiated the formation of NADH. Addition of tetraphenylboron caused a progressive inhibition of the rate of NADH formation.
Anion and Amine Uncoupling
80
Table 1 . Conccwtrurion (?/anion,fbr 50 y,:, uncoupling The extent of energy coupling was measured as rate of succinatc-linked pyridinc nucleotide reduction. The conditions are described in the experimental part and in Fig. 2. The rate of NADH formation was dctermined in each expcriment first arter addition of 2 m M ATP and then after thc anion. The rates obtaincd at various anion concentrations wereused to calculate the anion concentration requircd for 50 uncoupling. Amount of submitochondrial particles 1 mg/ml. Final volume 2 ml. In the experiment with tctraphenylboron 10 mM KC1 was also added
KBr
KCI
Addition
KNO,
KI
K SCN
Tetraphenylboron
mM
PM .~ -
-~ -
~
.__ ~
~
~
None
110
66
44
30
3.2
18.8
20 mM NH,CI
105
78
25
22.4
27
20.5
1 pM nigericin
98
56
16.5
16.5
2.5
16.3
B
A Tetraphenylboron-
0.5pM
20pM
a, rn m GI L
c 0
.~
1.5uM
u
~ L L M tetraphenylboron-
t
PrnM ATP
Fig. 2. Kinetics ol'rerraplit.riJ-lbur(>nqffkcf on R-unilinu- I -naphtltulrne sulplionure ,fluorescence und N A D H formution. (A) The medium containcd 0.2 M sucrose, 1 mM MgCI,, I 0 mM N-2-hydroxyethylpiperaxine-W-2-ethane sulphonate (Hepcs) pH 7.0,2 pgoligomycin, 5 pM 8-anilino-I -naphthalene sulphonate, and 0.35 mg protcin/ml of submitochondrial particles. Enegization was started with 1 mM succinate and tetraphenylboron was added after reaching the steady state. Each amount of tetraphcnylboron was added in independent experiments and thcn all the traces were redrawn together. (B) The medium contained 0.25 M sucrose, 10 m M Hepes pH 7.0, 5 m M succinate-Trk, 5 mM MgSO,, 200 pM NAD, 2 mM NaCN, 2 m M ATP and 1 mg/ml or subinitochondrial particles. The change in fluorescence was followed with an Eppendorf photometer. Final volume 2.5 ml
Again, even at the lowest tetraphenylboron concentrations there was no cyclic effect. Fig. 3 shows the effect of various tetraphenylboron concentration on ATP synthesis. The effect did not correspond to what predicted on the basis of stoichiometric energy utilization for anion uptake. Addition of 300 nmol of tetraphenylboron should have led to the utilization of 150 or 75nmol of ATP for anion uptake on the basis of a stoichiometry of 2 or 4 anion/ ATP. Instead, the amount of ATP not synthesized was of the order of 2000nmol. Again, as for the ex-
Calculated ( 2 A I P T P )
100 'LOO [Tetraphenylboron] ( K M )
3c0
Fig. 3. Lfjkct of tetraplrenylboron on ATP syzrlzrsk. Experimental conditions were as in Fig. 1. The same experiment was carried out in the absence and in the presence of 1 mM KCI + nigcricin, 330ngI mg protein. Final volume 2.5 ml. Amount of protein was 1.6 mg
periments of Fig. 1 and Table 1, the same values were obtained whether or not KC1 nigericin was added.
+
Bromthymol Blue Interaction and Uncoupling Bromthymol blue has been proposed as an indicator for internal pH [27] or as a lipophilic charge moving across the membrane [28]. Colonna et al. [7] observed in submitochondrial particles an energylinked uptake of the dye accompanied by a shift of the apparent pK,. They proposed that, as in the case of anilinonaphthalene sulphonate, the spectroscopic effect originates from the energy-linked uptake of the dye. Bromcresol purple behaves as bromthymol blue, although its affinity for the membrane is smaller. We have used bromthymol blue and bromcresol purple to study quantitatively the correlation between uptake and uncoupling. Fig. 4 shows the uncoupling effect caused by bromthymol blue and bromcresol purple on succinate-
G. F. Azzone. H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna
linked pyridine nucleotides reduction. About 50 ”/, uncoupling was observed at about 2.5 pM bromthymol blue and at 30 pM bromcresol purple. Also anilinonaphthalene sulphonate caused 50 % uncoupling at about 30pM (not shown). It is to be noticed that the sensitivity of the energy-linked reaction to uncoupling was about the same at pH 6.4, 1.2 and 8.0 in the case of bromthymol blue, and at pH 6.4 at 7.2 in the case
1
I
81
of bromcresol purple. Fig. 5A and B shows the active and passive binding of bromthymol blue and bromcresol purple under the same experimental conditions of Fig.4. In accord with previous findings [7] the binding of bromthymol blue and bromcresol purple was markedly pH-dependent. This is due to the fact that the binding occurs only with the unionized form of the dyes. Therefore the binding curve in function of pH mirrors the ionization curve of the dyes. Fig. 5A and B shows that the pH-dependent passive interactions of bromthymol blue and bromcresol purple corresponded essentially to a distribution between two phases. In the presence of ATP the interaction was slightly more marked. However also the energized particles gave no indication of the presence of a finite number of sites. At variance from the passive state, the ATP-induced enhancement of the bromthymol blue interaction was only slightly affected by the pH of the medium. Correlation between the data of Fig. 4 and 5 thus indicates that the uncoupling effect of bromthymol blue is independent of the extent of passive binding and is also accompanied by a negligible uptake.
I
-4 M) Fig. 4. Inhibitory [email protected] ?f hromthyniol blue and hromcresol purple on N A D H ,formation. Experimental conditions were as in Fig. 3. The empty and full circles and the full squares indicate, in the case of bromthymol blue, experiments carried out at pH 6.4,7.2 and 8.0. The empty and full triangles indicatc, in the case of bromcresol purple, experiments carried out at pH 6.4 and 7.2. Each point represents a separate experiment ; bromthymol blue or bromcresol purple were added about 30- 60 s after determination of the unhibited reaction rate following the addition of ATP -5
-6
[ D y e ] (log
Ejfjcect of Amines
The uncoupling effect of several amines, differing in respect to their lipophilicity is shown in Fig.6A and B. The inhibitory effect of the amines on ATP synthesis in Fig. 6A decreased in the order dibenzylmethylamine > dimethylbenzylamine > triethylamine > NH3. The inhibitory effect of the amines on succinate-linked pyridine nucleotide reduction, Fig. 6B,
10,
[ Brorncresoi purple]free ( p.M ) Fig. 5. I’ussive und uctive intrractioti of’bromthymol blue ( A ) arid bromcresol purple I B ) with suhmitochondrial particles at various p H . The mcdium contained 0.25 M sucrose, 10 mM Hepes pH 7.0,s mM succinate-Tris, 2 rnM NaCN, 5 mM MgSO, and 1 mg/ml of submitochondrial particles. The change in absorbance was recorded in a dual wavelength spectrophotometer after addition to submitochondrial particles of various dye concentrations in the absence and in the presence of 2 mM ATP. The extent of dye binding was calculated as described by Colonna et a/. [7] and the spectrophotometric data were confirmed by centrifugation experiments. Filled symbols, active ; open symbols, passive
Anion and Amine Uncoupling
82
I
I
I
3
-4
-2
[arn;ie] ( l o g
-1
M)
[arnine] (log M)
Fig. 6. C'orrelrrriori hetiwen atnine lipophilicit-v and uncoupling eflect. (A) The medium contained 0.1 M sucrose, 0.05 M cholincC1. 40 mM Pi pH 7.0, 10 mM MgSO,, 100 pM AMP, 200 pM ADP, 2.5 mg hexokinase (Sigma type IV), 20 mM glucose, and 2 mM succinate. Amount of submitochondrial particles was 1 m g protein/ml. Time of incubation was 10 min. In (8)thc succinate-linked pyridine nucleotide reduction was measured in amediuin containing 0.25 M sucrose, 10 mM Tris-C1 or Hepes pH 7.0,2 mM NaCN, 5 mM MgCI,, 5 mM succinate, 200 pM NAD and 0.8 mg protein/rnl. The reaction was initiated by the addition of 2 mM ATP and the formation of NADH was measured fluorimetrirally. Jn B was also added 10 pM tetraphenylboron
r------
dye
/NO
a 1 Gi
m a 3
a0 l,
6 m
c "m
0
0.
+.
aJ 1 x
W
0
I
I
20
40
[KC[]
10
(mM)
Fig. 7. Luck of-correlutiotl between antine uccurnulation und uncoupling. The extent of coupling was measured on the basis of the rate of the succinate-linked pyridine nucleotide reduction. Thc rate of NADH formation in the absence of acridine orange was taken as 100% coupling, while the rates at the various acridine orange and KCI concentrations were calculated as of the unhibited rate in order to determine the residual coupling. Experimental conditions as in Fig.6B. The acridine orange accumulation was measured from the absorbance change at 492- 550 nrn (0).The medium was identical to that used for the NADH formation except that 5 pM acridine orange was always present. 1 mg protein/ml. (0) No acridine orange; (A) 5 pM; (0)10 pM; (A) 20 pM acridine orange
'x
decreased in the order acridine orange > dibenzylmethylamine > dimethylbenzylamine > NH,. These are also orders of decreasing lipophilicity. In chloroplasts, NH4C1 is a good uncoupler and a dependence of uncoupling on amine lipophilicity has been observed [32a]. It has been proposed [32a] that the uncoupling by lipophilic amines be similar in nature to that of NH,CI. By applying this inter-
Fig. 8. Uncoupling induced by NH,Clplus vulinoniycin. Experimental conditions as in Fig. 6 B . The rates observed at the various valinomycin Concentrations are reported as a percentage of the basal rate determined in the absence of valinomycin. The basal rate was not affected by changes of the NH,CI concentration when valinomycin was omitted. Amount of submitochondrial particles was 1 mg protein/rnl. Valinomycin (0)1 pM; (0) 0.1 pM; (A)0.01 pM
pretation to the submitochondrial particles, the more marked uncoupling would be due to a higher rate of diffusion of the uncharged lipophilic amines, this leading to an uptake of the lipophilic but not of the hydrophilic amines. Fig. 7 shows a lack of correlation between absorbance change, which corresponds to acridine orange uptake [23,24] and uncoupling, in respect to the effect of KC1. Acridine orange uptake was completely C1- dependent while acridine orange orange uncoupling was C1- independent. Fig. 7 indicates that amine uptake and uncoupling are different processes. Fig. 8 shows that NH4Cl is a good uncoupler
G. F. Azzone, H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna
I
1
I
-7
-6
[Tetraphenyiboron]
(log
-5
M)
Fig. 9. Uncoupling ejjbct in presence o j Iipuphilic umines and unions. Experimental conditions as in Fig. 6A cxcept that the medium contained 0.2 M sucrosc and choline was omitted. ).( No addition; ( x ) 40 mM NH: ; (0) 0.4 FM acridine orange; (A) 1 pM acridine orange: (A) 5 pM acridine orange
83
in Fig. 6B. Fig. 9 shows that the inhibition by acridine orange of ATP synthesis was markedly enhanced by the addition of lipophilic anions. Tetraphenylboron enhanced the uncoupling effect of acridine orange and the effect was proportional to the tetraphenylboron concentration. Table 2 shows the concentration for 50 % uncoupling in the presence of various anions and two dyes, namely acridine orange and neutral red. The uncoupling effect was measured as release of the acridines taken up in the presence of 100 mM KCl. It is seen that the concentration of anions required for 50% abolition of the dye uptake decreased with the lipophilicity of the anions. Furthermore lower concentration of anions were always required to cause release of acridine orange as compared to neutral red. This corresponds to a higher lipophilicity of acridine orange in respect to neutral red. DISCUSSION Anion Uptake and Uncoupling
Table2. Abobtion oj'di'e uptake by lipophilic anions The uptake of acridine orange and neutral red was measured on the basis of the absorbance change following energization of submitochondrial particles. The medium contained 0.1 M KCI, 5 mM MgC12, 10 mM N-2-hydroxyethylpiperazine-W-2-ethane sulphonate (Hepes), 5 pM acridine orange or 10 pM neutral red and 0.6 mg protein/ml. pH was 7.0 in the case of acridine orange and 6.5 in the case of neutral red. Energization was started with 1 mM ATP. All experiments were carried out with pyrophosphate submitochondrial particles [26] Uptake of
Acridine orange Neutral red
Anion concentration for 50
inhibition
NO;
SCN-
tetraphenylboron
31 180
2 5.6
0.008 5
in the presence of valinomycin. 50% uncoupling was obtained at concentration of NH4Cl of 0.4, 5.0 and 40 mM, at concentrations of valinomycin of l , O . l and 0.01 pM, respectively. Thus in this experiment a decrease of valinomycin concentration of one order of magnitude caused an increase of NH4CI concentration for 50% uncoupling also of one order of magnitude. The dependence of the NH4Cl concentration for 50 % uncoupling on the valinomycin concentration suggests that the limiting step for uncoupling is the rate at which NH: moves across the membrane. By comparing the effect of amines in Fig. 6A and B it may be noticed that the degree of uncoupling was more marked in Fig. 6B than in Fig. 6A. This is due to the additional presence of 10pM tetraphenylboron
A stoichiometric energy utilization has been proposed to occur in submitochondrial particles in the presence of lipophilic anions plus nigericin - KCl or amines, where permeant anions are assumed to diffuse electrophoretically across the membrane and to be distributed at electrochemical equilibrium [4,33,34]. If the stoichiometric model would be correct, addition of permeant anions should cause two effects according to whether they are added in excess or in limited amounts. When present in excess energy should be conserved in form of a ApH and the system uncoupled by nigericin + KC1 or NH4Cl but not by the further addition of permeant anions. When added in limited amounts anions should cause, as for the addition of permeant cations to intact mitochondria, a transient uncoupling corresponding to a cycle of energy utilization. It may be predicted, in this latter case, that the original steady-state energy level of the system should not be affected and also the dimension of the cycle should be proportional to the amount of charges moved. The stoichiometric energy utilization model in submitochondrial particles is considered to be supported by the observation of Montal et al. 141 that addition of nigericin + KCl or of NH4Cl in the presence of lipophilic anions results in a loss of respiratory control. However in the experiments of Skulachev et ul. [2] the uncoupling effect of tetraphenylboron was observed in the absence of nigericin KCl or NH4Cl. Furthermore in the experiments of Montal et al. 141, 100% release of respiratory control was obtained with 7pM tetraphenylboron; however the uptake of 7 pM tetraphenylboron would have required the consumption of 2-4 natoms oxygen at a stoichiometry of 2-4 anionlhigh-energy bond.
+
84
In the present work, three evidences are brought against the view that the uncoupling by lipophilic anions in submitochondrial particles is equivalent to the energy utilization for cation uptake in mitochondria. First, the extent of uncoupling, in the presence of an excess of inorganic anions, is practically independent of the presence of NH4Cl or of nigericin + K C . The data are in substantial agreement with what observed in submitochondrial particles by Christiansen PI al. [3] and in cromatophores by Briller and Gromet Elhanan [14]. However the suggestion [14] that the lack of effect of NH,f be due to being the membrane impermeable to anions is not valid in the case of submitochondrial particles in view of the fact that lipophilic anions do uncouple at very low concentration. Second, the uncoupling effect of lipophilic anions is not cyclic and affects the steady-state energy level. The parameters for the assessment of the energy level of the system are not clearly established in the case of submitochondrial particles. However if the uncoupling effect of the anion would be due to a transient energy utilization some of the energy-linked reactions should be affected in a cyclic manner. Cycles of Hfuptake, proportional to the amount of added tetraphenylboron have been observed in chloroplasts [32]. Third, there is no correlation between uncoupling and uptake. A positive correlation is considered to be supported by the observation that inorganic anions, which bind very loosely, are poor uncouplers, while organic anions, which bind more tightly, are more powerful uncouplers. However the extent of uptake is minimal in respect to the amount required for uncoupling. Papa eta/. [ 8 ] reported an uptake of 13 nmol x mg protein-' in the presence of 10 mM SCN- (cfalso Fig. 1 and Table 1 for the uncoupling concentrations of SCN-). The lack of correlation between uptake and uncoupling is supported by two observations. One, the extent of inhibition of ATP synthesis is much higher than the amount of energy required for the uptake of tetraphenylboron. Two, the experiments with bromthymol blue indicate a negligible active uptake when there is a complete uncoupling. Indeed the enhancement of bromthymol blue uptake due to energization is pH-independent as is the uncoupling. On the other hand passive interaction and total uptake are both pH-dependent. In the case of bromthymol blue it appears therefore not correct to assume that all the dye, localized in the membrane, is actively bound [35]. Spectroscopic Shifts and Amine Localization
The energy-linked interaction of amines with submitochondria1 particles, chloroplasts and chromatophores, gives rise to several problems : firstly, the molecular mechanism for the spectroscopic shifts of the
Anion and Amine Uncoupling
acridines ; secondly, the localization of the amines ; thirdly, the mechanism for uptake and fourthly, the relationship between uptake and uncoupling. As to the interpretation of the spectroscopic shifts, two mechanisms have been proposed : transition from the base to the acid form of the dye [ 18- 20,221 and dye-dye interaction [21,23- 25,371. The former mechanism is however incompatible with two observations, namely : the quenching of acridine orange fluorescence where the protonation of the acridine ring causes enhancement [37] and the absence of base-acid transition in the absorbance spectra [18j. Fiolet rt ul. 1381 have also shown that the fluorescence changes of 9-aminoacridine in chloroplasts do not correspond to predicted on the basis of a ApH induced dye distribution. As to the localization of the accumulated dyes, also two mechanisms have been proposed : accumulation of the amines in the inner osmotic space and electrostatic binding to sites situated on the membrane. In the former case the uptake reflects a distribution of the amines determined by the dpH, while in the latter case the distribution is related to the degree of interaction of the charged amines with the membrane sites. The former mechanism is supported by the data on the distribution of methylamine in chloroplasts and by the inhibitory effect of nigericin KC1 and NH,CI [18,19]. The latter mechanism is supported by the immobilization of the accumulated dye 1371and the dependence of the spectroscopic shift on the protein/dye ratio [37]. It is possible that both accumulation of free dye and binding occur, depending on several parameters such as dye/protein ratio, type of membrane, lipophilicity of the dye etc.
+
Amine Uptake and Uncoupling
Crofts [I21 has proposed that in chloroplasts the uncoupling effect of the amines corresponds to a stoichiometric energy utilization for amine uptake. The mechanism requires the following steps : equilibration of NH, across the membrane; uptake of H + in the inner osmotic space; protonation of NH, to NH; and accumulation of NH; in the inner space; uptake of anions. In this mechanism the uptake of NH, is driven by d p H and that of anions by membrane potential. In submitochondrial particles the energy-linked uptake of amines [23,24] amounts to about 20, 40 and 150 nmol x mg protein-' in the case of NH: ,acridines and aminophenazines respectively. However the amines cause also uncoupling and the uncoupling increases with the amine lipophilicity. The question arises as to whether the amine uptake in submitochondrial particles can explain the uncoupling effect. If the uncoupling effect of the lipophilic amines would be due to a mechanism similar to that proposed by Crofts [I21 for the chloroplasts, there should be a
G. F. Azzone, H. Gutwcniger, E. Viola. E. Strinna, S. Massari, and R. Colonna
close correlation between extents of uptake and of uncoupling, and NH4C1 should be a good uncoupler. Neither of these predictions is verified. First, in the absence of C1- there is uncoupling without uptake. Second, the uncoupling effect of NH,Cl is negligible. It may be argued that the lack of uncoupling effect by NH,C1 be duc to a low permeation of the uncharged NH,. This is unlikely because NH3 is a good permeant in all biological membranes and becomes a good uncoupler also in submitochondrial particles in the presence of valinomycin. Uncoupling Mechanisms f o r Lipophilic Amines and Anions A mechanism for the uncoupling of lipophilic amines may be based on the cycling of NH: in the presence of valinomycin [4,14]. The uncoupling in this case is thought to involve: diffusion of the uncharged NH3 into the particle; protonation of NH3 to NHZ ; and release of NH: from the particle viu valinomycin. It is important to realize that a cycling mechanism is not necessarily dependent on the translocation of the amine across the membrane, but it may only involve the interaction of the uncharged amine with intramembrane proteus at the outer interphase. Whatever the molecular mechanism of penetration the limiting step is the diffusion of the charged amine. This is supported by the fact that 50% uncoupling requires a proportionally higher valinomycin concentration at decreasing NH4C1 concentrations. In the case of lipophilic amines the dependence of uncoupling on amine lipophilicity reflects the rate of permeation of the charged amines through the membrane. The uncoupling by lipophilic amines is enhanced by lipophilic anions. In this case the extrusion of charged amine is replaced by the release of the amineanion complex. The complex corresponds to an hydrophobic ion pair. The uncoupling involves in this case: uptake of H+ and anions; uptake of amine; formation of an hydrophobic ion pair between charged amine and anion; and release of the ion pair. As for the uncoupling by anions the following mechanism may be conceived: uptake of H’ and anions due to respiration; release of H f ; and release of anions. This mechanism resembles that of the classical uncouplers with the difference that the second step requires a membrane leaky for H + rather than a protonophoric effect of the uncouplers. The chemical properties of the anions may be important in establishing the degree of uncoupling. For example bromthyniol blue is a much stronger uncoupler than 8-anilino-lnaphthalene sulphonate [36]. This may be due to the fact that the bromthymol blue chromophore either operates also as a proton carrier or causes greater damage to the membrane structure because of its higher polarity.
85
A final question is why lipophilic amines and anions are so efficient uncouplers in submitochondrial particles and not in mitochondria. The reason is to be found in the leakiness of the submitochondrial particles membrane. This is partly due to the sonication procedure [39] and partly follows the uptake of the lipophilic amines and anions. This obviously raises the question as to the extent to which ion gradients can be maintained in submitochondrial particles.
REFERENCES 1. Azzone, G . F. & Massari, S. (1973) Biochim. Biophys. Acta, 301, 195-226. 2. Skulachev, V. P., Yaguzhinski, L. S., Jasaitis, A. A., Liberman, E. A., Topali, V. P. & Zofina, L. A. (1969) in Enerxy Level and Melubolic Control (Papa, S., Tager, 3 . M., Quagliariello, E. & Slater, E. C., eds) p. 283, Adriatica Editrice, Bari. 3. Christiansen, R. O., Loyter, A. & Racker, E. (1969) Bioc4im. BiOphyS. Acta, 180, 207-210. 4. Montal, M., Chancc, B. & Lee, C . P. (1970) J . Membrane Biol. 2, 201 -234. 5. Azzi, A,, Chance, B., Radda, G. K. & Lec, C. P. (1969) Proc. Nut1 Acad. Sci. U.S.A.62, 612. 6. Liberman, E. A. & Skulachev, V. P. (1970) Biochim. Biophys. Acta, 216, 30-42. 7. Colonna, R., Dell’Antone, P. & Azzonc, G. F. (1972) Arch. Biochem. Biophys. 151, 295 - 303. 8. Papa, S . , Guerrieri, F., Simone, S., Lorusso, M. & La Rosa, D. (1973) Biochim. Biophys. Acta, 292, 20- 38. 9. Shavit, N., Dilley, R. A. & San Pietro, A. (1968) Biochemi.stry, 7 , 2356-2363. 10. Krogman, D. W., Jagendorf, A. T.& Avron, M. (1959) Plant Phl$.siol.34, 272. 11. Good, N . E. (1960) Biochirn. Biophys. Acta, 40, 502. 12. Crofts, A. R. (1967) J. Biol. Chem. 242, 3352-3359. 13. Horio, T. & Yamashita, J. (1964) Biochim. Biophys. Acra, 88, 237. 34. Briller, S. & Gromet-Elhanan, Z. (1970) Biochim. Biophys. Aria, 205, 262 - 272. 15. Izawa, S . (1965) Biochim. Biophys. Actu, 102, 373-378. 16. Kraayenhof, R. (1970) FEBS Lett. 6 , 161- 165. 17. Eilerman, L. J. M. (1970) Biochim. Biophys. Acta, 216, 231 233. 18. Schuldiner, S., Rottenberg, H. & Avron, M. (1972) Eur. J . Biochem. 25, 64 - 70. 19. Gromet-Elhanan, Z . (1972) Eur. J. Biochem. 25, 84-88. 20. Deamer, D. W., Prince, R. C. & Crofts, A. R. (1972) Biochim. B i o p h ~ )Acta, ~ . 274, 323-335. 21. Colonna, R., Dell’Antone, P. & Azzone, G . F. (1970) FEBS Lett. 10, 13 - 16. 22. Lee, C. P. (1971) Biochemistry, 10, 4375-4381. 23. Dell’Antone, P., Colonna, R. & Azzone, G. F. (1972) Eur. J. Biochem. 24, 553 - 565. 24. Dell’Antone, P., Colonna, R. & Azzone, G. F. (1972) Eur. J . Biochem. 24, 565 - 576. 25. Dell’Antone, P. & Azzone, G . F. (1974) FEBS Lett. 39,67-72. 26. Racker, E. (1962) Proc. Null Acad. Sci. U.S.A.48, 1659- 1662. 27. Chance, B. & Mela, L. (1966) Proc. Natl Acad. Sci. U.S.A. 55, 1243- 1246. 28. Mitchell, P.. Moyle, J. & Smith, L. (1968) Eur. J . Biochem. 4, 9-17. 29. Azzone. G . F., Piemontc, G. & Massari, S. (1968) Eur. J . Biochem. 6,207-212.
86
G. F. Azzone, H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna: Anion and Amine Uncoupling
30. Rossi, E. & Azzone, G. F. (1970) Eur, J . Biochem. 12,319- 327. 31. Lee, C. P., Azzone, G . F. & Emster, L. (1964) Nature (Lond.) 201, 153-156. 32. Horton, A. & Packer, L. (1968) Arch. Biochem. Biophys. 128, 820 - 822. 32a. McCarty, R. E. &Coleman, C. H. (1970) Arch. Biochem. Biophys. 141,198-206. 33. Chance, B. & Montal, M. (1971)in Currenf Topicsin Membranes and Transport, vol. 2, pp. 99- 156, Academic Press, New York.
34. Skulachev, V. P. (1971) in Current Topics in Bioenergetics (Sanadi, D. R., ed.) vol. 4, pp. 127- 190, Academic Press, New York and London. 35. Nordenbrand, K. & Ernster, L. (1971) Eur. J . Biochem. 18, 258 270. 36. Layton, D. (1973) Ph. D. Thesis, Chelsea College, London. 37. Massari, S., Dell’Antone, P., Colonna, R. & Azzone, G . F. (1975) Biochemistry, I S , 1038- 1043. 38. Fiolet, J. W. T., Bakker, E. P. & Van Dam, K. (1974) Biochim. Biophys. Actu, 365, 432-445. 39. Azzone, G. F. & Massari, S. (1972) FEES Left. 28, 61 -64. ~
G. F. Azzone, H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna, Istituto di Patologia Generale,
Universita degli Studi, Via Loredan 16, 1-35100 Padova, Italy
J. Hiochcm. 62. 77-86 (1976)
Anion and Amine Uptake and Uncoupling in Submitochondrial Particles Giovanni Felice AZZONE. Heidi GUTWENIGER, Elena VIOLA, Ermanno STRINNA, Stefano MASSARI, and Raffaele COLONNA 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 Padova (Received September 5. 1975)
1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KC1 or NH,CI. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases. 2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion. 3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-I-napthalene sulphonate cause a pH-independent, 50 uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 pM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two phases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation. 4. Addition of amines to energized submitochondrial particles results in two types of effects: uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling. 5. While the uptake of amines is dependent on the presence of non-permeant anions, such as C1- , the uncoupling effect is independent of C1- . Furthermore the amine uncoupling is markedly enhanced by lipophlic anions. 6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.
When active transport takes place other energyAlthough the inlinked reactions are inhibited [l]. hibition is loosely indicated as “uncoupling” it has been realized that the uncoupling due to active transport is different in nature from the classical uncoupling. In fact the former is due to stoichiometric energy utilization for ion transport while the latter is due to catalytic energy dissipation. The former is accompanied by a stoichiometric cycle of energy utilization, denoted as state 4- state 3 -state 4 transition, while the latter is accompanied by a shift of the steady-state energy level. While the distinction between stoichiometric energy utilization, linked to ion uptake, and catalytic energy dissipation, due to damage or un-
coupler cycling, is clearly established in intact mitochondria, this is not the case in submitochondrial particles. Skulachev el al. [2] reported an uncoupling effect of lipophilic anions in submitochondrial particles and suggested it be equivalent to the energy utilization accompanying cation uptake in mitochondria. Christiansen et al. [3] observed uncoupling by lipophilic anions in submitochondrial particles. Montal et al. [4] reported that addition of nigericin + KCl or amines in the presence of lipophilic anions caused a release of respiratory control and attributed the effect to stoichiometric energy utilization. Other lipophilic anions, such as 8-anilino-1-naphthalene sulphonate, phenyl-
Anion and Amine Uncoupling
78
dicarbaundecaborane, bromthymol blue, and SCN- , are also accumulated in submitochondrial particles [5 - 81. Although it has been generally assumed that anion accumulation is equivalent to uncoupling, the correspondence between energy utilization and energy dissipation has never been analyzed. Also, it is not clear the extent to which anion uptake and uncoupling are dependent on the presence of amines or nigericin + KCl. A marked difference in sensitivity to nigericin KCI and amines has becn found between chloroplasts on one side and chromatophores and submitochondrial particles on the other, which has been tentatively attributed to a difference in anion permeability [9- 141. The uncoupling effect of amines in isolated chloroplasts is well established. Izawa showed that the uncoupling effect of amines in chloroplasts is accompanied by swelling [15]. Crofts [12] proposed a general mechanism for the uncoupling of the amines based on diffusion of NH3 across the membrane, down the pH gradient, and accumulation of NH; . The swelling in NH4Cl was attributed to uptake of NH4Cl in thc inner osmotic space. Kraayenhof [16] observed in chloroplasts an uncoupling effect of the lipophilic amine atebrine, which was accompanied by an uptake of the dye [17]. Schuldiner et al. [18] considered the uptake of the acridines similar to that of NH, and utilized the spectroscopic changes to determine the internal pH [19,20]. In the case of submitochondrial particles a large number of acridine and aminophenazine dyes has also been found to be accumulated after supply of energy [21-241. Recently however it has been reported [25] that addition of lipophilic anions cause release rather than uptake of acridine dyes. We will show below that in contrast with Montal et al. [4] and in accord with Christiansen et al. [3] the uncoupling effect of lipophilic anions is largely independent of the presence of nigericin KCI or of amines. Furthermore the uncoupling effect corresponds to a n energy utilization larger than required for the accumulation of the anions. In regard to the effect of amines in submitochondrial particles we will show that lipophilic amines act as uncouplers and the uncoupling effect increases parallel to the amine lipophilicity and also that, unlike the chloroplasts, the uncoupling effect is not related with the uptake of the amine. The view will be discussed that the uncoupling effect of anions and amines is catalytic rather than stoichiometric in nature and is due to charge cycling.
+
+
EXPERIMENTAL PROCEDURE Beef heart mitochondria1 were prepared according to the procedure previously described [13].Preparation of phosphorylating fragments was obtained by sonicating under nitrogen, at 0 "C,5 ml of beef heart mito-
chondria in 10 mi of the following medium: 0.25 M sucrosc, 5 mM ATP, 5 mM MgCI,, 10 mM MnCI,, 1 mM succinate and 10 mM Tris-CI pH 7.5. After sonication for 30 s the suspension was centrifuged at 7000 x g for 5 min. The pellet was resuspended in the same medium as before and again sonicated for 30 s. The suspension was again centrifuged for 5 min at 7000 x g. The supernatant of the two centrifugations were pooled and centrifuged at 105000 x g for 45 min. The particles obtained by this treatment were denominated as manganese submitochondrial particles and possessed a high rate of phosphorylative activity. However, the phosphorylative activity was rapidly lost and therefore all phosphorylation experiments were carried out within few hours after sonication. In the experiments of Table 2 the preparation of submitochondrial particles was carried out by sonication in 20 mM pyrophosphate [26]. The response of bromthymol blue was measured in a dual wavelength spectrophotometer as described previously [27 - 291. The response of 8-anilino-1-naphthalene sulphonate was measured in an Eppendorf fluorometer [ S ] . The rate of oxygen uptakc was measured polarographically with a Clark electrode. The phosphorylative activity was measured by adding 0.5-2 mg protein of sonic fragments to a standard incubation medium of 2.5 ml containing 40 mM P, pH 7.0, 1 or 10 mM MgCI2 l00pM AMP, 200pM ADP, 0.2 M sucrose, 2.5 mg hexokinase (Sigma type IV), 20 mM glucose and 1 mM NADH or 2 mM succinate. After 10 or 15 min the reaction was blocked with perchloric acid and the system analyzed for the presence of glucose 6-phosphate fluorimetrically [30]. In the experiments where NADH is the substrate since the amount of oxygen consumed corresponds to the amount of NADH added, it is possible to calculate the P/O ratio in the absence of direct oxygen measurements. The energy-linked transhydrogenase [311was measured in a medium containing 0.25 M sucrose, 10 mM Tris-C1, pH 7.2, 1 pg oligomycin, 50 pM NADP, 200 pM NADH, 500 yM oxidized glutathione, glutathione reductdse, 1 pM rotenone, 10 mM MgS04 and 1.6 mg submitochondrial particles. The reaction was initiated by the addition of 2 mM succinate and the disappearance of NADH was followed either fluorimetrically or in the dual wavelength spectrophotometer. The succinate-linked pyridine nucleotide reduction was measured in a medium containing 0.25 M sucrose. 10 mM Tris-CI or 10 mM N-2-hydroxyethylpiperazineN'-Zethane sulphate pH 7.0, 2 mM NaCN, 5 mM MgCI2, 5 mM succinate, 200pM NAD and 1.6mg SMP. The reaction was initiated by the addition of 2 mM ATP and the formation of NADH was followed either fluorimetrically or in a dual-wavelcngth spectrophotometer.
G. F. Azzone. H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna 2.0
1
I
With nigericin I
5
10
[KSCN] (mM)
Fig. 1. Effect of' SCN oti ATf .3jnthesis. The medium for ATP synthesis contained 0.2 M sucrose, 40 m M Pi pH 7.4, 10 mM MgSO,, 200 pM ADP, 0.05",, bovine serum albumin, 3 mM succinate, 1.5 mg manganese submitochondrial particles. The same experiment was carried out either in the absence or in the presence of 300 ng nigericinjmg protein. Incubation time was 15 min. Synthesis of ATP was determined as described in the Methods. Final volume 2.5 ml
RESULTS
+ KCI
In chloroplasts, ATP synthesis is uncoupled by nigericin KCl or amines [9,11,12]. The uncoupling effect has been interpreted as due to abolition of the ApH. In submitochondrial particles, Montal et al. [4] reported an enhancement of the uncoupling effect of lipophilic anions after addition of nigericin KCl or NH4Cl. This effect has not been confirmed in the present study. Fig. 1 shows that KSCN caused a marked inhibition of the ATP synthesis, About 50 "/o inhibition was obtained around 2 mM KSCN. However, the inhibition was practically unaffected by the addition of nigericin. Briller and Gromet-Elhanan [I41 to explain the lack of uncoupling of nigericin KCI and NH,Cl in bacterial chromatophores, assumed an impermeability of the membrane to anions. In the case of mitochondrial sonic fragments it has been suggested that the membrane be impermeable to C1- but permeable to NO; [4]. We have made also a comparison of the effects of various C1- and NO; concentrations in the presence of K ,K + nigericin, NH:, Na' ,choline' and Li'. There was an inhibition of phosphorylation at increasing anion concentrations, the effect being more marked with NO;. However, replacement of cations, which are assumed as uncapable of equilibrating across the membrane such as Li', K' or Na' or choline, with others which are assumed to equilibrate according to ApH such as K + in the presence of nigericin and NH: resulted only in a very slight increase of the uncoupling effect. The effect of nigericin + KC1 and of NH,CI are negligible at concentrations
+
+
+
+
+
where they cause a marked uncoupling in chloroplasts. It seems therefore that with either C1- or NO;, most of the inhibition of ATP synthesis in submitochondrial particles is due to the properties of the anion and it is independent of a ApH-driven equilibration of the cation across the membrane. By using different anions it was found that the inhibition of phosphorylation followed the Hofmeister series. Table 1 shows the effect of various anions on the succinate-linked pyridine nucleotide reduction. The rate of the energy-linked reaction was titrated against increasing concentration of K salts of various anions, and the concentration for 50 uncoupling is reported in the table. For comparison are also reported the concentrations for 50 uncoupling in the presence of 20 mM NH4Cl and 1 pM nigericin. It is seen that the uncoupling effect increased in the order C1- < Br< NO, < I- < SCN- < tetraphenylboron-, which is an order of increasing lipophilicity. However as in the case of the inhibition of phosphorylation either the presence of NH4Cl or of nigericin increased only slightly the uncoupling effect of the anions. Again, uncoupling seems to be due to the properties of the anions and not to that of the cations. Similar results were obtained when the anion effect was tested on the energylinked transhydrogenase. +
I
Eflcct of NH,CI and Nigericin
19
Stoichiometry oj'Anion Effect
In intact mitochondria addition of permeant cations in limited amounts results in a stoichiometric cycle of H + ion extrusion, stimulation of respiration, oxidation of respiratory carriers ere. However permeant cations do not affect the steady-state energy level of mitochondria after accumulation [l]. Fig. 2 shows the effect of increasing amounts of tetraphenylboron to sonic fragments. In Fig. 2A the particles were energized with succinate, which induced the usual enhancement of 8-anilino-I-naphthalene sulphonate fluorescence. After reaching the steady state, addition of tetraphenylboron caused a decrease of fluorescence which was proportional to the amount of tetraphenylboron. However, in no case there was a return to the initial level of fluorescence (cf [32]). Thus the effect of tetraphenylboron was not cyclic, and the presence of tetraphenylboron affected the energy level in the steady state. When the energy level of submitochondrial particles was monitored by bromthymol blue [7] similar results were obtained. Furthermore no cyclic effect was observed when the energy level was monitored through determination of the respiratory rate. In Fig. 2B the energy level of submitochondrial particles was followed through the rate of the succinate-linked pyridine nucleotide reduction. Addition of ATP initiated the formation of NADH. Addition of tetraphenylboron caused a progressive inhibition of the rate of NADH formation.
Anion and Amine Uncoupling
80
Table 1 . Conccwtrurion (?/anion,fbr 50 y,:, uncoupling The extent of energy coupling was measured as rate of succinatc-linked pyridinc nucleotide reduction. The conditions are described in the experimental part and in Fig. 2. The rate of NADH formation was dctermined in each expcriment first arter addition of 2 m M ATP and then after thc anion. The rates obtaincd at various anion concentrations wereused to calculate the anion concentration requircd for 50 uncoupling. Amount of submitochondrial particles 1 mg/ml. Final volume 2 ml. In the experiment with tctraphenylboron 10 mM KC1 was also added
KBr
KCI
Addition
KNO,
KI
K SCN
Tetraphenylboron
mM
PM .~ -
-~ -
~
.__ ~
~
~
None
110
66
44
30
3.2
18.8
20 mM NH,CI
105
78
25
22.4
27
20.5
1 pM nigericin
98
56
16.5
16.5
2.5
16.3
B
A Tetraphenylboron-
0.5pM
20pM
a, rn m GI L
c 0
.~
1.5uM
u
~ L L M tetraphenylboron-
t
PrnM ATP
Fig. 2. Kinetics ol'rerraplit.riJ-lbur(>nqffkcf on R-unilinu- I -naphtltulrne sulplionure ,fluorescence und N A D H formution. (A) The medium containcd 0.2 M sucrose, 1 mM MgCI,, I 0 mM N-2-hydroxyethylpiperaxine-W-2-ethane sulphonate (Hepcs) pH 7.0,2 pgoligomycin, 5 pM 8-anilino-I -naphthalene sulphonate, and 0.35 mg protcin/ml of submitochondrial particles. Enegization was started with 1 mM succinate and tetraphenylboron was added after reaching the steady state. Each amount of tetraphcnylboron was added in independent experiments and thcn all the traces were redrawn together. (B) The medium contained 0.25 M sucrose, 10 m M Hepes pH 7.0, 5 m M succinate-Trk, 5 mM MgSO,, 200 pM NAD, 2 mM NaCN, 2 m M ATP and 1 mg/ml or subinitochondrial particles. The change in fluorescence was followed with an Eppendorf photometer. Final volume 2.5 ml
Again, even at the lowest tetraphenylboron concentrations there was no cyclic effect. Fig. 3 shows the effect of various tetraphenylboron concentration on ATP synthesis. The effect did not correspond to what predicted on the basis of stoichiometric energy utilization for anion uptake. Addition of 300 nmol of tetraphenylboron should have led to the utilization of 150 or 75nmol of ATP for anion uptake on the basis of a stoichiometry of 2 or 4 anion/ ATP. Instead, the amount of ATP not synthesized was of the order of 2000nmol. Again, as for the ex-
Calculated ( 2 A I P T P )
100 'LOO [Tetraphenylboron] ( K M )
3c0
Fig. 3. Lfjkct of tetraplrenylboron on ATP syzrlzrsk. Experimental conditions were as in Fig. 1. The same experiment was carried out in the absence and in the presence of 1 mM KCI + nigcricin, 330ngI mg protein. Final volume 2.5 ml. Amount of protein was 1.6 mg
periments of Fig. 1 and Table 1, the same values were obtained whether or not KC1 nigericin was added.
+
Bromthymol Blue Interaction and Uncoupling Bromthymol blue has been proposed as an indicator for internal pH [27] or as a lipophilic charge moving across the membrane [28]. Colonna et al. [7] observed in submitochondrial particles an energylinked uptake of the dye accompanied by a shift of the apparent pK,. They proposed that, as in the case of anilinonaphthalene sulphonate, the spectroscopic effect originates from the energy-linked uptake of the dye. Bromcresol purple behaves as bromthymol blue, although its affinity for the membrane is smaller. We have used bromthymol blue and bromcresol purple to study quantitatively the correlation between uptake and uncoupling. Fig. 4 shows the uncoupling effect caused by bromthymol blue and bromcresol purple on succinate-
G. F. Azzone. H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna
linked pyridine nucleotides reduction. About 50 ”/, uncoupling was observed at about 2.5 pM bromthymol blue and at 30 pM bromcresol purple. Also anilinonaphthalene sulphonate caused 50 % uncoupling at about 30pM (not shown). It is to be noticed that the sensitivity of the energy-linked reaction to uncoupling was about the same at pH 6.4, 1.2 and 8.0 in the case of bromthymol blue, and at pH 6.4 at 7.2 in the case
1
I
81
of bromcresol purple. Fig. 5A and B shows the active and passive binding of bromthymol blue and bromcresol purple under the same experimental conditions of Fig.4. In accord with previous findings [7] the binding of bromthymol blue and bromcresol purple was markedly pH-dependent. This is due to the fact that the binding occurs only with the unionized form of the dyes. Therefore the binding curve in function of pH mirrors the ionization curve of the dyes. Fig. 5A and B shows that the pH-dependent passive interactions of bromthymol blue and bromcresol purple corresponded essentially to a distribution between two phases. In the presence of ATP the interaction was slightly more marked. However also the energized particles gave no indication of the presence of a finite number of sites. At variance from the passive state, the ATP-induced enhancement of the bromthymol blue interaction was only slightly affected by the pH of the medium. Correlation between the data of Fig. 4 and 5 thus indicates that the uncoupling effect of bromthymol blue is independent of the extent of passive binding and is also accompanied by a negligible uptake.
I
-4 M) Fig. 4. Inhibitory [email protected] ?f hromthyniol blue and hromcresol purple on N A D H ,formation. Experimental conditions were as in Fig. 3. The empty and full circles and the full squares indicate, in the case of bromthymol blue, experiments carried out at pH 6.4,7.2 and 8.0. The empty and full triangles indicatc, in the case of bromcresol purple, experiments carried out at pH 6.4 and 7.2. Each point represents a separate experiment ; bromthymol blue or bromcresol purple were added about 30- 60 s after determination of the unhibited reaction rate following the addition of ATP -5
-6
[ D y e ] (log
Ejfjcect of Amines
The uncoupling effect of several amines, differing in respect to their lipophilicity is shown in Fig.6A and B. The inhibitory effect of the amines on ATP synthesis in Fig. 6A decreased in the order dibenzylmethylamine > dimethylbenzylamine > triethylamine > NH3. The inhibitory effect of the amines on succinate-linked pyridine nucleotide reduction, Fig. 6B,
10,
[ Brorncresoi purple]free ( p.M ) Fig. 5. I’ussive und uctive intrractioti of’bromthymol blue ( A ) arid bromcresol purple I B ) with suhmitochondrial particles at various p H . The mcdium contained 0.25 M sucrose, 10 mM Hepes pH 7.0,s mM succinate-Tris, 2 rnM NaCN, 5 mM MgSO, and 1 mg/ml of submitochondrial particles. The change in absorbance was recorded in a dual wavelength spectrophotometer after addition to submitochondrial particles of various dye concentrations in the absence and in the presence of 2 mM ATP. The extent of dye binding was calculated as described by Colonna et a/. [7] and the spectrophotometric data were confirmed by centrifugation experiments. Filled symbols, active ; open symbols, passive
Anion and Amine Uncoupling
82
I
I
I
3
-4
-2
[arn;ie] ( l o g
-1
M)
[arnine] (log M)
Fig. 6. C'orrelrrriori hetiwen atnine lipophilicit-v and uncoupling eflect. (A) The medium contained 0.1 M sucrose, 0.05 M cholincC1. 40 mM Pi pH 7.0, 10 mM MgSO,, 100 pM AMP, 200 pM ADP, 2.5 mg hexokinase (Sigma type IV), 20 mM glucose, and 2 mM succinate. Amount of submitochondrial particles was 1 m g protein/ml. Time of incubation was 10 min. In (8)thc succinate-linked pyridine nucleotide reduction was measured in amediuin containing 0.25 M sucrose, 10 mM Tris-C1 or Hepes pH 7.0,2 mM NaCN, 5 mM MgCI,, 5 mM succinate, 200 pM NAD and 0.8 mg protein/rnl. The reaction was initiated by the addition of 2 mM ATP and the formation of NADH was measured fluorimetrirally. Jn B was also added 10 pM tetraphenylboron
r------
dye
/NO
a 1 Gi
m a 3
a0 l,
6 m
c "m
0
0.
+.
aJ 1 x
W
0
I
I
20
40
[KC[]
10
(mM)
Fig. 7. Luck of-correlutiotl between antine uccurnulation und uncoupling. The extent of coupling was measured on the basis of the rate of the succinate-linked pyridine nucleotide reduction. Thc rate of NADH formation in the absence of acridine orange was taken as 100% coupling, while the rates at the various acridine orange and KCI concentrations were calculated as of the unhibited rate in order to determine the residual coupling. Experimental conditions as in Fig.6B. The acridine orange accumulation was measured from the absorbance change at 492- 550 nrn (0).The medium was identical to that used for the NADH formation except that 5 pM acridine orange was always present. 1 mg protein/ml. (0) No acridine orange; (A) 5 pM; (0)10 pM; (A) 20 pM acridine orange
'x
decreased in the order acridine orange > dibenzylmethylamine > dimethylbenzylamine > NH,. These are also orders of decreasing lipophilicity. In chloroplasts, NH4C1 is a good uncoupler and a dependence of uncoupling on amine lipophilicity has been observed [32a]. It has been proposed [32a] that the uncoupling by lipophilic amines be similar in nature to that of NH,CI. By applying this inter-
Fig. 8. Uncoupling induced by NH,Clplus vulinoniycin. Experimental conditions as in Fig. 6 B . The rates observed at the various valinomycin Concentrations are reported as a percentage of the basal rate determined in the absence of valinomycin. The basal rate was not affected by changes of the NH,CI concentration when valinomycin was omitted. Amount of submitochondrial particles was 1 mg protein/rnl. Valinomycin (0)1 pM; (0) 0.1 pM; (A)0.01 pM
pretation to the submitochondrial particles, the more marked uncoupling would be due to a higher rate of diffusion of the uncharged lipophilic amines, this leading to an uptake of the lipophilic but not of the hydrophilic amines. Fig. 7 shows a lack of correlation between absorbance change, which corresponds to acridine orange uptake [23,24] and uncoupling, in respect to the effect of KC1. Acridine orange uptake was completely C1- dependent while acridine orange orange uncoupling was C1- independent. Fig. 7 indicates that amine uptake and uncoupling are different processes. Fig. 8 shows that NH4Cl is a good uncoupler
G. F. Azzone, H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna
I
1
I
-7
-6
[Tetraphenyiboron]
(log
-5
M)
Fig. 9. Uncoupling ejjbct in presence o j Iipuphilic umines and unions. Experimental conditions as in Fig. 6A cxcept that the medium contained 0.2 M sucrosc and choline was omitted. ).( No addition; ( x ) 40 mM NH: ; (0) 0.4 FM acridine orange; (A) 1 pM acridine orange: (A) 5 pM acridine orange
83
in Fig. 6B. Fig. 9 shows that the inhibition by acridine orange of ATP synthesis was markedly enhanced by the addition of lipophilic anions. Tetraphenylboron enhanced the uncoupling effect of acridine orange and the effect was proportional to the tetraphenylboron concentration. Table 2 shows the concentration for 50 % uncoupling in the presence of various anions and two dyes, namely acridine orange and neutral red. The uncoupling effect was measured as release of the acridines taken up in the presence of 100 mM KCl. It is seen that the concentration of anions required for 50% abolition of the dye uptake decreased with the lipophilicity of the anions. Furthermore lower concentration of anions were always required to cause release of acridine orange as compared to neutral red. This corresponds to a higher lipophilicity of acridine orange in respect to neutral red. DISCUSSION Anion Uptake and Uncoupling
Table2. Abobtion oj'di'e uptake by lipophilic anions The uptake of acridine orange and neutral red was measured on the basis of the absorbance change following energization of submitochondrial particles. The medium contained 0.1 M KCI, 5 mM MgC12, 10 mM N-2-hydroxyethylpiperazine-W-2-ethane sulphonate (Hepes), 5 pM acridine orange or 10 pM neutral red and 0.6 mg protein/ml. pH was 7.0 in the case of acridine orange and 6.5 in the case of neutral red. Energization was started with 1 mM ATP. All experiments were carried out with pyrophosphate submitochondrial particles [26] Uptake of
Acridine orange Neutral red
Anion concentration for 50
inhibition
NO;
SCN-
tetraphenylboron
31 180
2 5.6
0.008 5
in the presence of valinomycin. 50% uncoupling was obtained at concentration of NH4Cl of 0.4, 5.0 and 40 mM, at concentrations of valinomycin of l , O . l and 0.01 pM, respectively. Thus in this experiment a decrease of valinomycin concentration of one order of magnitude caused an increase of NH4CI concentration for 50% uncoupling also of one order of magnitude. The dependence of the NH4Cl concentration for 50 % uncoupling on the valinomycin concentration suggests that the limiting step for uncoupling is the rate at which NH: moves across the membrane. By comparing the effect of amines in Fig. 6A and B it may be noticed that the degree of uncoupling was more marked in Fig. 6B than in Fig. 6A. This is due to the additional presence of 10pM tetraphenylboron
A stoichiometric energy utilization has been proposed to occur in submitochondrial particles in the presence of lipophilic anions plus nigericin - KCl or amines, where permeant anions are assumed to diffuse electrophoretically across the membrane and to be distributed at electrochemical equilibrium [4,33,34]. If the stoichiometric model would be correct, addition of permeant anions should cause two effects according to whether they are added in excess or in limited amounts. When present in excess energy should be conserved in form of a ApH and the system uncoupled by nigericin + KC1 or NH4Cl but not by the further addition of permeant anions. When added in limited amounts anions should cause, as for the addition of permeant cations to intact mitochondria, a transient uncoupling corresponding to a cycle of energy utilization. It may be predicted, in this latter case, that the original steady-state energy level of the system should not be affected and also the dimension of the cycle should be proportional to the amount of charges moved. The stoichiometric energy utilization model in submitochondrial particles is considered to be supported by the observation of Montal et al. 141 that addition of nigericin + KCl or of NH4Cl in the presence of lipophilic anions results in a loss of respiratory control. However in the experiments of Skulachev et ul. [2] the uncoupling effect of tetraphenylboron was observed in the absence of nigericin KCl or NH4Cl. Furthermore in the experiments of Montal et al. 141, 100% release of respiratory control was obtained with 7pM tetraphenylboron; however the uptake of 7 pM tetraphenylboron would have required the consumption of 2-4 natoms oxygen at a stoichiometry of 2-4 anionlhigh-energy bond.
+
84
In the present work, three evidences are brought against the view that the uncoupling by lipophilic anions in submitochondrial particles is equivalent to the energy utilization for cation uptake in mitochondria. First, the extent of uncoupling, in the presence of an excess of inorganic anions, is practically independent of the presence of NH4Cl or of nigericin + K C . The data are in substantial agreement with what observed in submitochondrial particles by Christiansen PI al. [3] and in cromatophores by Briller and Gromet Elhanan [14]. However the suggestion [14] that the lack of effect of NH,f be due to being the membrane impermeable to anions is not valid in the case of submitochondrial particles in view of the fact that lipophilic anions do uncouple at very low concentration. Second, the uncoupling effect of lipophilic anions is not cyclic and affects the steady-state energy level. The parameters for the assessment of the energy level of the system are not clearly established in the case of submitochondrial particles. However if the uncoupling effect of the anion would be due to a transient energy utilization some of the energy-linked reactions should be affected in a cyclic manner. Cycles of Hfuptake, proportional to the amount of added tetraphenylboron have been observed in chloroplasts [32]. Third, there is no correlation between uncoupling and uptake. A positive correlation is considered to be supported by the observation that inorganic anions, which bind very loosely, are poor uncouplers, while organic anions, which bind more tightly, are more powerful uncouplers. However the extent of uptake is minimal in respect to the amount required for uncoupling. Papa eta/. [ 8 ] reported an uptake of 13 nmol x mg protein-' in the presence of 10 mM SCN- (cfalso Fig. 1 and Table 1 for the uncoupling concentrations of SCN-). The lack of correlation between uptake and uncoupling is supported by two observations. One, the extent of inhibition of ATP synthesis is much higher than the amount of energy required for the uptake of tetraphenylboron. Two, the experiments with bromthymol blue indicate a negligible active uptake when there is a complete uncoupling. Indeed the enhancement of bromthymol blue uptake due to energization is pH-independent as is the uncoupling. On the other hand passive interaction and total uptake are both pH-dependent. In the case of bromthymol blue it appears therefore not correct to assume that all the dye, localized in the membrane, is actively bound [35]. Spectroscopic Shifts and Amine Localization
The energy-linked interaction of amines with submitochondria1 particles, chloroplasts and chromatophores, gives rise to several problems : firstly, the molecular mechanism for the spectroscopic shifts of the
Anion and Amine Uncoupling
acridines ; secondly, the localization of the amines ; thirdly, the mechanism for uptake and fourthly, the relationship between uptake and uncoupling. As to the interpretation of the spectroscopic shifts, two mechanisms have been proposed : transition from the base to the acid form of the dye [ 18- 20,221 and dye-dye interaction [21,23- 25,371. The former mechanism is however incompatible with two observations, namely : the quenching of acridine orange fluorescence where the protonation of the acridine ring causes enhancement [37] and the absence of base-acid transition in the absorbance spectra [18j. Fiolet rt ul. 1381 have also shown that the fluorescence changes of 9-aminoacridine in chloroplasts do not correspond to predicted on the basis of a ApH induced dye distribution. As to the localization of the accumulated dyes, also two mechanisms have been proposed : accumulation of the amines in the inner osmotic space and electrostatic binding to sites situated on the membrane. In the former case the uptake reflects a distribution of the amines determined by the dpH, while in the latter case the distribution is related to the degree of interaction of the charged amines with the membrane sites. The former mechanism is supported by the data on the distribution of methylamine in chloroplasts and by the inhibitory effect of nigericin KC1 and NH,CI [18,19]. The latter mechanism is supported by the immobilization of the accumulated dye 1371and the dependence of the spectroscopic shift on the protein/dye ratio [37]. It is possible that both accumulation of free dye and binding occur, depending on several parameters such as dye/protein ratio, type of membrane, lipophilicity of the dye etc.
+
Amine Uptake and Uncoupling
Crofts [I21 has proposed that in chloroplasts the uncoupling effect of the amines corresponds to a stoichiometric energy utilization for amine uptake. The mechanism requires the following steps : equilibration of NH, across the membrane; uptake of H + in the inner osmotic space; protonation of NH, to NH; and accumulation of NH; in the inner space; uptake of anions. In this mechanism the uptake of NH, is driven by d p H and that of anions by membrane potential. In submitochondrial particles the energy-linked uptake of amines [23,24] amounts to about 20, 40 and 150 nmol x mg protein-' in the case of NH: ,acridines and aminophenazines respectively. However the amines cause also uncoupling and the uncoupling increases with the amine lipophilicity. The question arises as to whether the amine uptake in submitochondrial particles can explain the uncoupling effect. If the uncoupling effect of the lipophilic amines would be due to a mechanism similar to that proposed by Crofts [I21 for the chloroplasts, there should be a
G. F. Azzone, H. Gutwcniger, E. Viola. E. Strinna, S. Massari, and R. Colonna
close correlation between extents of uptake and of uncoupling, and NH4C1 should be a good uncoupler. Neither of these predictions is verified. First, in the absence of C1- there is uncoupling without uptake. Second, the uncoupling effect of NH,Cl is negligible. It may be argued that the lack of uncoupling effect by NH,C1 be duc to a low permeation of the uncharged NH,. This is unlikely because NH3 is a good permeant in all biological membranes and becomes a good uncoupler also in submitochondrial particles in the presence of valinomycin. Uncoupling Mechanisms f o r Lipophilic Amines and Anions A mechanism for the uncoupling of lipophilic amines may be based on the cycling of NH: in the presence of valinomycin [4,14]. The uncoupling in this case is thought to involve: diffusion of the uncharged NH3 into the particle; protonation of NH3 to NHZ ; and release of NH: from the particle viu valinomycin. It is important to realize that a cycling mechanism is not necessarily dependent on the translocation of the amine across the membrane, but it may only involve the interaction of the uncharged amine with intramembrane proteus at the outer interphase. Whatever the molecular mechanism of penetration the limiting step is the diffusion of the charged amine. This is supported by the fact that 50% uncoupling requires a proportionally higher valinomycin concentration at decreasing NH4C1 concentrations. In the case of lipophilic amines the dependence of uncoupling on amine lipophilicity reflects the rate of permeation of the charged amines through the membrane. The uncoupling by lipophilic amines is enhanced by lipophilic anions. In this case the extrusion of charged amine is replaced by the release of the amineanion complex. The complex corresponds to an hydrophobic ion pair. The uncoupling involves in this case: uptake of H+ and anions; uptake of amine; formation of an hydrophobic ion pair between charged amine and anion; and release of the ion pair. As for the uncoupling by anions the following mechanism may be conceived: uptake of H’ and anions due to respiration; release of H f ; and release of anions. This mechanism resembles that of the classical uncouplers with the difference that the second step requires a membrane leaky for H + rather than a protonophoric effect of the uncouplers. The chemical properties of the anions may be important in establishing the degree of uncoupling. For example bromthyniol blue is a much stronger uncoupler than 8-anilino-lnaphthalene sulphonate [36]. This may be due to the fact that the bromthymol blue chromophore either operates also as a proton carrier or causes greater damage to the membrane structure because of its higher polarity.
85
A final question is why lipophilic amines and anions are so efficient uncouplers in submitochondrial particles and not in mitochondria. The reason is to be found in the leakiness of the submitochondrial particles membrane. This is partly due to the sonication procedure [39] and partly follows the uptake of the lipophilic amines and anions. This obviously raises the question as to the extent to which ion gradients can be maintained in submitochondrial particles.
REFERENCES 1. Azzone, G . F. & Massari, S. (1973) Biochim. Biophys. Acta, 301, 195-226. 2. Skulachev, V. P., Yaguzhinski, L. S., Jasaitis, A. A., Liberman, E. A., Topali, V. P. & Zofina, L. A. (1969) in Enerxy Level and Melubolic Control (Papa, S., Tager, 3 . M., Quagliariello, E. & Slater, E. C., eds) p. 283, Adriatica Editrice, Bari. 3. Christiansen, R. O., Loyter, A. & Racker, E. (1969) Bioc4im. BiOphyS. Acta, 180, 207-210. 4. Montal, M., Chancc, B. & Lee, C . P. (1970) J . Membrane Biol. 2, 201 -234. 5. Azzi, A,, Chance, B., Radda, G. K. & Lec, C. P. (1969) Proc. Nut1 Acad. Sci. U.S.A.62, 612. 6. Liberman, E. A. & Skulachev, V. P. (1970) Biochim. Biophys. Acta, 216, 30-42. 7. Colonna, R., Dell’Antone, P. & Azzonc, G. F. (1972) Arch. Biochem. Biophys. 151, 295 - 303. 8. Papa, S . , Guerrieri, F., Simone, S., Lorusso, M. & La Rosa, D. (1973) Biochim. Biophys. Acta, 292, 20- 38. 9. Shavit, N., Dilley, R. A. & San Pietro, A. (1968) Biochemi.stry, 7 , 2356-2363. 10. Krogman, D. W., Jagendorf, A. T.& Avron, M. (1959) Plant Phl$.siol.34, 272. 11. Good, N . E. (1960) Biochirn. Biophys. Acta, 40, 502. 12. Crofts, A. R. (1967) J. Biol. Chem. 242, 3352-3359. 13. Horio, T. & Yamashita, J. (1964) Biochim. Biophys. Acra, 88, 237. 34. Briller, S. & Gromet-Elhanan, Z. (1970) Biochim. Biophys. Aria, 205, 262 - 272. 15. Izawa, S . (1965) Biochim. Biophys. Actu, 102, 373-378. 16. Kraayenhof, R. (1970) FEBS Lett. 6 , 161- 165. 17. Eilerman, L. J. M. (1970) Biochim. Biophys. Acta, 216, 231 233. 18. Schuldiner, S., Rottenberg, H. & Avron, M. (1972) Eur. J . Biochem. 25, 64 - 70. 19. Gromet-Elhanan, Z . (1972) Eur. J. Biochem. 25, 84-88. 20. Deamer, D. W., Prince, R. C. & Crofts, A. R. (1972) Biochim. B i o p h ~ )Acta, ~ . 274, 323-335. 21. Colonna, R., Dell’Antone, P. & Azzone, G . F. (1970) FEBS Lett. 10, 13 - 16. 22. Lee, C. P. (1971) Biochemistry, 10, 4375-4381. 23. Dell’Antone, P., Colonna, R. & Azzone, G. F. (1972) Eur. J. Biochem. 24, 553 - 565. 24. Dell’Antone, P., Colonna, R. & Azzone, G. F. (1972) Eur. J . Biochem. 24, 565 - 576. 25. Dell’Antone, P. & Azzone, G . F. (1974) FEBS Lett. 39,67-72. 26. Racker, E. (1962) Proc. Null Acad. Sci. U.S.A.48, 1659- 1662. 27. Chance, B. & Mela, L. (1966) Proc. Natl Acad. Sci. U.S.A. 55, 1243- 1246. 28. Mitchell, P.. Moyle, J. & Smith, L. (1968) Eur. J . Biochem. 4, 9-17. 29. Azzone. G . F., Piemontc, G. & Massari, S. (1968) Eur. J . Biochem. 6,207-212.
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G. F. Azzone, H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna: Anion and Amine Uncoupling
30. Rossi, E. & Azzone, G. F. (1970) Eur, J . Biochem. 12,319- 327. 31. Lee, C. P., Azzone, G . F. & Emster, L. (1964) Nature (Lond.) 201, 153-156. 32. Horton, A. & Packer, L. (1968) Arch. Biochem. Biophys. 128, 820 - 822. 32a. McCarty, R. E. &Coleman, C. H. (1970) Arch. Biochem. Biophys. 141,198-206. 33. Chance, B. & Montal, M. (1971)in Currenf Topicsin Membranes and Transport, vol. 2, pp. 99- 156, Academic Press, New York.
34. Skulachev, V. P. (1971) in Current Topics in Bioenergetics (Sanadi, D. R., ed.) vol. 4, pp. 127- 190, Academic Press, New York and London. 35. Nordenbrand, K. & Ernster, L. (1971) Eur. J . Biochem. 18, 258 270. 36. Layton, D. (1973) Ph. D. Thesis, Chelsea College, London. 37. Massari, S., Dell’Antone, P., Colonna, R. & Azzone, G . F. (1975) Biochemistry, I S , 1038- 1043. 38. Fiolet, J. W. T., Bakker, E. P. & Van Dam, K. (1974) Biochim. Biophys. Actu, 365, 432-445. 39. Azzone, G. F. & Massari, S. (1972) FEES Left. 28, 61 -64. ~
G. F. Azzone, H. Gutweniger, E. Viola, E. Strinna, S. Massari, and R. Colonna, Istituto di Patologia Generale,
Universita degli Studi, Via Loredan 16, 1-35100 Padova, Italy
