Dell'Antone, Paolo

Pregnolato, L.

Ronconi, G.

Volpato, Odillo

Elsevier - Publisher Connector 

H+ Gradient in submitochondrial particles generated by an electroneutral H+/Mn2+ exchange 

Volume 8 1, number 2 FEBS LETTERS September 1977 
H’ GRADIENT IN SUBMITOCHONDRIAL PARTICLES GENERATED BY AN 
ELECTRONEUTRAL H’/Mn2’ EXCHANGE 
Paolo DELL’ANTONE and Odillo VOLPATO, 
with the technical assistance of G. RONCONI and L. PREGNOLATO 
CNR Unit for the Study of Physiology of Mitochondria and Institute of General Pathology, University of Padova, Italy 
Received 11 July 1977 
1. Introduction 
It has been found that submitochondrial particles, 
obtained by sonication of mitochondria in the pres- 
ence of Mg”and ATP [l] or EDTA [2], can promote 
several respiration linked ion movements, in addition 
to proton uptake [3-91. 
Certain anions, such as NOJ- or PCB-, can be 
taken up by respiring submitochondrial particles [8,9]. 
Due to the electroneutral K’/H’ or Na’/H’ exchange 
system of the inner mitochondrial membranes [lo], 
K+or Na’uptake occurs [6-8,l l-121 at the expense 
of H’ uptake. However, at variance with mitochondria, 
ion fluxes have seldom been explored in respiration 
inhibited submitochondrial particles. In the present 
work, we give evidence of a Mn”/H’exchange, in a 
respiration inhibited Mn”-SMP preparation, mediated 
by the ionophore A23 187 [ 131, in the presence of 
EDTA. 
To monitor H’ translocation we have followed 
acridine absorbance [ 141 or fluorescence changes 
[ 14,151. The paramagnetic Mn”ion was followed by 
EPR technique, which has recently been shown to be 
useful in the study of divalent cation translocation 
in mitochondria [ 16- 191. 
Abbreviations: EPR, electron paramagnetic resonance; PCB-, 
phenyldicarbaundecaborane anion; SMP, submitochondrial 
particles; FCCP, p-trifluoromethoxycarbonyl cyanide phenyl- 
hydrazone; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethane- 
sulphonic acid; 9 AA, 9-aminoacridine 
North-Holland Publishing Company - Amsterdam 
2. Materials and methods 
Submitochondrial particles with a high Mn2+ content 
(Mn”-SMP) were prepared according to Hansen and 
Smith [20] and suspended in a medium containing 
0.2 M sucrose, 20 mM Hepes, pH 6.5. The final pH 
was 6.8-6.9. Protein was determinated by means of 
biuret procedure [21]. EPR spectra at X band 
(9 100 MC/S) were registered by means of a Varian 
V4502 spectrometer at room temperature. Samples of 
7.5 ~1 were placed in a flat quartz cell and closed with 
a teflon plug. The microwave frequency was approx. 
9.5 GHz. The modulation amplitude (M), recorder 
time constant (fc) and scanning rate (SR) were as 
follows: M 5 G, tc 0.1 s, SR 500 G/mm. 9-Amino- 
acridine fluorescence was measured with a Hitachi 
MP-2A spectrofluorimeter. The following wavelengths 
were employed: 400 nm for excitation and 440 nm 
for emission. The changes in acridine orange 
absorbance were recorded with a dual wavelength 
spectrophotometer made in the workshop of the 
Institute of Physics at the University of Padova. pH 
measurements were made by a Schott and Gen 
9259/8 combination electrode (Jena Glasswerk, 
Mainz, FRG). 
Acridine orange and 9aminoacridine were 
obtained from E. Merck, All other chemicals used 
were of the highest purity available commercially. 
When not otherwise specified, experiments were 
performed at room temperature. 
243 
Volume 8 1, number 2 FEBS LETTERS September 1977 
3. Results 
Figure 1 shows the response of the dye acridine 
orange to the addition of EDTA and A23 187 in the 
presence of Mn2’-SMP (pH 6.9). EDTA induced a 
quenching of the dye absorbance at 492 nm and a 
greater change was induced by the antibiotic A23 187. 
However, after incubation of the SMP for twenty or 
more minutes at room temperature, A23 187 was 
ineffective. Uptake was followed by a slow release 
of the dye, which could be stimulated by KC1 (see 
also fig.lc,d). A faster reversal was induced by 
nigericin, in the presence of K’ (fig. 1 a) while preincu- 
bation with nigericin abolished the effect (fig. lb). 
The reversal of the absorbance change was never 
complete since the change due to EDTA was not 
reversed. Contrary to nigericin, FCCP was ineffective 
in preventing the effect (not shown) or in stimulating 
its reversal (fig.1~). Figure Id shows that the acridine 
DTA 
bAd92-550 
decrease 
I 
O^ol 
” 
lKCl 
EDTA 
I 
---j (bl 
EDTA 
I’ 
A23187 
l -c- 
orange response is dependent upon the Mnf?,‘/Mnilt 
gradient. The ionophore A23 187, without EDTA in 
the medium, did not trigger the acridine orange 
response, which became evident only after the 
external divalent cations were chelated by EDTA. It 
must be observed that the change of the dye 
absorbance is in the same direction as that induced 
by ATP or respiratory substrates [ 141, which monitor 
dye uptake by submitochondrial particles. However 
the acridine orange energy-linked uptake required 
the presence of anions [14,22], while the experiments 
shown in fig.1 (except the experiment lb) were 
performed in a medium containing sucrose 0.2 M, 
without added anions. 
Similar results could be obtained with 9-amino- 
acridine (fig.2). The fluorescence quenching of 
this acridine derivative has been extensively used to 
measure ApH across membranes in liposomes [23], 
chloroplasts [24], chromatophores [25] and submito- 
,A 23187 
DTA 
Fig.1. Acridine orange response to A23187 and EDTA additions. The medium (2.5 ml) contained: sucrose 0.2 M, Hepes 10 mM, 
pH 6.9, Mn”-SMP 0.56 mg/ml, rotenone 2 PM, acridine orange 8 PM. In the experiment (b) KC1 10 mM and nigericin 1 pg were 
also present. Additions were: A23187 2 pg, nigericin 1 1.18, KC1 20 mM, EDTA (a-c) 1 mM, (d) 250 PM, FCCP 1 UM each 
addition. 
244 
Volume 8 1, number 2 FEBS LETTERS September 1977 
EDTA 
I E DTA, 
(4 
AA 
IVal 
(b) 
30 sec. 
4 z 
\A 
Fig.2. 9-Aminoacridine fluorescence response to EDTA and A23187 additions. The medium (2.5 ml) contained: sucrose 0.2 M, 
Hepes 10 mM, pH 6.9, Mn*‘-SMP 0.56 mg/ml, rotenone 2 PM. In (b) KC1 4 mM was also present. Additions were: 9-aminoacridine 
5 PM, EDTA 1 mM, A23187 2 pg, valinomycin 1 rg, nigericin 1 ccg. 
chondrial particles [ 1.51. The fluorescence quenching 
was about 20-30% of the initial fluorescence. For 
the transition de-energized-energized state in 
EDTA-SMP, a quenching has been observed [ 151 
ranging from 20-50%, depending upon the 
experimental medium. Figure 2b also shows that, 
valinomycin, in the presence of K’, did not stimulate 
the reversal of the fluorescence quenching. 
The quenching of the dye absorbance, induced 
by EDTA, prior to addition of A23 187, was further 
investigated. Figure 3a shows that Mn2+ in excess 
with regard to EDTA abolished the A23 187 as 
well as the EDTA effect on the acridine orange 
absorbance. Figure 3b shows that NH; abolished the 
A23187, but not the EDTA effect. Finally by 
supplementing the medium by choline chloride, it 
was possible to reduce the EDTA effect, without 
affecting the A23 187 effect (fig3c). 
The inhibition of the A23 187 effect by Mn2+ 
indicates again that the acridine response monitors 
the Mn2’/H’ exchange, which, in the presence of Mn2+ 
in the external medium, drives H’ release versus cations 
uptake. 
The effect of Mn2+ on the EDTA-induced acridines 
response will be discussed below. 
Figure 4 shows the EPR spectrum of a Mn”-SMP 
sample in the presence of 10 mM EDTA. Our EPR 
experiments, as compared to those of acridine, 
required a high protein content. This in turn 
compelled us to use higher EDTA concentrations 
(see legends of figures). The six line spectrum, shown 
in fig.4, with a line-width about 25 G, corresponds to 
free hexahydrated Mn2+ ions [ 171. This spectrum is 
practically identical to the one observed with different 
EDTA concentrations, unless not less than 2.5 mM. 
At lower EDTA concentrations the signal amplitude 
was larger, probably due to free external Mn’+ions. 
Prolonged incubation for twenty or more minutes, 
with EDTA at room temperature induced disappear- 
ance of the six line structure of the EPR spectrum 
245 
Volume 8 1, number 2 FEBS LETTERS September 1977 
Fig.3. Inhibitory effect of cations on the acridine orange response to EDTA and/or A23187 additions. The medium (2.5 ml) 
contained: sucrose 0.2 M, Hepes 10 mM, pH 6.9, Mn”-SMP 0.44 mg/mI rotenone 2 MM, acridine orange 10 PM. In (c) coline 
chloride 40 mM was present. Additions were: A23187 2 pg, EDTA 250 PM, (NH,),SO, 2 mM, MnCl, 1 mM. 
lcx3gauss 
-1 
(b) 
Fig.4. Mn*‘-SMP EPR spectra. Effect of A23187. Mn”-SMP 
were prepared as described in Materials and methods. EPR 
measurements were made without further dilution of the 
preparation (70 mg/ml protein), a few minutes after 10 mM 
EDTA was added. During the time preceding the measure- 
ments, the sample was maintained at 0°C. In (b) A23187 
2 c(g was present. 
246 
(not shown). It is therefore very likely that this 
signal corresponds to free Mn2+ ions inside the 
vesicles. Further support of this interpretation was 
given by the effect of the ionophore A23187 on the 
Mn” EPR spectrum, which in the above experiments 
was shown to catalyze the H’ uptake. In agreement 
with the acridine response, fig.4 shows that the 
ionophore A23 187 induces the complete disappear- 
ance of the six line structure. Control experiments 
showed no effect of A23 187 on the EPR Mn2+ 
spectrum in solution, at the ionophore concentration 
employed. This result suggests that the free internal 
Mn” is translocated to the external medium, where 
it is chelated by EDTA. A proton ionophore, such 
as FCCP, was not able to mimic the A23187 effect, 
when tested either with the acridine or the EPR 
technique (not shown). 
Moreover, in the experimental conditions of the 
Volume 8 1, number 2 FEBS LETTERS September 1977 
acridine response, attempts to observe pH changes 
of the external medium with a glass electrode (see 
Materials and methods) were unsuccessful. 
4. Discussion 
The EPR technique gives evidence that Mn-SMP 
(prepared according to [20]) retains a pool of free 
Mn*‘, which cannot be chelated by externally added 
EDTA, unless A23 187 is added to the medium. It 
seems therefore very likely to localize this pool in 
the interior of the SMP. 
The usefulness of acridines to measure ApH in 
different vesicular systems (liposomes, chloroplasts, 
chromatophores, SMP) has been extensively explored 
[22-24,151, though criticism as to quantitative 
estimation has been raised [27-291 due to binding 
properties of some acridines to membranes [ 14,301. 
Acridine responses, shown in the upper experi- 
ments, give evidence of an electroneutral Mn*‘-H’ 
exchange, mediated by the antibiotic A23187. This 
exchange trasmutes a Mn*+ gradient into a H’gradient. 
Since A23 187 is also a ionorphore for Mg*‘, though 
at a lower grade [ 131, Mg*+ could participate in the 
process. 
Indeed acridine orange monitors that a H’gradient 
is created, only after the external free Mn*+is chelated 
by EDTA. Moreover, the response is abolished by K’ 
plus nigericin or NH,‘. Other observations give support 
to the above suggestion: 
(i) The acridine orange (or 9-aminoacridine) response 
does not require anions. Strong anion requirement 
has always been observed, when H’ uptake was 
driven by ATP or respiratory substrates [14,21] 
by an electrogenic process [3]. 
(ii) FCCP does not prevent or abolish the acridine 
response. This suggests that the H’ gradient 
cannot be discharged unless cation counterflow 
occurs. 
(iii) Discharge of the H’ gradient is stimulated, also in 
the absence of a K’ ionophore, by K’ addition 
probably due to the natural K’/H’antiporter of 
the inner mitochondrial membrane [lO,ll]. The 
slower rate of discharge observed in the absence 
of added K+, could be due to proton leakage. 
However, one must also suppose that some other 
ions present in the medium, or delivered by the 
SMP, provide charge equilibration by permeating 
the membranes. Another possibility, suggested 
by the different rate of spontaneous release 
observed when the responses of the two probes, 
acridine orange and 9-aminoacridine are compared 
(fig.1 and 2), is that the probe itself contributes 
to ApH discharge. It is obvious that this point 
deserves further investigation. 
As mentioned above, FCCP was ineffective to 
induce the Mn*‘/H’exchange, whether tested by the 
acridine or the EPR technique. This could indicate 
that the submitochondrial preparation used in the 
present study does not possess the mitochondrial Mn*’ 
or Ca*+ carrier which, in the presence of a H’ ionophore 
should cause discharge of the Mn*’ gradient. 
Moreover the mitochondrial membrane seems 
unable to operate the Mn*‘/H’ exchange without 
A23 187, even if a Mn*+ gradient exists. Indeed the 
EDTA effect, in the absence of A23 187, does not 
monitor a ApH across the membranes, the interior 
more acidic, because the portion of the acridine 
response due to EDTA is not inhibited by K’ plus 
nigericin or NH6 (fig. 1 a and 3b). 
Our interpretation is that as EDTA complexes 
divalent cations, some negatively charged sites 
presumably of phospholipids could bind some dye 
molecules. This binding implies spectral effects as 
observed for acridine binding to negatively charged 
liposomes [3 11. In agreement with the upper inter- 
pretation, addition of Mn*+ by inhibiting the dye 
binding also abolished the absorbance response. A 
similar interpretation could be given for the choline 
effect. 
Finally, in agreement with previous findings [32], 
our results indicate that EDTA and/or Mn*+ equilibra- 
tion does occur, across the Mn*‘-SMP membranes, 
after prolonged incubation at room temperature. 
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Volume 8 1, number 2 FEBS LETTERS September 1977 
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H+ Gradient in submitochondrial particles generated by an electroneutral H+/Mn2+ exchange

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