320
N otizen
ATP Synthesis Driven by a
Valinomycine Induced K+ Diffusion Potential in
Liposomes Bearing C hloroplast A T P S ynthase
M. D inant and K K am inski
Institut de Botanique, U niversite de L iege, Sart T ilm an ,
Bat. B22, B-4000 Liege, Belgium
Z. N aturforsch. 39c, 3 2 0 -3 2 1 (1984);
received N ovem ber 25, 1983/January 20, 1984
ATP Synthesis, Valinom ycine, K+ D iffu sio n
Liposomes, C hloroplast A T P Synthase
P otential,
Partially purified chloroplast A T P synthase was re co n ­
stituted into asolectin liposom es. A v alinom ycine induced
potassium diffusion potential from o u tsid e to inside the
vesicles prom oted a m easurable A TP synthesis. If v a lin o ­
mycine was replaced by nigericine, p ractically no A T P w as
formed.
0.5 m M EDTA. The p hosphorylation reaction was
started by ad d itio n o f 0 . 8 ml reaction m ixture con­
taining 50 m M N a-T ricine (pH 8.0), 5 m M M gC l2,
5 m M N a-A D P, 2 m M p h o sp h ate (5 nCi 3 2 Pj) 0.25%
bovine serum album ine (defatted), 100 m M KC1,
20 m M glucose and 10 units hexokinase. A fter 5 m in
incubation at room tem p eratu re, the reaction was
stopped by ad d itio n o f 50 |il o f 50% trich lo racetic
acid. [32 P] ATP form ed was d eterm ined after
removal o f the 32Pj by the isobutanol-benzene ex­
traction o f the phospho m o ly b d ate com plex [9].
R adioactivity was counted w ith Lum agel scin tillato r
in a Packard scintillation counter.
In each series a control was run (trichloroacetic
acid was added before reaction m ixture) and its
radioactivity after extraction ( 1 0 -1 5 co u n ts/m in )
was negligeable. All the reagents used w ere o f
analytical grade.
Introduction
U nilam ellar liposom es containing A T P synthase
in their walls are usefull m odels for studying o x i­
dative- and photo-phosphorylations [ 1 -5 ] . T he
essential condition for A TP form atio n is the e n e r­
gization of the m em brane w ith a tran sm em b ran e
pH gradient, A pH or a tran sm em b ran e po ten tial
difference, A V [6 ]. In this paper, we show th a t a
K+ diffusion potential induced by th e ion o p h o re,
valinom ycine is sufficient to get m e asu rab le A T P
synthesis.
Experimental
Liposomes were p repared by sonication to clarity
o f soybean phospholipids (40 m g /m l) in 50 m M
N a-Tricine (pH 8.0) and 0.5 m M ED TA . A T P
synthase was isolated from spinach chloroplasts
according to [1]. The am m onium sulfate (37.5-45% )
precipitated fraction was reconstituted into lipoI phospholipids
,
\
.
^ c
somes ------------ :-------- w /w = 2 0 using th e freeze\
proteins
/
thaw technique [7] o r by a 10 m in in c u b atio n
at 20 °C.
The reconstituted vesicles (0.2 m l) w ere then
passed through a 1 ml S ephadex G 5 0 colum n [8 ]
equilibrated with 50 m M N a-T ricine (pH 8.0) and
Reprint requests to Dr. M. D inant.
0341-0382/84/0300-0320
Results and Discussion
The results are sum m arized in T able I. T he a d d i­
tion o f 1 |iM valinom ycine to the p h o sphorylation
m edium prom otes a m easurable A TP synthesis: the
values obtained are twice as high w hen the recon­
stitution is m ade by freeze-thaw co m p ared to
incubation at 20 °C. If valinom ycine is replaced by
nigericine, practically no A TP is form ed. T he sm all
quantity o f A TP observed (in case o f freeze-thaw
reconstitution) cannot be attrib u ted to p h o sp h o ry la­
tion. Indeed, in this case, the tran sm em b ran e K+
diffusion is accom panied by an an tip o rt p ro to n
movement, w ithout energization o f the m em brane.
Table I. ATP synthesis driven by a valinom ycine induced
K+ diffusion potential.
ATP, nm ol x mg p ro tein 1
Conditions
R econstitution by
freeze-thaw
10 m in 20 °C
R econstituted liposom es
0
0
R econstituted liposom es
+ 1 hm valinom ycine
30
15
Liposom es w ithout A TP
synthase
+ 1 (jm valinom ycine
0
0
Reconstituted liposom es
+ 1 |iM nigericine
3.5
0
$ 01.30/0
Unauthenticated
Download Date | 7/31/17 10:00 PM
321
N otizen
It was shown form erly [1] that the A T P synthesis
coupled to a transm em brane pH g rad ie n t driven by
an acid-to-base transition is enhanced by a K+
diffusion potential induced by valinom ycine. It is
found here that the energy o f the m em b ran e p o te n ­
tial alone is sufficient to get ATP synthesis.
The authors gratefully acknow ledge th e k ind help
and advices o f Dr. J. P. D ufour. T his research was
supported by a gran t from the Belgian G o v ern m en t
(Actions Concertees n 0 80/85-18).
[1] U. Pick and E. Racker, J. Biol. C hem . 254, 2793 (1979).
[2] G. D. Winget, N. K anner, and E. R acker, B iochim .
Biophys. Acta 460,490 (1977).
[3] G. Hauska, G. O rlich, D. Sam oray, E. H u rt, and P. V.
Sane, Proc. 5th Internat. Congr. P hotosynth. H a lk id ik i
1980, vol. 2, pp. 9 0 3 -9 1 4 (1 9 8 1 ).
[4] M. Rögner, K O hno, T. H am am oto, N. Sone, and
Y. Kagawa, Biochem. Biophys. Res. C om m un. 91,
362 (1979).
[5] P. G räber, M. R ögner, H. E. Buchw ald, D. S am oray,
and G. H auska, FEBS Lett. 145,35 (1982).
[6] P. M itchell, Science 206,1148 (1979).
[7] M. K asahara and P. C. H inkle, J. Biol. C hem . 252,
7384(1977).
[8] H. S. Penefsky, J. Biol. C hem . 252,2891 (1977).
[9] G. Hauska, in M ethods in Enzym ol. (S. P. C olow ick
and N. O. K aplan, eds.), vol. 69, pp. 6 4 8 -6 5 8 , A ca­
dem ic Press, N ew Y ork, L ondon 1980.
Acknowledgements
Unauthenticated
Download Date | 7/31/17 10:00 PM