BIOCHEMICAL SOCIETY TRANSACTIONS
Fig. 2. Lactose uptake and A$ at aerobic and anaerobic
conditions in cells of E. coli
Aerobically grown cells of E. coli were pretreated with
EDTA, washed and resuspended in 50 mM-potassium phosphate, pH 8.0, 5 mM-MgSO,, 50pg/ml chloramphenicol.
Then 1pM-nigericine was added to the cell suspension.
Lactose uptake (0,
O),A$ (A) and respiration rate (----)
were measured simultaneously at 30°C, in the same buffer
with 4 p ~ - T P P
+ ,200 pM-lactose (4.2Ci/mol). The cells were
present in a concentration of 0.8mg of protein/ml.
more pronounced for the Na /glutamate symport system.
This is an argument against the possible interpretation that
‘localized chemiosmosis’(Van Dam, 1978; Kell, 1979) plays
a role in these solute uptake systems. Moreover, according
to this hypothesis the lactose carrier of E. coli would have to
fit in perfectly into the local proton circuits of Rps.
sphaeroides (Elferink et al., 19836). The observations so far
fit into the chemiosmotic coupling scheme. However, in
addition to the energy transduction via the bulk phase
protonmotive force allosteric interactions between the
primary proton pump and secondary solute transport
system exist also. If one assumes that lactose is transported
with one proton under all conditions (Booth et al., 1979), the
direct interaction must also provide free energy to the transport process.
This direct interaction between the rate of electron
transfer and solute uptake causes a form of homoeostasis of
the magnitude of the protonmotive force in intact E. coli
cells. The increase in A$ upon transition from aerobic to
anaerobic conditions can only be observed when the cells
are in ‘optimal condition’, i.e. grown on a rich medium and
freshly harvested. When the cells are treated with dicyclohexycarbodi-imide this form of homoeostasis is lost.
value of the A$ increases slightly from 82mV in the
presence of oxygen to 88mV at anaerobic conditions. The
initial rate of uptake is lowered ftom 79nmol/min per mg ia
the presence of oxygen to 50nrnol/min per mg after exhaustion of the oxygen. Giutamate uptake in the presence of
oxygen is linear until all the oxygen is consumed. Subsequently, it leaks out slowly. Under anaerobic conditions it
was not possible to measure any glutamate uptake. From
these data it is clear that the direct interaction between an
electron transfer chain and transport carriers in E. coli is not
restricted to H +-symportsystems but also occurs and is even
Booth, I. R., Mitchell, W. J. & Hamilton, W. A. (1979) Biochem. J .
182, 687496
Elferink, M. G. L., Friedberg, I., Hellingwerf, K. J. & Konings, W.
N. (1983~)Eur. J . Biochem. 129, 583-587
Elferink, M. G. L., Hellingwerf, K. J., Nano, F. E., Kaplan, S . &
Konings, W. N. (19836) FEBS Lett. 164, 185-190
Kell, D. B. (1979) Biochim. Biophys. Actu 549, 55-99
Konings, W. N., Elferink, M. G. L. & Hellingwerf, K. J. (1983)
Sixth International Congress on Photosynthesis in the press
Lolkema, J. S., Hellingwerf, K. J. & Konings, W. N. (1982)
Biochim. Biophys. Actu 681, 85-94
Van Dam, K., Wiechmann, A. H. C. A., Hellingwerf, K. J.,
Arents, J. C. & Westerhoff, H. V. (1978) Fed. Eur. Biochem. SOC.
Symp. 45, 121-132
+
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Time (min)
Kinetic studies on the ATP synthesis reaction in liposomes containing bacteriorhodopsin
and ATPase-complex
ROB L. VAN DER BEND, JOHN PETERSEN and
JAN A. BERDEN
Laboratory of Biochemistry, B.C.P. Jansen Institute,
University of Amsterdam, P.O. Box 20151, 1000 H D
Amsterdam, The Netherlands
The process of oxidative and photophosphorylation in mitochondria, chloroplasts and bactexja is mediated by an
electrochemical proton gradient ( A ~ H +as) predicted by the
chemiosmotic theory (Mitchell, 1966). To obtain a better
understanding of the mechanism of the ATP synthesis reaction as caialysed by the ATPase complex, the relation
between A ~ H and
+ kinetic parameters of the ATP synthesis
reaction can be studied. For this purpose we used liposomes
containing bacteriorhodopsin and the yeast mitochondrial
ATPase complex. Use of the liposome system has several
favourable aspects: (1) the ATP-ase complex can be studied
in a ‘clean’ environment; (2) A ~ H can
+ easily be varied by
varying light intensity; (3) no specific protein interaction
between bacteriorhodopsin from Halobacterium halobium
and the yeast mitochondria1 ATPase complex may be
expected.
Bacteriorhodopsin and the ATPase complex [purified
according to Rott & Nelson (1981)l were reconstituted in
liposomes using a procedure which resulted in an optimal
functional coupling between both proteins (R. L. van der
Bend, J. B. W. J. Cornelissen, J. A. Berden & K. van Dam,
unpublished work). First bacteriorhodopsin and soya bean
phospholipids were sonicated at bacteriorhodopsin and
phospholipid concentrations of 2 mg/ml and 20 mg/ml
respectively, followed by incubation of liposomes and
ATPase complex with cholate (1.1% w/v) at phospholipid
and ATPase complex concentrations of lOmg/ml and
0.2mg/ml respectively. Cholate was afterwards removed by
a quick gel filtration method (Berden & Henneke, 1981).
ATP synthesis was measured by trapping ATP, formed
during illumination, as glucose 6-phosphate using hexokinase. Glucose 6-phosphate was afterwards determined
using an assay with glucose 6-phosphate dehydrogenase
(Bergmeyer, 1970). Lineweaver-Burk plots are constructed
for l/[ADP] versus l / v (v = ATP synthesis per min per mg of
ATPase-complex protein) at constant phosphate concentration (33mM).
The dependence of the ATP synthesis reaction on A;H+
was studied in two ways: (1) by comparing LineweaverBurk plots obtained at different light intensities (Fig. l a ) ;
(2) by comparing Lineweaver-Burk plots obtained at different concentrations of uncoupler (Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone (Fig. 1b). Parallel lines
were obtained for different light intensities (decreasing
K,(ADP) and VmaX,(ADP)
at decreasing light intensities,
Fig. la), but titration of carbonylcyanide-p-trifluoro1984
509
606th MEETING, CORK
I
0
I
0 05
010
0
0 05
0 10
I/lADPI (PM ')
Fig. 1. Lineweaver-Burk plots for l / [ A D P ]us l / v for the ATP synthesis reaction in
bacteriorhodopsin- and A TPase complex-containing liposomes
( a )Varying light intensity using neutral density filters. 100%(m), 50% (O),25% ( 0 )
and 10% (0)
transmission. (6) Titration of uncoupler. OnM- (m), 1 7 0 n ~(0)
- and
3 4 0 n ~ -( 0 )carbonyl cyanide-p-trifluoromethoxyphenylhydrazone.K,(ADP at
100% transmission and 0 nM-carbonyl cyanide-p-trifluoromethoxyphenylhydrazone
was 8 . 3 (Fig.
~ ~ la) and 6 . 4 (Fig.
~ ~ lb) respectively; VmaX,(ADP)
was 109 and
65nmol/min per mg respectively. N o phosphorylation in the dark could be
observed.
methoxyphenylhydrazone resulted in lines intersecting in
the second quadrant (decreasing V,,,,(ADP) but increasing
K,(ADP) at higher carbonyl cyanide-p-trifluoromethoxyphenylhydrazone concentrations, Fig. 1b). These results
may lead to the following consideration. If energy transduction proceeds by a bulk-bulk chemiosmotic process and
uncoupler functions as a protonophore, then the results are
not consistent with this. An explanation may be, however,
that uncoupler also binds to the ATPase complex or may
react with protons on the ATPase complex which may alter
the kinetic properties. Results found by others for chloroplasts (Aflalo & Shavit, 1983) and submitochondrial
particles (Hatefi et al., 1982) are, however, in contradiction
with this explanation. Increasing K,(ADP) and decreasing
V,,,,(ADP) were found also when nigericin plus valinomycin was used. In this case no binding to the ATPase
complex may be expected. The binding of uncoupler to the
ATPase complex, as has been studied for the beef heart
mitochondria1 ATPase complex (Berden & Henneke, 1981),
will be investigated further.
When light intensity was varied, also for chloroplasts
parallel Lineweaver-Burk plots could be found (Aflalo &
Shavit, 1983) for the light-driven ATP synthesis reaction.
The similar behaviour displayed by both chloroplasts and
the liposome preparation (also when uncoupler was used)
indicates that the ATP synthesis reaction in the liposome
system proceeds via the same mechanism as the ATP
synthesis reaction in chloroplasts (which reflects the
situation in oivo more closely). Further analysis will be
performed to construct a model for the reaction mechanism
for the ATP synthesis and hydrolysis, respectively, in which
the above-mentioned findings will fit.
Aflalo, C. & Shavit, N . (1983) FEBS Lett. 154, 175-179
Berden,J.A.&Henneke,M.A.C.(1981)FEBSLPtr. 126,211-214
Bergmeyer, H. U. (ed.) (1970) Methoden der enzyrnatischen Anulyse,
2nd end, 2 vol., Verlag Chemie, Weinheim
Hatefi, Y., Yagi, T., Phelps, D. C., Wong, S.-Y., Vik, S. B. &
Galante, Y. M. (1982) Proc. Natl. Acud. Sci. U.S.A. 79, 1 7 5 6
1760
Mitchell, P. (1966) Biol. Rev. 41, 44-502
Rott, R. & Nelson, N. (1981) J. Biol. Chem. 256, 9224-9228
Direct interaction between ATP synthase and the respiratory chain in beef heart
submitochondrial particles
MARGA A. HERWEIJER, J A N A. BERDEN and
ALBERTUS KEMP
Laboratory of Biochemistry, B.C.P. Jansen Institute,
University of Amsterdam, P.O. Box 20151,
I000 H D Amsterdam, The Netherlands
According to Mitchell's chemiosmotic theory, coupled
energy transport over membranes is catalysed by a primary,
Vol. 12
energy-producing pump and a secondary, energy-requiring
proton pump. The primcry pump generates a APH+over the
membrane and this A,uH+ is the driving force for the
secondary proton pump (Mitchell, 1966). In oxidative phosphorylation the respiratory chain is the primary proton
pump and the ATP synthase the secondary. If A j i ~ +is, as
Mitchell proposed, a fully delocalizej gradient of chemiosmotic protons, we expected this ApH+ to behave like an