vol. 83, No. 4, I 978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
August 29,197B
Pages 1496-1501
COUPLINGBETWEEN H+ ENTRYAND ATP FORMATIONIN Escherichia
coZi
Peter C. Maloney The Johns Hopkins University Received July
School of Medicine,
Baltimore,
Maryland 21205
19, 1978 SUMMARY
ATP synthesis driven by a pH gradient was studied in wild type coZi and in a mutant lacking functional membrane bound Ca++, Mg++-stimulated adenosineS'-triphosphatase (BF,F,; EC 3.6.1.3). In the wild type, ATP synthesis required the presence of mobile ions other than H+; ATP synthesis was not observed in the mutant. Direct measurement of internal pH also showed that when ATP synthesis occurred in the wild type, acidification of the cell was more rapid than in the mutant. These observations provide direct evidence in support of the idea that BFoFl catalyzes an obligatory coupling between H+ entry and ATP synthesis.
Escher-ichia
INTRODUCTION The chemiosmotic
theory
of Mitchell
of H+ couples redox reactions phorylation. this
respiration
view [reviewed
in the electrochemical
potential
It is also known that
a difference
Since this
the driving
by H+ entry,
that
as required
direct
31.
[4,5]
using Escherichia
For example, it
coli
catalyzed
to the hydrolysis
of a difference
potential by BF,F,
for H+ [8-121.
of ATP [5,13],
it
mediated by BFoFl is also accompanied theory.
such ATP synthesis
The work given here
is linked
to the inward
movement of H+. METHODS Most experiments used E. coli strains AN180 (wild type) and its derivative, AN120 (uncA), which lacks functional BF,F, [14]. Strain 0006-291X/78/0834-1496$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
have
membrane [6,7].
in the electrochemical
by the chemiosmotic
evidence that
phos-
is known that
and the maintenance
force for ATP synthesis
ATP synthesis
a circulation
during oxidative
for H+ across the cell
enzyme couples H+ extrusion
has been inferred
studies
in ref.
leads to H+ extrusion
can provide
proposes that
and ATP synthesis
Genetic and biochemical
supported
provides
[1,2]
1496
BIOCHEMICAL
Vol. 83, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
SASX76 (hml) [15] was also used. Cells were grown and starved of metabolizable reserves as described [lo]. After starvation, 0.2 M potassium phosphate (pH 8) was used for washing and resuspension. ATP was measured as in earlier experiments [lo], and internal pH was estimated [16] from the distribution of salicylic acid (pK = 2.98). In such cases, cells were placed in 0.2 M potassium phosphate (pH 8) along with 5 nM ['4C]salicylic acid (New England Nuclear Corp.) at a density equivalent to 0.25 1-11cell water per ml of suspension. Addition of 2N acid lowered outside pH to pH 3.5, after which 0.2 ml samples were withdrawn and rapidly filtered using prewetted, prechilled filters (0.6 n pore size). This was followed by a brief wash with 3 ml of iced buffer (0.2 M potassium phosphate adjusted from pH 8 to pH 3.5 with sulfuric acid). Internal pH was calculated after correction for the low level of nonspecific binding assessed by filtration before the addition of acid. In a control experiment using both AN180 and AN120, the label found within cells after addition of sulfuric acid (e.g. Fig. 2) was extracted with ethanol and chromatographed on silica gel in the presence of unlabelled salicylic acid, using hexane:acetic acid: chloroform (85:15:10, v/v). All radioactivity cochromatographed with the authentic salicylic acid.
RESULTSAND DISCUSSION The general synthesis
plan was to impose a pH gradient
in a wild type strain.
Proton entry which reflected
between H+ movements and ATP synthesis comparison between the internal lacking
functional
BF,F,.
for such experiments
capable of driving
Strains
a coupling
could then be identified
pH of the wild
type strain
ATP
by a
and a mutant
AN180 and AN120 (~4)
were suitable
since they show the same passive permeability
of the
membrane to H+ [lo]. Figure
1 shows internal
(AN120) cells
after
these conditions, will
arise
external
as H+ enters
shown, this
the cell,
a membrane potential by both specific
would block net formation
"back potentiall'
chloride
that ATP synthesis
had no effect
or anions.
on ATP synthesis
1497
inside)
and nonspecific of ATP if
this
routes. synthetic
charge.
In the
by efflux
of
or by entry of an external, but not sulfate
was observed in the wild
in the presence of mobile cation valinomycin
or nitrate,
Under
(positive
would be relieved
K+ in the presence of valinomycin,
permeant anion (either is clear
pH was lowered from pH 8 to pH 3.5.
is coupled to the net inward movement of positive
experiment internal
of ATP in wild type (AN180) and mutant
one expects that
Such a "back potential" reaction
levels
[17,18]).
type strain,
It
but only
(Other work showed that when the pH jump was given with
Vol. 83, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
AN180
RESEARCH COMMUNICATIONS
AN120
MINUTES
Internal levels of ATP after addition of acid. Starved cells Figure 1. of strains AN180 and AN120 were suspended at pff 8 in 0.2 M potassium phosphate. After sampling for basal levels of ATP, external pH was lowered to pH 3.5 by addition of the indicated acid (2N). Where shown, valinomycin was also present at 10 u final concentration.
either
hydrochloric
mutant,
confirming
indicate
that
or nitric earlier
between ATP synthesis Further
studies
the reaction
experiments
ATP formation
acids.)
[8,10].
catalyzed
was not found in the
Taken together,
these results
by BF,F, in E. coZi requires
and the net inward movement of positive of this
was blocked by a proton
kind
(not given)
conductor
demonstrated
a coupling
charge.
that ATP synthesis
(carbonylcyanide-p-trifluoromethqxy-
phenylhydrazone). To determine acid equivalents, labelled
salicylic
whether ATP synthesis similar acid,
studies so that
was also linked
were performed
cases, the internal sulfuric
pH could be monitored.
experiments.
In individual
pH of AN180 and AN120 was compared after
acid and either
hydrochloric
acid
of
in the presence of
changes in internal
The data given in Figure 2 summarize 6 separate
to the entry
(3 experiments)
a pH jump using or nitric
acid
(3 experiments). When sulfuric extent
acid was used to impose the pH gradient,
of acidification
of the cell
were identical
1498
the rate and
for strains
AN180 and
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Vol. 83, No. 4, 1978
4
6 MINUTES
Internal pH after addition of acid. Starved cells of strains Figure 2. AN180 (circles) and AN120 (triangles) were suspended in 0.2 M potassium phosphate along with 5 1.04labelled salicylic acid. After addition of the indicated acid (2N), samples were taken for measurement of internal pH as described in Methods. Mean values from 6 separate experiments are given, and error bars indicate f. 1 SEM. In all experiments, samples were taken Those data confirmed the at 1 min intervals to determine ATP levels. Differences in internal pH of AN180 results presented in Fig. 1. Inset: and AN120 when sulfuric (O), hydrochloric (0) or nitric (a) acid was were added. Mean values for the standard errors of the paired differences 0.05, 0.08 and 0.07 pH units, respectively.
AN120. [lo],
Since membrane permeability the similar
to H+ is the same for the two strains
rates of net acidification
power is also the same for the two cell differences net proton
in internal
pH of the two strains
in net proton
was imposed with hydrochloric into
that
internal
Consequently, must reflect
buffering
measured
differences
in
entry.
Such differences
entry
types.
indicate
the wild
or nitric
type was faster
AN180 and AN120 do not differ
entry were observed when the pH gradient acids.
than into
in their
relative
1499
In both instances, the mutant. permeabilities
proton
Assuming that to either
Vol. 83, No. 4, 1978
BIOCHEMICAL
the chloride
anions,
or nitrate
may be attributed by BF,F,.
the inset
to Figure 2.
This "ATPase-specific"
of the wild
to synthesize
ATP.
when nitric
type strain
when hydrochloric
cases showed about the same twofold
values at earlier
the inhibitor increase
rate
of proton
carbodiimide
used cells
blocked ATP synthesis
entry
into
caused an increase
not encountered
this
(Fig.
1).
for
in the two
As well,
from their
than hydrochloric previously
both the
maximal
acid was used.
exposed to N,N'[19].
Although
was also an apparent
of the cell The inhibitor
occurred
as fast
also increased
posen [20] had previously in H+ permeability,
difficulty.
capacity
of acidification
of ATP synthesis
in AN180, there
controls.
AN120.
by
the increased
with its
of BF,F, in B. coZi
for acidification
than in the untreated
rates
of ATP
is indicated
directly
of ATP fell
rather
an inhibitor
in H+ permeability,
or faster
rates
and levels
(not given)
dicyclohexylcarbodiimide,
synthesis
observed in AN180 was about
difference
times when nitric
Other experiments
during
seen in AN180
acid was used, and about 1.4 pH units/min
The initial
pH difference
entry
also suggest that
correlates
the increment
acid was used.
ATPase-specific
proton
A comparison of the initial
shows that
RESEARCH COMMUNICATIONS
pH difference
Other observations
acidification
0.6 pH units/min
the increased
to the entry of acid equivalents
catalyzed
to two strains
AND BIOPHYSICAL
noted that
but others
[21,22]
The reason for these discrepant
the this have
findings
is not clear. The presence of functional demonstration
of proton
entry
cytochromes was not required coupled to ATP synthesis.
SASX76, was grown under conditions capable of electron
transport.
that prevented
acid,
but not sulfuric
preparations
As with AN180, net synthesis
The difference
presented
BFoFl catalyzes
here give positive a coupling
of cytochromes of ATP was
using hydrochloric
in internal
was the same as than observed for AN180 (Fig.
The results view that
acid.
The hemA mutant,
formation
observed when a pH jump (pH 8 to pH 3.5) was performed
for the
pH of the two 2).
evidence in support
of the
between the entry of H+ and the
1500
BIOCHEMICAL
Vol. 83, No. 4, 1978
synthesis
of ATP, as proposed by the chemiosmotic
stoichiometry
of such coupling
kinase,
If E. coli,
[6]. this
estimate
such calculations the biochemical
buffering
as Streptococcus
RESEARCH COMMUNICATIONS
theory.
in these experiments
about 10 H+/ATP, assuming internal Hamilton
AND BIOPHYSICAL
may be calculated
reaction
Zactis
overestimate
because of internal
as
power as given by Collins [23],
is reduced to about 5 H+/ATP.
may significantly
The apparent
has active
adenylate
As noted earlier
the true stoichiometry
reactions
and
[23], of
which consume ATP.
ACKNOWLEDGMENTS The technical assistance of F.C. Hansen, III is gratefully This work was supported by a research grant from the National Health (GM 24195).
acknowledged. Institutes of
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
Mitchell, P. (1961) Nature 191,144-148. Boyer, P.D., Chance, B., Ernster, L, Mitchell, P., Racker, E. and Slater, E.C. (1977) Ann. Rev. Biochem. 46,955-1026. Harold, F.M. (1977) Curr. Top. Bioenerg. 6,83-149. Lawford, H.G. and Haddock, B.A. (1973) Biochem. J. 136,217-220. Hertzberg, E.L. and Hinkle, P.C. (1974) Biochem. Biophys. Res. Commun. 58,178-184. Collins, S.H. and Hamilton, W.A. (1976) J. Bacterial. 126,1224-1231. Ramos, S. and Kaback, H.R. (1977) Biochem. 16,848-854. Maloney, P.C., Kashket, E.R. and Wilson, T.H. (1974) Proc. Nat. Acad. Sci. USA 71,3896-3900. Grinius, L., Slusnyte, R. and Griniuviene, B. (1975) FEBS Lett. 57,290-293. Wilson, D.M., Alderete, J.F., Maloney, P.C. and Wilson, T.H. (1976) J. Bacterial. 126,327-337. Tsuchiya, T. and Rosen, B. (1976) Biochem. Biophys. Res. Commun. 68,497-502. Tsuchiya, T. and Rosen, B. (1976) J. Bacterial. 127,154-161. West, I.C. and Mitchell, P. (1974) FEBS Lett. 40,1-4. Butlin, J.D., Cox, G.B. and Gibson, F. (1971) Biochem. J. 124,75-81. Sasarman, A., Surdeanu, M. and Horodniceanu, T. (1968) J. Bacterial. 96,1882-1884. Maloney, P.C., Kashket, E.R. and Wilson, T.H. (1975) in "Methods in Membrane Biology", (E. Korn, ed.) Vol. 5, pp l-49, Plenum Press, New York. Mitchell, P. and Moyle J. (1956) in "Bacterial Anatomy", (E.T.C. Spooner and B.A.D. Stocker, eds.) Sot. Gen. Microbial. (London), Cambridge Univ. Press, London. Flagg, J.L. and Wilson, T.H. (1977) J. Membr. Biol. 31,233-255. Fillingame, R.H. (1975) J. Bacterial. 124,870-883. Rosen, B. (1973) Biochem. Biophys. Res. Commun. 53,1289-1296. Altendorf, K., Harold, F.M. and Simoni, R.D. (1974) J. Biol. Chem. 249,4587-4593. Patel, L., Schuldiner, S. and Kaback, H.R. (1975) Proc. Nat. Acad. Sci. USA 72,3387-3391. Maloney, P.C. (1977) J. Bacterial. 132,564-575.
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