F1F0-ATPase from Acetobacterium woodii contains F-type and V-type rotor subunits and is capable of ATP synthesis Michael Fritz and Volker Mu¨ller Molecular Microbiology & Bioenergetics,
Trang 1F1F0-ATPase from Acetobacterium woodii contains F-type and V-type rotor subunits and is capable of ATP synthesis Michael Fritz and Volker Mu¨ller
Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany
Membrane-bound, multisubunit, ion-translocating ATP
synthases⁄ ATPases are present in every domain of life
They arose from a common ancestor, but evolved into
three distinct classes of ATP synthases⁄ ATPases: the
F1F0-ATP synthase present in bacteria, mitochondria
and chloroplasts, the A1A0-ATP synthase present in
archaea, and the V1V0-ATPase present in eukarya
[1,2] A common feature of ATP synthases⁄ ATPases
is their organization into two domains, a soluble and
a membrane-bound domain, which are connected by
(at least) two stalks, one central and one to two
peripheral [3–5] The hydrophilic, cytoplasmic domain
catalyzes ATP hydrolysis [6,7], whereas the membrane
domain translocates ions from one side of the
membrane to the other against their electrochemical
gradient [8,9]
ATP synthases⁄ ATPases are rotary machines that
work as a pair of coupled motors, a chemically
driven (F1⁄ A1⁄ V1) motor and a membrane-embedded, ion gradient-driven motor (F0⁄ A0⁄ V0) [10–12] The membrane-embedded motor is composed of a stator and a rotor The stator is composed of subunits a and b, and the rotor is composed of multiple copies
of subunit c They form an oligomeric ring of non-covalently linked subunits, and rotation of the c ring
is obligatorily coupled to ion flow across the mem-brane [13,14]
Subunit c of F1F0-ATP synthases has a molecular mass of around 8 kDa, and folds in the membrane like a hairpin, with two transmembrane helices that are connected by a cytoplasmic loop [15] Each monomer contains an ion-binding site, and as 10–15 subunits constitute the rotor (depending on the spe-cies), it has a total of 10–15 ion-binding sites [12,16– 19] This gives a H+ (Na+)⁄ ATP stoichiometry of 3.3–5, a value required for ATP synthesis, given a
Keywords
Acetobacterium woodii; ATP synthase;
F-type; rotor subunits; V-type
Correspondence
V Mu¨ller, Molecular Microbiology &
Bioenergetics, Institute of Molecular
Biosciences, Johann Wolfgang Goethe
University Frankfurt ⁄ Main,
Max-von-Laue-Str 9, 60438 Frankfurt, Germany
Fax: +49 69 79829306
Tel: +49 69 79829507
E-mail: vmueller@bio.uni-frankfurt.de
(Received 23 March 2007, revised 2 May
2007, accepted 8 May 2007)
doi:10.1111/j.1742-4658.2007.05874.x
Previous preparations of the Na+F1F0-ATP synthase solubilized by Triton X-100 lacked some of the membrane-embedded motor subunits [Reidlinger
J & Mu¨ller V (1994) Eur J Biochem 233, 275–283] To improve the subunit recovery, we revised our purification protocol The ATP synthase was solu-bilized with dodecylmaltoside and further purified to apparent homogeneity
by chromatographic techniques The preparation contained, along with the
F1subunits, the entire membrane-embedded motor with the stator subunits
a and b, and the heterooligomeric c ring, which contained the V1V0-like subunit c1 and the F1F0-like subunits c2 and c3 After incorporation into liposomes, ATP synthesis could be driven by an electrochemical sodium ion potential or a potassium ion diffusion potential, but not by a sodium ion potential This is the first demonstration that an ATPase with a V0–F0 hybrid motor is capable of ATP synthesis
Abbreviations
DY, membrane potential; DlNa + , electrochemical sodium ion potential; DpNa, sodium ion potential.
Trang 2transmembrane electrochemical ion gradient of
around 200 mV The c subunit of V1V0-ATPases
arose by duplication and fusion of the bacterial c
subunit, giving rise to a 16 kDa protein with two
hairpins [20] Most important, the ion-binding site is
not conserved in hairpin 1 If one assumes the same
number of hairpins in V0 and F0, the rotor of V1V0
-ATPases has only half the number of ion-binding
sites This is seen as the reason for the apparent
inab-ility of V1V0-ATPases to catalyze ATP synthesis
in vivo Indeed, V1V0-ATPases have evolved to be
effi-cient ion pumps, a function required by the
physiol-ogy of the eukaryotic cell [21]
The operon encoding the Na+ F1F0-ATP synthase
from the anaerobic, acetogenic bacterium Acetobacterium
woodii is unique and encodes nine F1F0-like subunits
along with one gene encoding a V1V0-like subunit The
atp operon has one homolog each of a gene encoding
the F1F0 subunits a, b, c, d, e, a, and b, but it has
three genes encoding differently sized c subunits [22]
Subunits c2and c3have a molecular mass of 8.18 kDa,
are identical at the amino acid level, and are similar to
csubunits from F1F0-ATP synthases Like other F1F0
-ATP synthase c subunits, they are predicted to span
the membrane like a hairpin and to have one
ion-bind-ing site In contrast, subunit c1 is similar to the c
sub-units of V1V0-ATPases and predicted to have four
transmembrane helices with only one ion-binding site
A woodii is, so far, the only organism known with
V1V0- and F1F0-like c subunit genes in one ATPase
operon This poses the obvious questions of whether
the heterooligomeric c ring can promote ATP
synthe-sis, whether the c subunit stoichiometry is variable,
and, if so, whether a variation of the c1⁄ c2 ⁄ 3 ratio may
change the function of the enzyme from an ATP
syn-thase to an ATPase [23]
These questions have not so far been addressed, due
to the lack of a purified, intact ATP synthase Despite
the clear genetic evidence for different c subunits, as
well as for the presence of subunits a and b, they were
not detected in previous preparations of the enzyme
[24,25] Later, separation of gently solubilized
mem-brane protein complexes by blue native PAGE and
N-terminal sequencing of polypeptides present in the
gel revealed both types of c subunit, as well as
sub-units a and b, in the ATPase complex [26] These
stud-ies prompted us to revise our purification scheme, with
the aim of obtaining an intact ATP synthase from
A woodii We here present a protocol yielding a
com-plete Na+ F1F0-ATP synthase complex, including the
entire membrane motor Most important, both types
of c subunits were present This made it possible to
readdress the longstanding question of whether an
ATPase with F0- and V0-type rotor subunits is able to synthesize ATP
Results
Purification of a complete Na+F1F0-ATP synthase from A woodii
The Na+ F1F0-ATP synthase purified previously was solubilized from membranes with Triton X-100 [24,25]
To improve the recovery of subunits, we analyzed the effectiveness of dodecyl-b-d-maltoside in solubilizing the entire ATP synthase When used at 1% (w⁄ v) and
1 mg of detergent per mg of protein, dodecyl-b-d-maltoside solubilized about 85% of the membrane-bound ATPase activity The ATP synthase was then purified by gel filtration to apparent homogeneity This procedure resulted in a 16-fold enrichment, but was accompanied by loss of 70% of the activity (Table 1) The molecular mass of the complex as determined by gel filtration was 590 kDa As can be seen from Fig 1, the enzyme preparation contained 12 polypeptides The identity of the peptides was established using MALDI-TOF or western blot analyses These studies revealed that the 58 kDa fragment corresponds to sub-unit a, the 54 kDa fragment to subsub-unit b, the 35 kDa fragment to subunit c, the 19 kDa fragment to subunit
d, the 18 kDa fragment to a mixture of subunits a and
b, the 16 kDa fragment to subunit e, the 14 kDa frag-ment to subunit c1, and the 10 kDa fragment to sub-unit c2⁄ 3 (Fig 1A) The 42 kDa fragment reacted with antibobies against c2⁄ c3 (which also recognize c1) and with antibodies against c1 (which do not recognize
c2 ⁄ 3; Fig 1B) These data demonstrate that the 42 kDa fragment represents the SDS-resistant, heterooligomeric
c ring of the Na+F1F0-ATP synthase When the pre-paration was heated to 120C for 5 min, the c oligo-mer was disrupted, and the monooligo-mers could be detected immunologically (Fig 1B) In summary, these experiments clearly demonstrated the presence of sub-units a, b, c1 and c2⁄ 3 in the membrane-embedded rotor of the purified Na+ F1F0-ATP synthase from
A woodii
Table 1 Purification of the Na+F 1 F 0 -ATP synthase from A woodii.
Step
Protein (mg) Volume (mL) Activity (U) Activity (U ⁄ mg)
Purification (fold)
Yield (%)
Concentrated solubilizate
Trang 3Characterization of the Na+F1F0-ATP synthase
from A woodii
The specific ATPase activity of the complete enzyme
was in the range 5–9.9 UÆmg)1 protein, depending on
the batch This is in the same range as the one
deter-mined previously with the incomplete enzyme [25]
Next, we compared the enzymatic properties of the
complete enzyme with those of the preparations
stud-ied previously The basic biochemical parameters,
such as temperature and pH dependence, as well as
the kinetic data for ATP hydrolysis were identical
(data not shown) Of special interest was the effect of
Na+ on activity, as Na+ is known to interact with
the membrane-embedded motor As seen before, ATP hydrolysis was clearly Na+ dependent (Fig 2), and
Na+ could be substituted by Li+ Furthermore, the
Km for Na+ or Li+ (0.5 mm, 2.0 mm) was compar-able to the values determined before As described before, the stimulation by Na+ was less pronounced
at more acidic pH values, indicating competition of
Na+ and H+ for a common binding site (data not shown) Furthermore, inhibition by N¢,N¢-dicyclo-hexylcarbodiimide was abolished in the presence of
Na+ (Fig 3) In summary, the biochemical parame-ters of the complete preparation were indistinguish-able from those of the preparation described previously [25]
α β γ
c-oligomer
c2/3
ε
c1
δ a/b
3 4 5 6 7
1 2
a
c it n
1
t
n i a n it c2/
3
66
45
30
20
14
66
30
20
14
45
94
66
- 45
- 30
- 20
- 14
Fig 1 Subunit composition of the Na + F 1 F 0 -ATP synthase from A woodii Proteins were separated by SDS⁄ PAGE and stained with SERVA Blue G (Serva GmbH, Heidelberg, Germany) (A) or blotted against specific antibodies (B) Lane 1: molecular mass marker Lane 2: ATP syn-thase preparation was denatured by incubation at 80 C for 10 min Lane 3: ATP synthase was heated for 5 min at 120 C prior to SDS ⁄ PAGE to disrupt the c oligomer and blotted against c 1 antibodies Lane 4: ATP synthase was incubated for 10 min at 80 C, and blotted against c 1 antibodies Lane 5: the sample was incubated for 10 min at 80 C and hybridized with antibody specific for the a subunit Lane 6: ATP synthase was incubated for 5 min at 120 C and hybridized with antibodies against subunit c 2 ⁄ 3 (which also detect subunit c1) Lane 7: ATP synthase was denatured by boiling for 15 min, and blotted against c2⁄ 3 antibodies.
A
0 1 2 3 4 5
-3 -2 -1 0 1 2 3 4 5 6 7 8 0.1
0.3 0.5 0.6 0.7 0.8
0 1 2 3 4 5
1.0
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.1
0.3 0.5 0.7 0.9
B
Fig 2 Ion dependence of ATP hydrolysis by
the Na + F1F0-ATP synthase from A woodii.
ATPase activity was measured at 30 C
using the assay described in Experimental
procedures NaCl (A) or LiCl (B) was added
from stock solutions to the concentrations
indicated The insert shows the same data
plotted by the method of Lineweaver–Burk.
Trang 4ATP synthesis catalyzed by the Na+F1F0-ATP
synthase from A woodii
Next, we investigated whether the enzyme is capable
of ATP synthesis despite its heterooligomeric c ring
Liposomes were prepared from lipids extracted from
chicken egg, and the complete Na+ F1F0-ATPase
was reconstituted into these liposomes The rate of
ATP hydrolysis as catalyzed by these proteoliposomes
was 2.8 UÆmg)1 To analyze whether the enzyme
was reconstituted in a functionally coupled state that
allows for ATP synthesis, the following experiments
were performed In a fully coupled system, ATP
hydrolysis is accompanied by ion transport into the
proteoliposomes, and the membrane potential
estab-lished slows down or even inhibits further ion
trans-port and thus ATPase activity This thermodynamic
control can be overcome by addition of ionophores
After addition of the Na+ionophore
N¢,N¢,N¢,N-tetra-cyclohexyl-1,2-phenylenedioxydiacetamide to the
proteo-liposomes, ATP hydrolysis was stimulated seven-fold
Stimulation, but to a lower extent, was also observed with the protonophore tetrachlorosalicylanilide These experiments demonstrated coupling of ATP hydrolysis
to the generation of a membrane potential in our pro-teoliposome system
Next, we applied artificial driving forces to the pro-teoliposomes The general strategy is shown in Fig 4 When the proteoliposomes were loaded with 200 mm NaCl and incubated in the presence of 200 mm KCl, thus creating a sodium ion potential (DpNa), there was
no ATP synthesis (Fig 5) Upon addition of 2 lm vali-nomycin, a membrane potential (DY, inside positive) was created in addition by the influx of K+ into the liposomes (potassium ion diffusion potential) and in the presence of both DY and DpNa, ATP was synthes-ized at a rate of about 40 mol ATPÆ(mol pro-tein))1Æmin)1 When a DY was applied separately, ATP was synthesized at a rate comparable to that with electrochemical sodium ion potential (DlNa+) as the driving force ATP synthesis was strictly dependent on the presence of ADP, the coupling ion Na+, and the presence of a DY (data not shown)
In summary, these data demonstrate not only that subunits a and b are required to confer the ability to synthesize ATP, but equally important, that the pres-ence per se of subunit c1 does not abolish ATP synthe-sis by the Na+F1F0-ATP synthase from A woodii
Discussion
The Na+F1F0-ATP synthase from the anaerobic, ace-togenic bacterium A woodii purified here contained all the subunits deduced from the operon sequence Most importantly, it contained the membrane motor sub-units a and b, which were absent in previous prepara-tions Because the ATP synthase preparations from the close relatives Moorella thermoautotrophicum and Moo-rella thermoacetica were also devoid of subunits a and
b, it was suggested in the literature that the ATP synthases of acetogenic bacteria may be simpler in architecture than other F1F0-ATP synthases [27,28]
0
25
50
75
100
Fig 3 Inhibition of the Na + F1F0-ATP synthase from A woodii by
N ¢,N ¢-dicyclohexylcarbodiimide and relief of inhibition by Na +
ATPase activity was measured at 30 C and pH 7.5 using the assay
described in Experimental procedures The samples were incubated
with 5 mM (m) or 100 lM NaCl (j) for 30 min
N¢,N¢-Dicyclohexyl-carbodiimide was then added, and the samples were incubated for
another 25 min The reaction was started by addition of 5 mM ATP.
One hundred per cent activity corresponds to 9 UÆmg)1.
Fig 4 General scheme for the application of artificial driving forces to the proteoliposomes For explanation, see text.
Trang 5However, this argument was difficult to understand, as
the genes encoding subunits a and b were embedded
in the atp operons of M thermoacetica and A woodii
[22,29], and one would have to envision a mechanism
that post-trancriptionally or post-translationally
specif-ically removes these subunits Furthermore, these
sub-units are essential for motor function From the results
presented here, it is evident that the critical step in
purification is the solubilization procedure The Triton
X-100 used in previous studies apparently did not
solu-bilize the stator subunits a and b Whether or not our
previous preparation contained both types of c subunit
is difficult to retrace However, Fig 3 in Reidlinger &
Mu¨ller [25] shows a faint band at about 17 kDa, which
appeared when the enzyme was autoclaved At that
time, c1 was unknown, and an N-terminal sequence of
this fragment was not obtained Therefore, it is not
only not excluded that it was the c1 subunit but likely,
as the c ring is rather stable, and the use of Triton
X-100 should not lead to removal of c1only
The basic biochemical properties of the complete
enzyme are similar to those of the enzyme studied
before It is Na+ dependent, and Na+ and
N¢,N¢-di-cyclohexylcarbodiimide compete for binding to a
com-mon site It should be noted that the incomplete
enzyme isolated previously was capable of ATP-driven
Na+ transport As ATP-driven Na+ transport would
also require the stator subunits, one has to assume that
previous preparations had substoichiometric amounts
of subunits a and b This is in accordance with the low
Na+⁄ ATP ratio determined previously The important difference is that the stator-depleted enzyme was unable
to synthesize ATP in a proteoliposome system [30], whereas the entire enzyme used here was competent in ATP synthesis This underlines the essential function of the stator subunits in driving ion gradient-driven ATP synthesis As observed before with the Na+F1F0-ATP synthase from Propionigenium modestum, DlNa+ as well as DY but not DpNa were sufficient as driving forces [31–33] Most importantly, the Na+ F1F0-ATP synthase from A woodii was able to catalyze ATP synthesis despite its different c subunits Although the c subunit stoichiometry has not yet been solved, at least one copy of c1must have been present Further discus-sion of the coupling efficiencies has to await the deter-mination of the rotor subunit stoichiometry
In summary, we have solved a longstanding problem, the purification of the Na+ F1F0-ATP synthase from
A woodii, including the membrane-embedded motor Most importantly, we have demonstrated that the enzyme as isolated from fructose-grown cells is compet-ent in ATP synthesis, despite its unusual and unique membrane-embedded motor This is the starting point for a detailed analysis of the stucture and function of the rotor of the Na+F1F0-ATP synthase from A woo-dii, the first containing V0- and F0-like c subunits
Experimental procedures
Growth of cells and isolation of membranes
A woodii(DSM 1030) was grown in 20 L vessels to midex-ponential growth phase as described previously [34] Fruc-tose (20 mm) was used as the carbon and energy source The cells were harvested by continuous centrifugation (Heraeus centrifuge, Stratos, HCF 22.300 rotor), and stored
at ) 70 C until used For the isolation of membrane vesi-cles, 20–25 g of cells (wet mass) was suspended in 200 mL
of 50 mm Tris⁄ HCI, 10 mm MgCI2, and 420 mm sucrose (pH 7.5) (buffer A) After addition of 1 g of lysozyme (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), the suspension was incubated at 37C for 60 min All sub-sequent steps were carried out at 4C unless otherwise indicated The resulting protoplasts were collected by cen-trifugation (16 000 g, 10 min, Beckman Avanti J25, JA14 rotor) and suspended in one volume of buffer A containing
a few crystals of DNase (Sigma-Aldrich Chemie GmbH) and phenylmethanesulfonyl fluoride (final concentration 0.5 mm) The protoplasts were disrupted by two passages through a French pressure cell at 42 MPa Cell debris and unbroken cells were removed by two sequential centrifuga-tion steps at 6000 g for 15 min (Beckman Avanti J25, JA14 rotor) The supernatant was diluted with one volume of
0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 1 0
0
0
0
0
0
0
0
0
0
0
m i
t e ( s )
Fig 5 ATP synthesis by Na + F 1 F 0 -ATP synthase-containing
proteo-liposomes The artificial driving forces DlNa+(j), DY (m) or DpNa
(r) were applied to the proteoliposomes as described in
Experi-mental procedures In one assay (d), a DlNa + was applied but
ADP was omitted.
Trang 650 mm Tris⁄ HCl (pH 7.5), 10 mm MgCl2, and 17% (v⁄ v)
glycerol (buffer B), and centrifuged at 130 000 g for 60 min
(Beckman L100K, 50.2 Ti rotor) to collect the membranes
The membranes were washed once with buffer B, and
resuspended in 60 mL of buffer B
Purification of ATP synthase
The membranes were diluted to 10 mgÆmL)1with buffer B,
and solubilization was accomplished by the addition of
1% dodecyl-b-d-maltoside (w⁄ v) (Sigma-Aldrich Chemie
GmbH) After 60 min on ice, the extract was centrifuged at
130 000 g for 30 min (Beckman L100K, 50.2 Ti rotor) The
solubilized ATP synthase was concentrated to a volume of
15 mL (Vivaspin 20 columns, 300 kDa cutoff; Vivascience,
No¨rten-Hardenberg, Germany), and applied to a gel
filtra-tion column (Sephacryl S-400, 2.6⁄ 100 cm; GE-Healthcare,
Freiburg, Germany) The column was equilibrated with
col-umn buffer (50 mm imidazole, 50 mm NaCl, 25 mm MgSO4,
0.5 mm phenylmethanesulfonyl fluoride, 0.1% reduced
Tri-ton X-100 (pH 7.5) (Sigma-Aldrich Chemie GmbH), at a
flow rate of 0.5 mLÆmin)1 The purified ATP synthase was
found in three fractions These fractions were pooled and
concentrated to 5 mL (Vivaspin 20 columns, 100 kDa cutoff;
Vivascience) All preparations were routinely analyzed by
SDS⁄ PAGE, using the buffer system of Scha¨gger & von
Jagow [35] Polypeptides were visualized by staining with
SERVA blue G250 (Sigma-Aldrich Chemie GmbH) [36] or
silver [37]
Determination of ATPase activity
The ATPase activity was assayed in buffer C (100 mm Tris
base, 100 mm maleic acid, 5 mm MgCl2) The pH was
adjus-ted to 7.5 with KOH The characterization of the enzyme
was performed at 30C by a discontinuous assay following
the ATP-dependent formation of inorganic orthophosphate,
according to the method of Heinonen & Lahti [38] as
des-cribed previously [39] The assay contained 5 mm MgCI2
when carried out at pH 7.5, and 50 mm MgCl2 at pH 5.3
For inhibitor studies, the samples were incubated with the
inhibitor for 30 min before the reaction was started by
addi-tion of ATP N¢,N¢-Dicyclohexylcarbodiimide
(Sigma-Ald-rich Chemie GmbH) was added as an ethanolic solution,
and controls received solvents only
Western blot analysis
After separation by SDS⁄ PAGE, the ATP synthase
sub-units were blotted onto a nitrocellulose membrane as
des-cribed previously [40] Western blot ECL detection
reagents were either purchased from PerkinElmer Life
Sci-ences (Boston, MA, USA) or made in-house [solution A
(200 mL containing 0.1 m Tris⁄ HCl, pH 6.8, 50 mg of
luminol), and solution B (10 mL of dimethylsulfoxide con-taining 11 mg of p-hydroxycoumaric acid)] Blot mem-branes were incubated in a mixture of 4 mL of solution
A, 400 lL of solution B and 1.2 lL of H2O2 for 2 min before exposure to WICORex film (Typon Imaging AG, Burgdorf, Switzerland)
Reconstitution of ATPase into proteoliposomes
A suspension of 60 mgÆmL)1 l-a-phosphatidylcholine type II-S (Sigma-Aldrich Chemie GmbH) in buffer D (100 mm Tris, 100 mm maleic acid, 20 mm NaCl, 5 mm MgCl2,
pH 7.5) was sonicated on ice at 120 W and 20% (Ultra-sonic Disintegrator, type MK2, Crawley, England) until the creamy suspension became translucent To the purified ATP synthase, the liposomes were added to a final lipid concentration of 30 mgÆmg protein)1 The proteoliposomes were prepared by the method of Knol et al [41], and the detergent was removed by stirring in the presence of Bio-Beads (Bio-Rad, Mu¨nchen, Germany) for 12 h at 4 C The proteoliposomes were collected by gel filtration using a
10 mL column filled with Sephadex 25 (Bio-Rad) matrix equilibrated with buffer D and driven by gravity More than 85% of the ATPase activity applied for the reconstitu-tion experiments was found in the liposome fracreconstitu-tion (0.7 mg proteinÆmL)1), indicating almost complete incor-poration into the proteoliposomes ATP synthesis was determined via a standard luciferin⁄ luciferase assay, monit-oring the emitted light with a chemiluminometer (Lumac,
AC Landgraaf, The Netherlands)
To generate aDlNa+, the proteoliposomes were first loaded with Na+to create a DpNa The vesicles were incu-bated in buffer D containing, in addition, 200 mm NaCl for 12 h at 4C After this, the Na+-loaded vesicles were collected by gravity-driven gel filtration using a 10 mL pip-ette filled with Sephadex 25 matrix and equilibrated with buffer D containing, in addition, 200 mm KCl and 5 mm
KH2PO4 The synthesis reactions were carried out at 30C with 2 mL of proteoliposome solution from gel filtration and by adding 5 mm ADP The synthesis reaction was star-ted by addition of 2 mL of valinomycin (Sigma-Aldrich Chemie GmbH) to induce a DY Samples (10 mL) were withdrawn every 30 s and immediately added to 250 mL of
an ATP determination buffer (5 mm NaHAsO4, 4 mm MgSO4, 20 mm glycylglycine, pH 8) After the addition of
5 mL of firefly lantern crude extract (Lumac), light emis-sion was measured Calibration was done with standards of
a known ATP content
To apply a DY only, the proteoliposomes were incubated for 12 h with buffer D After this, the vesicles were collected
by gravity-driven gel filtration using a 10 mL pipette filled with Sephadex 25 matrix and equilibrated with buffer D containing, in addition, 200 mm KCl and 5 mm KH2PO4 The synthesis reactions were carried out at 30C with 2 mL
Trang 7of proteoliposome solution from gel filtration and by adding
5 mm ADP The synthesis reaction was started by the
addi-tion of 2 mL of valinomycin to induce a DY
Furthermore, when only a DpNa was to be applied, the
vesicles were incubated in buffer D containing, in addition,
200 mm NaCl for 12 h at 4C After this, the Na+
-loaded vesicles were collected by gravity-driven gel filtration using
a 10 mL pipette filled with Sephadex 25 matrix and
equili-brated with buffer D containing, in addition, 200 mm KCl
and 5 mm KH2PO4 The synthesis reactions were carried
out at 30C with 2 mL of proteoliposome solution from
gel filtration, and started by addition of 5 mm ADP
MALDI-TOF analysis
Proteins were separated by SDS⁄ PAGE, and all bands
vis-ible by Coomassie staining were entirely cut out and
subjec-ted to in-gel digestion protocols [42,43], which were adapsubjec-ted
for use on a Microlab Star digestion robot (Bonaduz,
Switzerland) Samples were reduced, alkylated and
subsequently digested overnight using bovine trypsin
(sequencing grade; Roche, Mannheim, Germany) The gel
pieces were extracted, and the extracts were dried in a
vacuum centrifuge and stored at) 20 C until use ⁄ analysis
MALDI-TOF MS experiments were performed on an
Ultraflex TOF⁄ TOF mass spectrometer (Bruker Daltonics
Inc., Billerica, MA, USA) The samples were prepared as
described previously [44] Spectra were externally calibrated
with a Sequazyme Peptide Mass Standards Kit (Applied
Biosystems, Foster City, CA, USA), and internally
calibra-ted on a tryptic auto digestion peptide (m⁄ z 2163.0564)
The spectra were processed in flexanalysis version 2.2
(Bruker Daltonics) using the SNAP algorithm
(signal-to-noise threshold 3; maximal number of peaks 150; quality
factor threshold 80) Proteins were identified by mascot
(Matrix Science, Boston, MA, USA) (peptide mass
toler-ance 50 p.p.m.; maximum missed cleavages 1) using the
NCBInr database (2 543 645 sequences; date 6 July 2005)
Proteins with a score of 77 or higher were considered to be
significant (P < 0.05)
Acknowledgements
This work was supported by a grant from the
Deut-sche Forschungsgemeinschaft (SFB472) The help of
Dr O Klimmek in preparing the proteoliposomes is
gratefully acknowledged
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