Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF ATPase ACTIVITY
RECONSTITUTION
FROM THE ISOLATED a, 8, AND y
SUBUNITS OF THE COUPLING FACTOR, Fl,
OF ESCHERICHIA COLI
M. Futai Section of Blochemistry,.Molecular Cornell University, Ithaca, Received
November
and Cell Biology New York 14853
14,1977
(a, 8 and y) of the coupling factor, Fl SUMMARY: The three major subunits ATPase, of Escherichia coli were separated and purified by hydrophobic column chromatography after the enzyme was dissociated by cold inactivation. The ability to hydrolyze ATP was reconstituted by dialyzing the mixture of subunits against 0.05 M Tris-succinate, pH 6.0, containing 2 mM ATP and 2 mM MgC12. A mixture containing a, 8 and y regained ATP hydrolyzing activity. Individual subunits alone or mixtures of any two subunits did not develop ATPase activity, except for a low but significant activity with a plus 8. The reconstituted ATPase had a Km of 0.23 mM for ATP and a molecular weight by sucrose gradient density centrifugation of about 280,000. ATPase membranes (for
complexes
are
similar
reviews,
membrane units, cedures
for
the inhibitor be essential
from many sources.
were
obtained
the binding
in pure of Fl
of ATPase activity for
binding
the Fl
Recently
Sternweis
y by passing
"4-subunit"
ATPase
Copyright All rights
cytoplasmic
in energy
is
extrinsic
metabolism to the
The functions
antibody
to E (13).
to Fl depleted elegant
studies
0 1977 by Academic of reproduction in any
of Fl has been done
and the assembly
(12).
very
role
of the complex
and reconstitution
of each subunit
essential
binding
portion
a central
to as a, 8, y, 6 and E (3) have been studied
subunits
linked
and play
and bacterial
of its
sub-
by various
pro-
(4-12).
the role
plasts
The Fl
and has been purified
Dissociation
minor
in structure
see l-3).
referred
mitochondria
of chloroplasts,
This
membranes Sagawa
form
(7-10)
to depleted (7,8).
complex and it
membranes The 6 subunit
from Streptococcys obtained (lacking "3-subunit"
understand
molecule.
The two
was shown that (8,
9) and that
has also
faecalis
(11)
6)
through ATPase
(14)
a column required of energy
reconstituted
shown to
and chloroonly
with both
6 is E is
been
an ATPase containing
and reconstitution
and coworkers
Press, Inc. form reserved.
of this
to further
a, $, and covalently
S and E for
functions. ATPase
In
from isol-
1231 ISSN
0006-291X
Vol. 79, No. 4, 1977
ated
subunits
plete this
BIOCHEMICAL
of a thermophilic
resolution
It
y) from E. __ coli
Fl
in this
one can obtain
rich
in y (16)
dissociated
of interest
(EFl)
since
extensive
Previous
the Bsubunit
I report
EFl by hydrophobic by dialyzing
only
to obtain
workers
column
their
procedure
the very
the three studies
have
a mixture
active
isolation
for
genetic
(15),
in reconstitutively paper
was reconstituted containing
is
organism.
tion
In this
However,
bacterium.
of Fl may be applicable
bacterium.
possible
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
stable
major
for
enzyme from
subunits
(a,
of the complex
shown that
by cold
of a and 8 (16)
com-
B and
are
dissocia-
and a fraction
form.
and purification
of a, 8 and y from cold-
chromatography.
The activity
the mixture
of three
subunits
against
of ATPase buffer
ATP and Mg 2+ at 22-25'C. MATERIALS
AND METHODS
Phenyl sepharose and butyl agarose were obtained from Pharmacia Co. and Miles-Yeda Ltd., respectively. Recycled materials were not used. Other chemicals were of the highest grade commercially available. Protein was assayed Pure EFl (about 100 units per mg) was obtained as previously described (18). from 3. coli K12h or ML308-225 by a published procedure (17). One unit of ATPase is expressed as the amount hydrolyzing 1 umole of ATP/min (17). Modification of the previous procedures (15, 16) were used to dissociate EFl: Pure EFl from K12A (6 depleted) was precipitated with (NH4)2SO4 (65% saturation), dissolved in buffer A (0.05 M Tris-succinate, pH 6.0, 1.0 M KCl, 0.1 M KN03, 0.1 mM DTT (dithiothreitol) and 1 mM EDTA) and dialyzed against the same buffer overnight at 4". After dialysis the dissociated enzyme was rapidly frozen in a dry ice-ethanol bath and stored at -80". All procedures were carried out at 4O and all Isolation of y subunit. precipitates were collected by centrifugation at 10,000 x g for 10 min. The dissociated enzyme (33 mg in 9 ml of buffer A) was precipitated with (NB4)2SO4 (60% saturation) and dissolved in 0.8 M (NH4)2SO4 in buffer B (0.05 M Ttissuccinate , pH 6.0, 1 mM DTT and 1 mM EDTA) and applied to a column of butyl agarose (1.2 x 20 cm) previously equilibrated with the same buffer. The column was washed extensively with the same buffer (about 40 ml) and successively with 0.7 M (NH4)2S04 in buffer B (about 20 ml), until no material with absorbancy at 280 nm eluted from the column. Fractions (0.8 and 0.7 M eluate) were combined and concentrated to about 7 ml by ultrafiltration (Amicon filter, DMlO), and precipitated with (NH4)2SO4 (0.60 saturation). The precipitate was dissolved in 1 ml of 0.8 M ammonium sulfate in buffer B and applied to a butyl agarose column (0.7 x 13 cm) after removal of any insoluble material. About 0.74 mg of y subunit was obtained in 5.5 ml of eluate with 0.8 M (NH4)2SQ in buffer B, and was stored at -80'. Isolation of a and f3 subunits. All procedures were carried out at 23'. Dissociated enzyme (12 mg in 2 ml of buffer A) was applied to a nhenyl senharose column (0.7 x 9 cm) equilibrated with buffer A.--After extensive washing with the same buffer, the 8 subunit was eluted with 10 mM Tris-HCl, pH 8.0, followed by 2 mM Tris-HCl, pH 8.0, and the a subunit was eluted with 25% ethylene glycol in 2 mM Tris-HCl pH 8.0. All buffers used above contained 0.1 mM
1232
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
About 2.4 mg of a and 4.0 mg of 8 was obtained Each subunit was frozen and stored at -80'.
DTT. form.
as practically
homogeneous
RESULTS Purification served
of three
repeatedly
form aggregates that
major
that
subunits
when the
previous
workers
exchange
column
series
of alkyl
agarose
in high
columns
and the y subunit
1) using
phenyl
the
subunits
and Mg
2+
gels.
of ATPase
individual
ions.
ATPase
0.1-0.25 2+ ATP and Mg ions
by dialyzing
fractionated Maximal
units
(a, not
subunits reconstitution
8 and
give
Properties was 0.23
significant activity
This
value
(both
l-2
against
(15,
16).
11).
after about
Fig.
ATP could
except
(Fig. of
activity
was reATP
initiation
of
4 hours
of dialysis.
was
The presence work
on parti-
by ADP or AMP. of three
or combinations
the mixture
pH 6.0
No activity
previous
not be replaced
subunits
pro-
Tris-succinate 1).
by
containing
by the combination
that
form
5% as judged
of pH above 8.0.
confirming
Individual
the a and B
of a, 8 and y (total
I,
buffer
only
than
a
the separation
above,
buffer
against
mM) (Table
the reconstitution,
sub-
of any two
of a + 8 gave about
obtained. ATPase.
was measured is
after
was dialyzed
activity,
of reconstituted mM when it
mg/ml)
for
The ATPase
when the mixture
peak
Contamination
linearly
was reconstituted
was observed
y) (Table
10% of the maximal
almost
to
may be the reason
homogeneous
was less
subunits.
increased
the subunits for
given
respectively.
fraction
tend
We have tried
column
of a, 8 and Y against
was observed
of ATP was essential
(2:l).
activity
concentration,
obtained
did
a mixture
reconstitution
containing
ally
from
sepharose
agarose,
We have ob-
in a single
Fl.
in practically
subunit
and maximum activity
Optimal
obtained
and butyl
by dialyzing
dialysis
tein
sepharose
of subunits
By the procedure
of polyacrylamide
Reconstitution constituted
were
This
dissociated
and a phenyl
in each purified
staining
a mixture
Fl.
in the cold,
was lowered.
of cold
strength.
E. coli
once dissociated
16) obtained
ionic
8 and y)from
concentration
chromatography
subunits
other
(a,
of EFl,
salt
(15,
in ion
of subunits
subunits
essentially
The Km of the reconstituted
by keeping
the ratio
the same as for
1233
ATPase
for
ATP
of ATP to Mg 2+ constant
native
ATPase
(17).
The re-
Vol. 79, No. 4, 1977
BIOCHEMICAL
A
-*
B
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
E
D
C
t -
(,
-DV
c
Front
Fig. 1. Polyacrylamide gel electrophoresis of the three isolated major subunits from E. coli Fl. Approximately 5~10 pg of isolated u subunit (c), 8 subunit (D) and y subunit (E) were applied to 10% polyacrylamide gels in the presence After electrophoresis gels were stained with of 0.1% sodium dodecyl sulfate(21). As controls native enzyme from E. e strain Mh308Coomassie Brilliant Blue. 225 and from K12(X) (minus S) are shown in (A) and (B), respectively.
TABLE L Requirements
for
Reconstitution Subunits
of ATPase
of E. coli
from
Activities unitsfmg
reconstitution
Major
F1.
ATPase Standard
Three
Reconstituted protein
33.9
buffer
- ATP
0.0
- MgC12
6.4
- ATP + ADP
3.7 0.0
- ATP + AI@
The three major subunits (cI‘, 12.6 ug; 8, 14.8 pg; y, 2.2 pg) were mixed in 200 pl of O.OlM Tris-succinate pH 6.0 containing 0.2 M KCl, 10% glycerol and 0.1 mM DTT and were dialyzed for about 8 hours against 400 ml of standard reconstitution solution (0.05 M Tris-succinate pH 6.0 containing 10% glycerol, 0.1 mM DTT, 0.1 mM EDTA, 2 mM ATP and 2 mM MgC12) or the same solution with Concentrations of ADP, and AMP subtraction or addition as specified above. were the same as that of ATP.
1234
BIOCHEMICAL
Vol. 79, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE II Reconstitution
of ATPase Major
from Various Subunits
Combinations
of --E. coli
of the Three
Fl
ATPase Activity unitslmg a
0.1
8
0.3
Y
Reconstituted protein
0
a+!3
3.1
B+Y
0.2
a+Y a+B+y
0 30.7
The three of Table I.
constituted
major
subunits
ATPase
in sucrose
weight
stituted
ATPase
molecules
sedimented
gradient
molecular
were mixed
as a single
centrifugation from
is
this
and dialyzed
(Fig.
sharp 2).
a homogeneous
molecular
of the a and B subunits.
peak,
more rapidly
A rough
run was 280,000.
These species
Detailed
in the Legend
as described
than
estimation
results
(19)
suggest
and contains
studies
catalase
of the
that
recon-
two to three
of the assembly
are
in
progress. DISCUSSION The procedure vantage
used
for
of a difference
of the present a y-rich relatively
mild
(16)
thermophilic
and other may remove
bacterial
of our Fl
results (TFl)
bination
of g and y from EFl
from TFl
gave ATPase
reconstitution
all
are
which
from EFl
(b)
possible
from those
from
of Kagawa
interesting
but
f3 subunit
and
Our procedure
is
sources.
and coworkers
while
for
TF . 1
ad-
(15)
be noted: this
of ATP was required not
took
Combination
other
and should
Presence
1235
study
contamination.
to F 1 ATPases
subunits
this
gave the
showed no reconstitution
activity.
of ATPase
in
of the subunits.
procedures
and may be applicable
The differences
of subunits
in the hydrophobicities
procedure
fraction
the separation
These
(14)
on
(a) A com-
combination for
the
differences
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fraction
PH
Numbor
Fig. 2. Effects of pH on the reconstitution of ATPase from the three major sub units of E. coli Fl. A mixture of subunits as described in Table I was dialyzed for 8 hours against standard reconstitution solution with 0.05M of different buffers and ATPase activity was assayed as described in the text. Buffers used were Tris-succinate (O), Tris-HCl (o), and glycine-NaOH (0). Fig. 3. Centrifugation of the ATPase reconstituted from the three major subReconstituted ATPase (about 30 yg protein in units, in a sucrose gradient. 0.1 ml) was applied to a 11 ml linear sucrose gradient (5 to 20%) containing 0.05 M Tris-HCl pH 8.0, 0.1 mM DTT, 1 mM ATP, 0.1 mM EDTA. After centrifugawere collected tion in the SW40 rotor for 14 hours at 31,000 rpm, 41 fractions from the bottom of the tube and ATPase activity (13) and catalase (shown by an arrow) were assayed (20).
may be due to species procedures
used
fractionated ution
for
the
following
(data
not
A detailed
separation
standing
of the mechanism
from
major
with
of the assembly
here
three
the minor
is also subunits
the native
for
TFl were
inactive
in the
partially for
reconstit-
shown). study
It
used
or to differences
The EFl subunits
of subunits.
the procedure
isolated
molecule.
among Fl molecules
differences
of EFl molecules
subunits
of energy
of interest
(8,
to compare
enzyme
the
is of interest
transduction
and "three-subunit"
5-subunit
10)
from
of this ATPase
ATPase
for complex
reconstituted
obtained
subunits
major
an underE. __ coli from the
by removing
6 and e
(13).
Acknowledgment - The work reported in this paper was undertaken during the tenure of an American Cancer Society - Eleanor Roosevelt - International CanIt was cer Fellowship awarded by the International Union Against Cancer. for carried out in the laboratory of Dr. Leon A. Heppel to whom I am indebted Support by National Science Foundation Grant No. advice and encouragement. Institutes of Health is BMS 75-20287 and Grant No. AM-11789 from the National also acknowledged. I also thank Ms. Sharon Johnston for technical assistance.
1236
Vol. 79, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3.
Senior, A. E. (1973) Biochem. Biophys. Acta 301, 249-277. Harold, F. M. (1977) Curr. Top. Bioenerg. 5, 83-149. Racker, E. (1966) A New Look at Mechanisms in Bioenergetics, Academic Press, New York, N.Y. 4. Nelson, N., Deters, D. W., Nelson, H. and Racker, E. (1973) J. Biol. Chem. 248, 2049-2055. 5. Deters, D. W., Racker, E., Nelson, N-and Nelson, H. (1975) J. Biol. Chem. 250, 1041-1047. 6. McCarty, R. E. and Fagan, J. (1973) Biochemistry 12, 1503-1507. 7. Nelson, N., Nelson,H. and Racker, E. (1972) J. Biol. Chem. -247. 7657-7662. 8. Smith, J. B. and Sternweis, P. C. (1977) Biochemistry 16, 306-311. H. and Karny, 0. (1977) FEBS Letter 70 249-253. 9. Nelson, 10. Sternweis, P. C. and Smith, J. B. (1977) Biosemistry 16, 4020-4025. 11. Abrams, A., Jensen, C. and Morris, D. H. (1976) Biochem. Biophys. Res. Commun. 9, 804-811. 12. Younis, H. M., Winget, G. D. and Racker, E. (1977) J. Biol. Chem. 252,1814-l8l8. 13. Sternweis, P. C. (1977) Ph.D. Thesis, Cornell University 14. Yoshida,M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485. G. and Steinhart, R. (1976) Biochemistry 5, 208-216. 15. Vogel, R. J. and Smith, J. B. (1977) Biochemistry I& 4266-4270. 16. Larson, M., Sternweis, P. C. and Heppel, L. A. (1974) Proc. Nat. Acad. Sci. 17. Futai, USA 71, 2725-2729. N. J., Farr, A. L. and Randall, 18. Lowry, O.H., Roseborough, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. Martin, R. G. and Ames, B. N. (1961) J. Biol. Chem. 236, 1372-1379. 20. Beers, R. J., Jr., and Sizer, I. W. (1952) J. Biol. Chem. 195, 133-140. 21. Laemmli, U. K. (1970) Nature -227. 680-685.
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