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.

1237

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.

Vol. 79, No. 4, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS OF ATPase ACTIVITY RECONSTITUTION FROM THE ISOLATED a, 8, AND y SUBUNIT...
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