Reconstitution of acetylcholine receptor from Torpedo californica with highly purified phospholipids: Effect of α-tocopherol, phylloquinone, and other terpenoid quinones

Vol.

93, No.

March

28,

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 409-414

1980

RECONSTITUTION

OF ACETYLCHOLINE

RECEPTOR FROM 2'0&%Z

WITH HIGHLY PURIFIED EFFECT OF u-TOCOPHEROL, Patricia

CALIFURNICA

PHOSPHOLIPIDS:

PHYLLOQUINONE, AND OTHER TERPENOID QUINONES

L. Kilian,

Carolyn

R. Dunlap,

and Paul

Mueller

Department of Molecular Biology Eastern Pennsylvania Psychiatric Institute Henry Avenue and Abbottsford Road Philadelphia, Pennsylvania 19129 Mark A. Schell,

Richard

and L. Huganir,

and Efraim

Racker

Section Molecular

of Biochemistry and Cell Biology Wing Hall Cornell University Ithaca, New York 14853

Receive61

February

1,198O

receptor from Torpedo caZifomica can be incorSUMM4RY: Acetylcholine porated by the cholate dialysis procedure into liposomes prepared with crude Vesicles reconstituted with asolectin desoybean phospholipids (asolectin). pleted of neutral lipids or with a mixture of pure phospholipids, are less active in catalyzing carbamylcholine-sensitive Na+ flux. Inclusion of ct-tocopherol or certain quinones such as coenzyme Q1o or vitamin K1 during reconstitution yi'eldsvesicles with carbamylcholine-sensitive Na+ flux which, under was considerably higher than that observed with vesicles optimal conditions, reconstituted with crude phospholipid mixtures. The lipid organ have

environment

of the acetylcholine

may be an important shown that

of the receptor inhibits

ion

are often

determinant

of the receptor

for

(1,2)

fluxes

difficult

For example,

products. fornica

electroplax approach

ments

Among the

differentiating

Na+ efflux

to

whether

the role

treatment

with

Experiments of side prepared

by unsaturated

fatty is

required

for

the affinity

phospholipase

by hydrolysis

from i"Or#0 (3).

reconstitution

biological

is

A2

phospholipases

effects

acids

of such experiments is

Studies

in

with

from vesicles

of phospholipids

limitations a lipid

to changes

because

electric

properties.

leads

(3).

however,

was inhibited

alternative (4).

and that

in membrane vesicles to evaluate,

in the

of the receptor's

delipidation agonists

receptor

caliAn experi-

the difficulty activity

in or for

reconstitution. 0006-291X/80/060409-06$01.00/0 409

Copyright @ 1980 by Academic Press, Inc. All rrghts of reproduction in any form reserved.

Vol.

93, No.

2, 1980

BIOCHEMICAL

In the present receptor

paper

we show that

by the procedure

a crude

mixture

However,

enzyme QIo or certain

and Racker

phospholipids,

to obtain other

RESEARCH

active

(5),

vesicles

have

COMMUNICATIONS

of the acetylcholine originally

can be carried

quinones

MATERIALS

BIOPHYSICAL

the reconstitution

of Epstein

of soybean

phospholipid.

AND

out with

either

to be present

performed purified

a-tocopherol, during

with

co-

reconstitution.

AND METHODS

Crude soybean lipids (asolectin) and coenzyme QIo were purchased from Sigma Chemical Co., St. Louis, MO. Chromatographically pure plant phosphatidylethanolamine and phosphatidylcholine and bovine phosphatidylserine were obtained from Avanti Biochemical, Birmingham, AL. Synthetic a-dltocopherol, phylloquinone (vitamin KI),menadione , phytol, and isophytol were gifts from Hoffmann-La Roche, Nutley, NJ. Tocopheryl quinone was prepared by oxidation of 22Na-sodium chloride (carrier-free) a-tocopherol in gold chloride solution (6). was purchased from New England Nuclear, Boston MA. Partially purified soybean phospholipids were prepared by washing asolectin with dry acetone and by extracting with ether (7). Preparation of liposomes, reconstitution of acetylcholine receptor and uptake of 22Na measurements were performed as described All assays were performed at the "10 set" point with carbamylpreviously (5). choline concentration of 2 x 10m4M. Lipid concentrations were 25 mg/ml and the ratio of lipid to protein was 16 (w/w). RESULTS AND DISCUSSION In the original stitution

experiments

of the acetylcholine

extraction

of asolectin of reconstituted

extract,

when added

Purification

of the active

great

in activity

losses neutral

lipid

tion

(vitamin isophytol,

Kl),

was used

for

We have now observed to remove

neutral

was greatly

ingredient

reduced.

difficult,

and inhibition

that

after

(7),

the Na+flux

The neutral

procedure,

proved

handling

lipids

the recon-

restored however,

lipid activity.

because

of Na+ flux

Fig.

1, a-tocopherol

the neutral

Several

other

lipid

quinones

and tocopherylquinone,

and butylated

when

hydroxytoluene

were

at high fraction

concentrations

(20 to

during

the reconstitu-

such as coenzyme

Q, phylloquinone

also

menadione,

active,

but

or hydroxyaniline

had little

phytol, or no

activity. Appreciable vesicles

carbamylcholine-stimulated

reconstituted

of

was used.

replaced

of vesicles.

asolectin

the reconstitution

during

As can be seen from 40%) effectively

acetone

vesicles

during

crude

receptor.

with

activity

excess

(5)

with

Na

phosphatidylethanolamine

410

+

fluxes

were

observed

with

and phosphatidylserine

Vol.

93,

No.

2, 1980

BIOCHEMICAL

AND

Q-Tocopherol Rig.

1.

a ratio

lipids

of

and

when

3:l

The

in

in

the

titrations the

of

1,

reconstituted

the were

these

II).

to 60%

case

necessary

from

concentrations

in crude

Table neutral because

30

to

70% of

Qlo

were

by

a-tocopherol

though

some

u-tocopherol

or

lipid

fractions

from

excesses

markedly

impaired

with

increased during quinones

variability in 40% 10%

of

activity

or

example,

40%

411

the

incorporated

while

vesicles.

batches

markedly

toxic,

I,

highly

different

however,

For

concentrations. was

with

was,

coenzyme

stimulation

u-tocopherol

of

varied

vesicles

This

optimal

shown

activity

or

appropriate

experiments In

toxic.

at

respect Fig.

(% of total lipid wgt.)

ranging

phylloquinone

observed

shown

The

preparations

(Table

with

I).

activity

reconstitution

noted

COMMUNICATIONS

in presence of carbamylcholine without carbamylcholine

(Table

a-tocopherol,

always

influx influx

membrane

asolectin.

RESEARCH

Effect of a-tocopherol on reconstitution of acetylcholine receptor with partially-purified soybean phospholipids. Reconstitution and assay were performed with partially purified phospholipids as described under Materials and Values for crude asolectin were 80 and 25 nmoles Methods. Na+/mg protein with and without carbamylcholine, respectively. # Na+ 0 Na+

in

BIOPHYSICAL

was was

the was

experiments tolerated;

phylloquinone

asolectin, the

was careful

activity

of

PCfPE+PS (1.5:1.5:1)

*2x10

-

-

-

-

19

-

A

M carbamylcholine

15

149

87

17

39

14

+carb*

34

10

21

10

10

104

-carb Na+

30

115

36

10

195

10

influx,

+carb

20

-

-

-

174

-

A

I

total

31

29

25

25

35

10

protein

+carb

-

21

-

-

-

25

A

-carb

12

15

10

18

20

10

47

16

18

43

147

10

+carb

a-Tocopherol (20%)

weight

of

-

35

-

-

25

127

A

lipid was constant and reconstitution and Methods. Na+ influx for asolectin and without carbamylcholine, respectively.

10

27

16

25

10

10

nmoles/mg

-carb

lipid

on reconstitution purified phospholipids

Phylloquinone (10%)

% of

tocopherol highly

Sum of weight of phospholipid and neutral was performed as described under Materials were 74 and 23 nmoles Na+/mg protein with

-4

20

138

96

PC+PS(3:1)

PC

20

16

PE+PS(3:1)

PC+PE(l:l)

19

PE

-carb

Phylloquinone (5%)

lipid,

Neutral

NOW2

and with

Effect of phylloquinone acetylcholine receptor

TABLE

24

10

28

10

24

10

and assay vesicles

-carb

89

18

114

10

40

10

+carti

a-Tocopherol 140%)

65

-

86

-

16

-

A

s.-

Vol.

93, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

TABLE II Effect of a-tocopherol, phylloquinone and coenzyme Ql, on reconstitution of the acetylcholine receptor with phosphatidylethanolamine (PE) and phosphatidylserine (PS) at a ratio of 3:l. Na+ influx (nmoles x 10 set -1 x mg protein-l) - carb + carb* a

% of total lipid weight Asolectin

21 24

111 113

a9

PE + PS (3:l)

12 19

48 50

33

+ a-Tocopherol

10 20 30 40

17 25 24 21

123 198 196 204

106 173 172 183

+ Phylloquinone

2 4 6 8 10

24 18 30 29 26

92 157 198 138 128

68 139 168 109 102

4 6 8

20 31 32

144 167 207

124 136 175

+ Coenzyme Q,,

-~*

2 x 1o-4 M carbamylcholine

Desensitization curare

(5 )

was observed

with

of carbamyl-sensitive

proteoliposomes

Na+ flux

and sensitivity

reconstituted

with

purified

or hydroxyaniline

were

ineffective,

to

lipids

and quinones. Since

butylated

seems unlikely

hydroxytoluene

that

a-tocopherol

centration

of a-tocopherol

that

these

neutral

This

interpretation

required black

lipid

lipids

affect supported

stitution

of the vesicles

results.

This

between

an effect

activity

'of the

approach

that

Attempts

needs

to incorporate completed

to be explored

of the quinones

have not further

on the reconstitution

receptor

protein

413

(4).

con-

reconstitution

of the phospholipid

by the observation

has been

reconstituted

for

suggest bilayer.

a-tocopherol

is

Material

into

of the Excitability-Inducing

(9).

The high

(8).

required

the packing

incorporation

membranes

as an anti-oxidant

or the quinones

is

Eor the

acts

it

the quinone

after

yielded

reproducible

in order process

also

recon-

to differentiate or on the

Vol.

93, No.

2, 1980

BlOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGEMENTS This work was supported by Grant CA-08964 (E.R.), awarded by the National Cancer Institute, DHEW and Grant GM-25256 (P.M.) awarded by the Institute of General Medical Sciences, DHEW. We thank Mrs. P. Lewis for technical assistance.

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9.

Briley, M.S. and Changeux, J.P. (1978) Eur. J. Biochem. 84, 429-439. Chang, H.W. and Bock, E. (1979) Biochemistry 18, 172-179. Andreasen, T.J., Doerge, D.R., and McNamee, M.G. (1979) Arch. Biochem. Biophys. 194, 468-480. Racker, E. (1977) J. Supramolecular Structure 6, 215-228. Epstein, M. and Racker, E. (1978) J. Biol. Chem. 253, 6660-6662. Karrer, P., Escher, R., Fritzoche, H., Keller, H., Ringier, B.H., and Salomon, H. (1938) Helv. 21, 939-953. Kagawa, Y. and Racker, E. (1971) J. Biol. Chem. 246, 5477-5478. Bieri, J.G. and Farrell, P.M. (1976) Vitamins and Hormones 34, 31-75. Mueller, P. and Rudin, D.O. (1968) J. Theoret. Biol. 18, 222-258.

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