Single-phase butanol extraction: A new tool for proteolipid isolation

Vol. 74, No. 1, 1977

BIOCHEMICAL

SINGLE-PHASE

BUTANOL EXTRACTION:

A NEW TOOL FOR PROTEOLIPID

Hans Sigrist: Kristine Sigrist-Nelson*=& of Membrane Research, Weizmann Institute

Departmsnt

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

November

ISOLATION

Carlos Gitler of Science, Rehovot,

Israel

2,1976

SUMMARY

An alternative method to the proteolipids is presented. plasmic reticulum membranes extraction and subsequently step procedure described is of the dicyclohexylcarbodi-imide particles.

chloroform/methanol extraction of The proteolipid fraction from sarcois isolated by a single-phase n-butanol precipitated by diethyl ether. The two successfully applied in the purification binding protein from submitochondrial

INTRODUCTION The isolation pered ion

by the absence methods

system is

of suitable

utilizing

organic

was always

extraction

of highly

using

present.

as a pronounced

lipid-protein

interactions

of aqueous

solutions,

of the solubilized lipid binding

from

describes for

isolated

membrane

for

phase

system,

technique

favors detailed

and facil

two step

extracta biphasic proteolipid n-Butanol

system.

components,

and the mitochondrial

in a rapid

classical

a new technique

of n-butanol

a two-phase

By means of the

has been ham-

(l-6),

membrane

The miscibility

While

often

employed

in a single

agent

forming

reticulum

have been

techniques.

solvent

(7-9). without

sarcoplasmic

protein

as organic

proteins

have been widely

report

solubilizing

fraction.

membrane

nonaqueous solvents

This

n-butanol

known

hydrophobic

with an here

disorganizing small

amounts separation

easy

the proteo-

membrane

IXCD-

procedure.

METHODS AND MATERIALS Sarcoplasmic reticulum membranes were prepared from rabbit skeletal muscle (10) in the presence of $uM phenylmethylsulfonyl fluoride. The final membrane fraction was suspended in 1OOmM KCI, 5mM imidazole pH 7.4 and stored at -7OOC. Before isolation of the proteolipid fraction, sarcoplasmic reticulum membranes (15-20 mg/ml) were sedimented by centrifugation at 24,000 g for 60 minutes. The membrane pellet was suspended in 5mM CaC12 or water, conserving the initial protein concentration. Isolation of rat liver mitochondria was carried out according to Johnson and Lardy (11). Submitochondrial particles were prepared with a Branson sonifier in a medium containing 21nM EDTA pH 8.5 and were subsequently twice washed (105,000 g, The iso30 minutes) with a buffer containing 25OmM sucrose, 1OmM Tris-HCl pH 7.5. lated membranes were stored at -7OOC. Submitochondrial particles were incubated ATP'ase activity is dewith DCCD (2jug per mg protein) for 18 hours at O°C (6,12). fined and assayed as in reference 6 in the absence of an ATP regenerating system. The labeled membranes were washed twice with sucrose-Tris buffer and collected by Abbreviations: DCCD - N,N'-Dicyclohexylcarbodi-imide SDS - Sodium dodecylsulfate *Swiss

National

Science

Foundation

Copyright 0 I977 by Academic Press, Inc. All rights of reproduction in any form reserved.

fellows 178

Vol. 74, No. 1, 1977

Fig.

BIOCHEMICAL

1. Schematic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

representation

of the proteolipid

isolation

procedure.

centrifugation at 105,000 g for 30 minutes. The final pelleted membranes were suspended in water (6-8mg/ml). Protein was determined in the presence of 0.1% SDS according to Lowry et. al (13). Samples containing organic solvents were dried under nitrogen before assaying for protein. Total phosphate was measured by the method of Chen et. al (14). SDS gel electrophoresis was carried out in accordance with Weber and Osborn The electrophoresis was performed in 0.1% SDS, 50mM sodium phosphate buffer (15). pH 7.2 using 10% polyacrylamide gels. Gels were stained with coomassie brillant blue and destained with 7% acetic acid. For molecular weight determinations bovine pancreatic insulin (MW 5,700) and egg white lysozyme (MW 14,300) served as standards in the co-electrophoretic analysis. When "C-LECD-labeled samples were electrophoressamples were applied to separate gels. One gel was stained; the ed, identical duplicate gel was immediately cut into 2mm slices. The individual gel slices were extracted overnight with 1OmM Triton X-100. Scintillation fluid was added and the slices were courted for radioactive content. All chemicals and reagents were of the highest purity commercially available. "C-DCCD (45tii/n@l) was a generous gift of Dr. H.R. Kaback, Roche Institute of Molecular Biology, Nutley. RESULTS Isolation

procedure: The isolation

n-butanol

followed

The widely Step

I:

by addition

applicable

Using formed

at 12,000 Step cooled

II:

is

ether

described

concentration

of the membrane to the butanol

suspension

fraction

into

(fig.

1).

as follows:

the membrane

is separated

suspension

of the aqueous by repeated

is

injected

component

centri.fugation,

is

into 2%.

usually

n-butanol The immedithree

times

10 minutes.

The n-butanol supernatant is The finely dispersed

to -2OOC.

when centrifuged

injection

of diethyl

syringe

Final

precipitate

g for

entails

procedure

a tuberculin

at room temperature. ately

procedure

at 12,000

g for

combined with 5 volumes precipitate (proteolipid

10 minutes

179

(4OC).

of diethyl fraction)

The sedimentation

ether, presediments of the sarco-

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

P

B

0

I. 2. Protein patterns of fractions obtained during the sarcoplasmic reticulum proteolipic isolation. SDS gel electrophoresis, staining and destaining were performed as described in METHODS. The amount of protein applied is given in brackets. precipitate (4Oug); A. Sarcoplasmic reticulum membranes (60,ug); B. Butanol C. Ether precipitate (proteolipid) (5,ug); D. Insulin (12,ug).

plasmic

reticulum

diethyl drial

ether,

proteolipid whereas

proteolipid

Sarcoplasmic

is

fraction

(fig.

proteolipid

lipid,

2). could

The proteolipid

All

performed

the n-butanol/diethyl stored

ether

purification

major

proteins

be identified

in

was present

periments the amount

reduced it

was noted of proteolipid

mixture

after

containing

the addition

of

the mitochon-

at -2OOC overnight.

in

was routinely

extent that

followed

of the sarcoplasmic

the gel gels

pattern

in gels

made of the ether

of low into

precipitate. concentrations When Ca++

butanol.

180

precipitate

by SDS gel

electrophore-

excepting precipitate (fig.

the proteo(fig. ZC).

ZB). In the

of 9,000 daltons was determined. was present in all gels although

of the butanol

the presence solubilized

reticulum,

of the butanol

gel system employed, an apparent molecular weight A faint band attributable to the lipid components to a greatly

immediately

fraction:

The proteolipid sis

is

In preliminary

ex-

(5mM) of Ca++ increased was absent

during

the ad.

Vol. 74, No. 1, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Isolation

of proteolipid

and carbodi-imide total

fractions

- treated

protein

from sarcoplasmic

submitochondrial

Sarcoplasmic

Ether

Protein

reticulum

precipitation: precipitation:

dition

precipitation:

of sarcoplasmic

were detected

in

The recoveries proteolipid

,umoles

%

membranes

78.02

100.0

58.01

100.0

Precipitate

75.01

96.2

2.93

5.0

Superna tant

1.03

1.3

53.47

92.1

Precipitate

0.12

0.15

0.44

0.7

Supernatant

2.40

3.0

54.38

93.5

6.03

100.0

4.65

100.0

5.77

95.6

0.50

10.7

fectively

precipitated

total ly tent

in

treated

in

successful

3.65

78.4

to n-butanol,

small

amounts

membranes protein

of ether

served

and total

the ether

lipids

II

of proteolipid

96% being

butanol

in

not be detected

with

fraction

fig.

ef-

the butan-

of sarcoplasmic

particles supernatant.

94% of were

the

identical-

The protein

con-

due to the simultaneous

the Lowry

was confirmed

(see also

in

leaving

overestimated

to interfere

in this

n-butanol

supernatant

the ether

probably

of the

submitochondrial present

fraction

When submitochondrial most

the isolation

electrophoresis,

the proteolipid remained

is

known

of protein

could

by gel

to the separated

phosphate

during

and the DCCD-labeled

approximately

supernatant. step

phosphate

reticulum

to precipitate

proteolipid

viously-characterized

5.4

protein,

the absence

purification

0.32

As demonstrated

of concentrated

The versatility

Supernatant

all

in

Mitochondrial

9.4

almost

total

bands

83.8

0.44

I.

78% of the

ditionally, where protein

3.90

1.9

sarcoplsamic

of the supernatant

presence

3.4

0.12

table

Addition

membranes

phosphate

0.20

Precipitate

both

from

listed

01 precipitate. reticulum

Supernatant

precipitate.

of

fraction are

Phosphate %

reticulum

the butanol

particles

of

w

DCCD-treated submitochondrial particles Butanol precipitation: Precipitate Ether

membranes

Recoveries

and phosphate.

Fraction

Butanol

reticulum

particles:

determination by gel

(16).

electrophoresis

3C).

fraction: of the proteolipid of

isolation

the DCCD-binding

as a proteolipid

procedure

protein

by Catell

181

was demonstrated

from mitochondrial et.

al.

(5).

Under

membranes the described

by the precon-

Ad-

Vol. 74, No. 1, 1977

BfOCHEMfCAL

AND BlOPHYSfCAL RESEARCH COMMUNfCATfONS

30

00

00

10

20

Slice

Fig.

1 I 30

40

number

protein from submitochondrial particles: 3. Isolation of the 14 C-DCCD-binding ibution of protein and radioactivity in polyacrylamide gels. ,"f"B C-CCCD-treated submitochondrial particles (40,ug); B. Butanol precipitate (proteo C. Ether supernatant (equivalent to 4pg); D. Ether precipitate (32p9l; lipid) (Spg) . 182

BIOCHEMICAL

Vol. 74, No. 1, 1977

ditions

nonradioactive

the ATP'ase out

inhibitor.

(6)

and Cattell

drial ly

reported

ive, ing

(5).

DCCD-labeled

protein

being

electrophoresis.

in

DCCD-binding

of 11,000

daltons;

ref.

daltons

(fig.

complete

As previous-

totally 3B).

(fig.

was found

quantitat-

Protein

(fig.

(molecular

of the

30)

all

weight

weights

subjected

at the posit-

precipitation

molecular

band-

3C) were

on the gels

precipitate

was determined

5, 13,000-14,000

super-

the submitochondrial

was not

supernatant

al.

of submitochon-

band in

An apparent

band.

et.

and ether

precipitate

confirming

protein

by Steckhoven

analysis

proteolipid

In the ether

with-

14C-DCCD labeling.

one protein

the butanol

inhibited

suspension

(proteolipid),

no radioactivity

by ether.

reported

SDS gel

of the ether

mobility,

the corresponding

protein

6, 10,000

in

samples

relative

radioactivity

was present

found

protein)

to a membrane

of corresponding

selectively

Moreover,

of the proteolipid's

protein-bound

3 presents precipitate

of the DCCD-binding

when concentrated

2/ug/mg membrane

the data

ether

highly

Extraction

with

Figure

as the patterns

DCCD labeled 3A).

was absent

(both

compared

are in agreement

precipitate,

as well

(fig.

to SDS gel ion

butanol (5,6)

particles

results

and co-workers

fractions,

as 14C-IXCD

97% and 98% respectively,

These

particles,

natant

DCCD as well

activity,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

radioactivity for

the

reported

in ref.

daltons).

DISCUSSION: In various functions

(17-19),

DCCD-binding

in

fraction.

of membrane simplicity

proteins

transmembrane

with

submitochondrial

rapidity

times.

n-Butanol,

of all

globular

(5,6)

a weakly

method

and purity

protic

solvent

The use of a single-phase

extraction

additionally

two-phase

denaturated

systems

in

that

and resulting

such as the proteolipids a nonaqueous

of the proteolipid greater

extent

described

environment.

to an aqueous than

by other

be used to form injected Indeed,

Since

preliminary

studies

interfacial here

phase,

treatments.

liposomes

(20)

by the present

and lipids protein

is

183

procedure

of endogenous utilizing

rapid

(2-4)

of the

or

preparation precipitation

the organic

phase.

classical

hydrophobic

this

extensive (step

with exposure

may be retained

supernatant lipid

complexes

upon associations avoids

conformation butanol

procedures

absent.

dependent

extraction

authors

into

of highly

may be critically

composed

Other

and longer

the problems

properties

Concentrated

class

of

membranes

was used to effect

the functional

of this

the advantages

techniques

avoids

functional the present

presents reticulum

the proteolipids

Conformation

here

more laborious

nonaqueous

of

was developed. fractions.

extracting

the significance aspects

sarcoplasmic

proteins,

of a

of the sarcoplasmic

(4) emphasize

of the isolated

from either involve

The identification

and structural

described

respiratory-linked

involvement

translocation

isolation

impared

permeability.

the functional

procedure

extraction

particles

to restore

proton

ion

study

isolation

proteolipid

reported

(5) and the possible

an alternative

combined for

by decreasing

To further

The proteolipid reported

DCCD is

as a proteolipid

proteolipid

protein

systems

presumably

protein

reticulum this

membrane

to a I)

can

and proteolipid. technique

indicate

that

Vol. 74, No. 1, 1977

the proteolipid vestigations data

from for

Finally, plast

sarcoplasmic

on the butanol

reported

been further

BIOCHEMICAL

extracted

the ionophoric the general

demonstrated

membranes

reticulum

applicability personal

does possess

proteolipid

activity

may

of the of

by the successful

( N. Nelson,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

help

ionophoric

activity.

to elucidate

sarcoplasmic

reticulum

the proteolipid

isolation

isolation

of a proteolipid

Further

in-

the conflicting proteolipid technique fraction

(2,4,21) has from

chlorog

communication).

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Folch, J. and Lees, M. (1951) J. Biol. Chem. 191, 807-817. MacLennan, D., Yip, C., Iles, G. and Seeman, P. (1972) Cold Spring Harbor Symp. Quant. 37, 469-478. Biol. P. and Barrat, M. (1975) Arch. Biochem. Biophys. 170, 92-101. Lawn=-, Racker, E. and Eytan, E. (1975) J. Biol. Chem. 250, 7533-7534. Cattell, K. J., Lindop, C. R., Knight, I. G., and Beechey, R. B. (1971) Biochem. J. 125, 169-177. Steckhoven, F. S., Waitkus, R. F. and van Moerkerk, H. (1972) Biochemistry 11, 11441150. Maddy, A. H. (1972) Biochim. Biophys. Acta 288, 255-262. Lenaz, G., Parenti-Castelli, G., Sechi, A. M. and Masotti, L. (1972) Arch. Biochem. Biophys. 148, 391-399. Lockshin, A. and Burris, R. H. (1966) Proc. Natl. Acad. Sci. (USA) 56, 1564-1570. Martonosi, A. (1968) J. Biol. Chem. 243, 71-81. Johnson, D. and Lardy, H. (1967) Methods in Enzymology 10, 94-96. Robertson, A. M., Holloway, C. T., Knight, I. G. and Beechey, R. B. (1968) Biochem. J. 445-456. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem, 193, 265. Chen, P. S., Toribara, T. and Warner, H. (1956) Anal. Chem. 28, 1756-1758. Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412. Eichberg, J. and Mokrash, L. (1969) Anal. Biochem. 30, 386-390. Racker, E. and Horstman, L. L. (1967) J. Biol. Chem. 242, 2547-2551. McCarty, R. E. and Racker, E. (1967) J. Biol. Chem. 242, 3435-3439. Patel, L., Schuldiner. S. and Kaback, H. R. (1975) Proc. Natl. Acad. Sci. (USA) 72, 3387-3391. Batzri, S. and Korn, E. D. (1973) Biochim. Biophys. Acta 298, 1015-1019. Laggner, P. and Graham, D. E. (1976) Biochim. Biophys. Acta 433, 311-317.

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