Isolation of phospholamban and a second proteolipid component from canine cardiac sarcoplasmic reticulum

Vol. 99, No. 3,198l April

BIOCHEMICAL

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15, 1981

Pages 796-803

ISOLATION

OF PHOSPHOLAMBAN AND A SECOND PROTEOLIPID

COMPONENT FROM CANINE CARDIAC SARCOPLASMIC RETICULUM John H. Collins, Evangelia Louise M. Bilezikjian Department University

Received

January

G. Kranias, and Arnold

Anita S. Reeves Schwartz

of Pharmacology and Cell Biophysics of Cincinnati College of Medicine 231 Bethesda Avenue Cincinnati, Ohio 45267

27,1981

Summary: We have isolated two proteolipid fractions from canine cardiac sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B, and Sephadex LH-60. One, "fraction B", is phosnhorylated by cyclic AMPdependent protein kinase and was identified as phospholamban, the activator of cardiac sarcoplasmic reticulum. The other, "fraction A", is not phosphorylated and has an amino acid composition very similar to those of proteolipids we previously isolated from (Na,K)-ATPase. Introduction: The Ca2+-ATPase stimulated,

of cardiac

upon phosphorylation

of a small,

has been named phospholamban phospholamban

tein,

further

kinases

is also

by an endogenous, kinase.

pholamban dence

protein

phospholamban

protein

It

According

this

form

reported

that

,

IAbbreviations: monophosphate;

by either (2-5).

phospholamban

SR, sarcoplasmic

it

(6,

however,

gel

has been sites

796

that years

that

reported

(6-8)

on the pro-

calmodulin-stimulated electrophoresis, presented

subunits.

a proteolipid.

reticulum,

several

has recently

9) of Mr%ll,OOO is

protein

is

or exogenous

Ca 2+ -dependent,

dodecylsulfate.

:Z> 1981 b-v Academic Press, Inc. in any form reserved.

of reproduction

endogenous

to SDS-polyacrylamide

is a dimer

for

at different

0006-291X/81/070796-08$01.00/0 Copyright All righis

known

Recently,

phosphorylated,

One group,

SDS, sodium

and Ca*+ transport

membrane-bound

has been

membrane-bound,

has Mr%22,000.

that

(1).

can be phosphorylated

@&'-dependent that

SR1 is activated,

CAMP, cyclic

These

Proteolipids adenosine

phoseviauthors are very 3',5'-

BIOCHEMICAL

Vol. 99, No. 3,198l

hydrophobic

proteins

methanol

form: covalently lipid that

suggested

the

for

Experimental

(12).

isolation

SR, one of which

was being

isolated

not

The finding be related

from

lamb kidney here

of two proteolipid we have

identified

completed,

Le Peuch,

--et al.

phospholamban

which

differs

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solubility

all, that

might

We describe

RESEARCH

by their

in some, but

(11). it

BIOPHYSICAL

characterized

(10);

to us that

procedures for

are

lipids

we had recently

cedures

tion

mixtures

attached

tographic

diac

which

AND

cases

they

from

is a proteo-

to the Mrs12,000

proteolipids

(Na,K)-ATPase

by column

the application

of these

components

published that

contain

phospholamban

from canine

as phospholamban. (13)

in chloro-

reported

While

an amino

acid

chromapro-

carthis

work

composi-

here.

Procedures:

SR from dog cardiac muscle was prepared by a modification of the Harigaya and Schwartz procedure (14) as previously described (15). The final yield of SR protein ranged from 0.6 to 0.8 mg per gram of wet cardiac tissue. The purity and homogeneity of the preparations was checked by SDS-polyacrylamide gel electrophoresis and by various enzyme marker activities. Sarcolemmal and mitochondrial contamination were low, as judged from 5'-nucleotidase (0.043 + 0.015 pm01 mg-l min- l), H3-ouabain binding (4-10 pmoles/mg) and cytochrome 2 oxidase (0.0058 pmol mg-l min -I-) activities. The SR Ca2+-ATPase (50 pg of protein/ml) was assayed in 25 mM histidine buffer (pH 7.0) containing 5 mM MgC12, 100 mM KCl, 5 mM NaN3, 100 UM EGTA and 100 uM CaC12 (10 pM free Ca2+) and 5 mM ATP; the specific activity was 0.67-1.00 pmol Pi mg-l min -I at 37'C. The rate of Ca2+ uptake was determined at 20°C in 40 mM Tris/maleate buffer (pH 6.8) containing 100 mM KCl, 10 mM MgC12, 5 mM NaN3, 5 mM Trisoxalate, 5 mM ATP, 100 JJM EGTA, and 100 pM CaC12 (10 pM free Ca2+) and found to be 0.33-0.42 pmol mg-1 min -1.

Phosphorylation of SR was carried out at 30°C for 5 min in 50 mM phosphate buffer (pH 7.0) containing 10 mM MgC12, 10 mM NaF, 1 mg/ml SR vesicles and 500 pM ATP (including [v-~~P]ATP), in the presence of CAMP (1 uM) and CAMP-dependent protein kinase (0.1 mg/ml). The reaction mixture was then centrifuged at 105,000 x g for 30 minutes. The pellet was homogenized gently in ice-cold 20 mM Tris/maieate buffer (pH 6.8) containing 100 mM KC1 to a concentration of 7-12 mg protein/ml. Then solid SDS (10 mg per mg of protein) and B-mercaptoethanol (final concentration 1%) were added, and the mixture was stirred for 1 hour at 37'C. The solubilized SR was then dialyzed overnight at room temperature vs column buffer (see Fig. 1) and 40-100 mg of protein, at a concentration of 2-4 mg per ml, was applied to a 5 x 195 cm column of Sepharose CL-6B. Fractions from this column were lyophilized, and the 32P-containing fraction was dissolved in 3-4 ml of 88% formic acid and applied to a 2 x 195 cm column of Sephadex LH-60. The LH-60 column was run in a 3:l (v/v) mixture of 95% ethanol and 88% formic acid, a solvent very similar to that developed by Gerber, --et al. (16) for separation of hydrophobic peptides.

797

Vol. 99, No. 3,198l

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AND

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1000

1:

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2000 Elution

Figure

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Volume,

ml

Separation of the components of soluhilized canine cardiac SR on a 5 x 195 cm column of Sepharose CL-6B. Solvent: 5 mM sodium phosphate, pH 8.0, containing 0.1% SDS, 1 mM 2-mercaptoethanol and 0.01% sodium azide. Fractions of 10 ml each were collected at a flow rate of 80 ml/hr. Horizontal bars indicate fractions pooled.

Amino acid analyses were carried out on a Glenco MM-60 amino acid analyzer by standard procedures. Samples for analysis were evacuated and hydrolyzed at 1lO'C for 20 hr in 6N HCL containing 0.1% phenol. Phospholipids were detected by the presence of large amounts of serine and ethanolamine in hydrolyzed samples. ethanolamine is eluted between (On our amino acid analyzer, lysine and ammonia. Accurate quantitation of lysine was not possible in the presence of a large excess of ethanolamine). Protein concentrations were determined by amino acid analysis. SDS-polyacrylamide gel electrophoresis was carried out according to the method of Laemmli (17).

Results

and Discussion: The results

CL-6B

are

of gel

shown

in Fig.

and has been obtained tions

indicated

amide

gel to

protein

recovered

be

the

accounted

for

with

that

seven

and

stained essentially

5% of with all

the

total

Coomassie of

the

large

accounted major

blue

recovered after

phosphoprotein.

798

Fraction for

component

peak at the void

protein

reproducible Each of

analysis.

(18),

a single

E, the

is very

on 0.1% SDS-7.5%

acid

protein

SR on Sepharose

preparations.

by electrophoresis

contained

Fraction

cardiac

pattern

different

and by amino

Ca'+-ATPase

only

of solubilized

The separation

was examined

of MrQOO,OOO.

contained

1.

electrophoresis

presume

ponents

filtration

volume and

electrophoresis.

polyacrylA, which

33% of (at

the frac-

least

the

we

total

90% pure)

of the column,

contained

no

com-

Fraction

D

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Vol. 99, No. 3,198l

AND

BIOPHYSICAL

RESEARCH

0.6

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2400

0.4 .!. 8 UN 0.3

02

0.1

0

0

100

200

Elution

Figure

2:

The results

of Sephadex

material

designated

A and B, were

is

that

we observe

gels

were

stained

with

be observed

tion

is

not

Extremely on stained

homogeneous.

kidney

solvent Na,K-ATPase

that

band

bands

as phospho-

on autoradiograms

B again

of Figure

we used previously

SR.

a very

weakly

the

(not

that

shown)

1 with

same

When the

a single staining

and Mr%20,000

could

this

frac-

we extracted

the chloroform-

to isolate

proteolipids

When the chloroform-methanol

extract

799

is

showed

A, indicating

experiments

fraction

This

of Mrs15,000

of fraction

fractions,

of 32 P-labeled

A revealed

In earlier

(12).

of Mr%22,000

fraction

fraction

gels

proteolipid

electrophoretograms.

blue,

faint

the phosphoprotein-

B was identified

electrophoresis

while

the phosphoprotein-containing methanol

after

of

Two major

Fraction

gel

Coomassie

band of Mr~22,000,

also

2.

of a single

(7)

band of r~ll,OOO.

chromatography

obtained.

polyacrylamide

band

major

LH-60

shown in Figure

by the appearance

of 0.1% SDS-12.5%

(ml)

Chromatography of fraction D from Figure 1 on a 2 x 95 cm column of Sephadex LH-60. Solvent: 88% formic acid:95% ethanol (1:3, v/v). Fractions of 3 ml each were collected at a flow rate of 20 ml/hr. Horizontal bars indicate fractions pooled.

containing

lamban

Volume

from from

lamb

cardiac

Vol. 99, No. 3,198l

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AND

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RESEARCH

TABLE I Amino Acid Compositions of Proteolipids Cardiac Sarcoplasmic Reticulum.a Fraction Asp Thr Ser GlU Pro GlY Ala Vsl Met Ile Len Tyr Phe LYS His ArR

A

Fraction

a.24 5.66 7.05 11.2

5.88

Yield=

residues per 100 shown in Figure . Cys not , gal., ~g recovered per

obtained

the fractions

A and B shown

fraction

A were

Figure

2 are

contains

most

protein indication

The low

of homogeneity. and then

composition

did

not

composition

acid,

glycine,

histidine Most Recently,

6.5 1.7 4.9 9.5 2.1 3.4 4.9 2.0 4.4 5

residues. Fractions A and B 2. Cys and Trp were not determined. present., Trp not determined (13). mg of SR protein.

using

the

in Figure

same solvent,

2, although

I.

compositions It

of the phosphoprotein,

was pooled,

ferent

LH-60

acid

in Table

recovered.

11.0

we also

the yields

of

50% lower. and amino

listed

5.5 5.5 11.4 5.5 10.6

10

on Sephadex

The recoveries

11.1

8.63

SR was chromatographed

about

"Phospholamban ,tb

B

co.1

46

"Expressed as bare the peaks From Le Peuch CExpressed as

from

a.93 4.79 5.75 16.7 2.34 1.85 8.55 5.08 5.90 9.77 13.3 2.29 3.68 2.51

7.69 8.22 5.69 2.33 4.68 10.6 3.57 5.42 6.65 2.30 4.89

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is

noteworthy

represents

content

methionine,

than

third

B, particularly

B, which

20% of the

in fraction

Fraction

total

B is a further

of the

fraction

its

amino

B peak acid

A has a distinctly in

the content

and the basic

isoleucine,

A and B of

fraction

on the same column,

significantly.

fraction

less

the middle

rechromatographed

from

that

of histidine

When only

change

of fractions

amino

acids

dif-

of glutamic lysine,

and arginine. groups however,

report

that

Le Peuch,

phospholamban et al.

(13)

800

is

a nrotein

reported

of

Mr~22,000.

the purification

of canine

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vol.99,No.3,1981

AND BIOPHYSICAL

RESEARCH

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TABLE II Statistical

Comuarisons

with

Sarcoplasmic

of Reticulum

Proteolipids Associaied and (Na,K)-ATPase.

aIndividual differences in moles% for each amino acid (excluding Cys and Trp) were squared and summed to obtain Lower numbers are indicative the tabulated values (20). of greater similarity in amino acid composition, with values below 50 indicating a high probability of amino acid sequence homology. bPractions A and B of cardiac SR are the peaks shown in Figure 2. CFrom canine cardiac SR (13). dThe 11,700-dalton phosphorylated protein isolated from beef eThe two proteoheart (Na,K)-ATPase by Dowd, et al. (19). lipid fractions from lamb kidney (Na,K)-ATPase (12).

cardiac In

phospholamban

the

first

dimeric the

duce

dimension,

form

second

after

of phospholamban)

ferent

--et al.

from

It

is

material

able

electrophoresis;

In with

the

I> is

compared II

the

"phospholamban"

similar

A, plus

their

to fraction acid of

Le

The reason

our

isolated

for

this

of dif-

of material

B, and perhaps

on Sephadex Coomassie that

Sur-

discrepancy

mainly

of fraction

finding

to repro-

A and very

consists

with

For

preparation

fraction

by chromatography

with

was

the

electrophoresis.

"phospholamban"

amount

preparation

form

call

not been able

on gel

preparation

a small

consistent

amino

of SDS.

to our B.

their

we have removed

is

in the presence

We have

of the

fraction that

to stain this

Table

Table

fraction

which

not

weakly

composition

possible

to our

They were

very

(see

they

Mr%ll,OOO

and SDS.

technique.

of Mrs22,000(which

of phospholamban

our phosphorylated

is unknown. equivalent

acid

electrophoresis

monomeric,

X-100

dissociation

the amino

Le Peuch,

was isolated

in Triton

apparent

gel band

the presumed

dissociation this

a two-dimensional a radioactive

dimension,

prisingly,

other

by

blue

fraction

LH-60. after

gel

A stains

B. compositions Peuch

-et --al

801

of

(131,

fractions

A and

two proteolipids

B are

compared

(~1 and y2)

Vol. 99, No. 3,198l

of Mrsl2,OOO

BIOCHEMICAL

that

and a Mr~l1,700 bovine

cardiac

fraction

we recently

isolated

phosphorylated

protein

(Na,K)-ATPase

B appear

amino

acid

AND

(19).

to be very

sequence

to unambiguously

homology

intersting

to compare

associated

with

their

lamb kidney

all

(Na,K)-ATPase

function

any speculation

purity.

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from

further

Nevertheless,

sequences

except

regarding

since

it

of these

(12),

isolated

of the proteolipids

be premature

acid

RESEARCH

of unknown

similar,

the amino

membrane

from

While

would

establish

BIOPHYSICAL

possible work

will

is

needed

be very

and other

proteolipids

ATPases.

Acknowledgements: This tion,

work

a grant

Association, grants

from

by a grant

the Southwestern

by grant

AM-20875,

recipients

was supported

78-1167

HLr22619(IIB,

of NIH Research

Ohio

from

Chapter

of the American IVA)

Career

the Muscular

Development

of the American

Heart

and HL-07382,

Dystrophy

Association, J.H.C.

Heart and by NIH

and E.G.K.

Awards.

References: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Tada, M., Kirchberger, M.A., Iorio, J.M,, and Katz, A.M. (1973) Circulation 48: Suppl. 4, 25. LaRaia, P.J., and Morkin, E. (1974) Circ. Res. 35: 298-306. Wray, H.L., and Gray, R.R. (1977) Biochim. Biophys. Acta 461: 441-459. Schwartz, A., Entman, M.L., Kaniike, K., Lane, L.K., Van Winkle, W.B.. and Bornet, E.P. (1976) Biochim. Biophys. Acta 426: 57-72. Kirchberger. M.A., and Tada, M. (1976) J. Biol. Chem. 251: 725-729. (1979) Biochem. Le Peuch, C.J,, Haiech, J., and DeMaille, J.G. 18: 5150-5171. Kranias, E.G., Bilezikjian, L.M., Potter, J.D., Piascik, M.T. and Schwartz, A. (1980) Ann. N.Y. Acad. Sci.: in press. Bilezikjian, L.M., Kranias, E.G., Potter, J.D. and Schwartz, A. (1980) Fed. Proc. 39: 1663. Le Peuch, C.J., Le Peuch, D.A.M., and DeMaille, J.G. (1980) Fed. Proc. 39: 1817, Folch, J., and Lees, M, (1951) J. Biol. Chem. 191: 807-817. Folch-Pi, J., and Sakura. J.D. (1976) Biochim. Biophys. Acta 427: 410-427. (1980) Biochem. Reeves, A.S., Collins, J.H., and Schwartz, A. Biophys. Res. Commun., 95: 1591-1598. (1980) Le Peuch, C.J., Le Peuch, D.A.M., and DeMaille, J.G. Biochemistry 19: 3368-3373.

802

Associa-

are

Vol. 99, No. 3,198l

14. 15. 16. 17. 18. 19. 20.

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Harigaya, S., and Schwartz, A. (1969) Circ. Res, Sumida, M., Wang, T., Mandel, F., Froehlich, J.P., (1978) J. Biol. Chem. 253: 8772-8777. Gerber, G.E., Anderegg, R.J., Herlihy, W.C., Gray, and Khorana, H.G. (1979) Proc. Natl. Acad. Sci., 227-231. Laemmli, U.K. (1970) Nature 227; 680-685. Van Winkle, W.B., Pitts, B.J.R., and Entman, M.L. Chem. 253: 8671-8673. Dowd, F.J., Pitts, B.J.R., and Schwartz, A. (1976) Biophys. 175: 321-331. Weltman, J.H., and Dowben, R.M. (1973) Proc. Natl. 70: 3230-3234. U.S.A.

803

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25: 781-794. and Schwartz,

A.

C.P., Biemann, U.S.A. 70: (1978)

J.

Biol.

Arch.

Biochem.

Acad.

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H.