Identification of bovine brain Ca2+-binding proteins

Vol.

128,

May

16.

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1985

IDENTIFICATION David

M.

Waisman*,

Maasaki

Department

Received

OF BOVINE

of

BRAIN

Ca2+-BINDING

Tokuda, Stephen and Tim Clark

Medical Calgary,

J.M.

1138-1144

PROTEINS

Morys,

Leona

Biochemistry University Alberta T2N 4Nl CANADA

of

T.

Buckland

Calgary,

2, 1985

April

In a previous communication (Waisman, D.M., Smallwood, J.I., Lafreniere, D. 116, 435-441) we reported that and Rasmussen, H. (1983) Biochem. Biophys. Res. Commun. chromatography of bovine brain 100,000 x g supernatant oniethylaminoethyl (DEAE) cellulose and analysis of resultant fractions by chelex competitive calcium binding assay, resolved three peaks of calcium binding activity. Gel permeation chromatographic analysis of each peak resolved apparent Mr 40,000 (Peak I), Mr 75,000, Mr 230,000 and In the present communication the calcium Mr 420,000 (Peak II), and M, 38,000 (Peak III). binding proteins responsible for the calcium binding activity peaks resolved by gel permeation chromatography, have been purified and identified as caligulin, (Mr 4O,OUU), calcineurin, (Mr 230,000) and calmodulin, (Mr 38,000). In addition, a novel calcium binding protein(M, 48,000 by SDS PAGE) has been identified from the Mr 75,000 calcium 0 1985 Academic Press, Inc. binding activity peak.

In used

a previous to

assay

brain.

the

calcium

Chromatography

revealed Peak

three II

of

at M,

40,000

at

Peak

binding

of

III

Peak

which

A comparison

whom

activity

that

all

with of

calmodulin

correspondence

of

0.18

M,

and

Peak

75,000,

of

binding

(Peak

should

III)

II

III

eluting peak

at

5,5'-dithiobis-(2-nitrobenzoic High performance liquid Dithioerythritol Diethylaminoethyl Sodium Dodecyl Sulfate

Mr

binding

responsible

binding three

420,000.

resolved for

only

acid) chromatography

Gel

1138

about

on calcium

be addressed.

0006-291X/85 $1.50 Copyright 0 1985 by Acadetntr Press, Inc. All righrs of reproduction in any form reserved.

at

M NaCl.

activity

was

bovine

0.05

M NaCl,

Chromatoactivity peaks

of

Chromatography peak

of

Mr

38,000

activity. peaks

Polyacrylamide

of

0.25

Abbreviations: DTNB HPLC DTT DEAE SDS-PAGE

assay

cellulose

6B resolved

230,000,and

activity was

I eluting

on Sepharose

calmodulin

binding

on diethylaminoethyl Peak

a calcium

peak

calcium

x g supernatant

100,000

a major

M,

resolved

major

the

Peak

resolved

of

calcium

activity,

M NaCl, G-150

competitive

x g supernatant

binding

G-150

the

chelex

100,000

calcium

at

the

the activity

chromatography

on Sephadex

co-eluted

suggested

about

the

I on Sephadex and

calcium

of

(1) binding

of

peaks

eluting

graphy

*To

communication

Electrophoresis

on DEAE cellulose a small

percentage

of

the

Vol.

128,

total

calcium

tion

BIOCHEMICAL

No. 3, 1985

binding

chromatography

chelators, II

were

with

these

of

Peak

I and

of

the

Peak

activity

possibility

binding

proteins

was

calmodulin

activity

correlated

possibility

that

In

the

present

several

of

peaks

reported

binding

Peak

the

protein

(Mr

activity

peak.

This

Materials

and

Methods

Peak

II

that

responsible

III

calcium

with contained

48,000

by

protein

performed

calcium

in

the

binding the

therefore,

the

calcium

binding

activity

used

binding

45Ca2+

responsible

SDS PAGE)

In

detectable

of

of

of

calmodulin

Peak

could

calcium

the

purified M,

75,000

although III

the

not

binding

45Ca2+

calcium I and

for

we have

permea-

Furthermore,

proteins

the

gel

Peak

(2)

for by

of

autoradiography

addition

responsible

absence

association

activity.

calcium

Since

proteins

binding

(1).

was not

the

COMMUNICATIONS

x g supernatant.

calcium

other

study

was

RESEARCH

for

proteins

earlier

BIOPHYSICAL

100,000

and

we have

binding

our

brain

proteins,

communication

in

the

existed

calmodulin

AND

to

be ruled

out

identify activity

a novel calcium

calcium binding

autoradiography.

45 CaC12 (specific activity 28.4 mCi/mg) was obtained from New England Nuclear. Proteolytic inhibitors were obtained from the following sources: leupeptin, pepstatin A and antipain were obtained from Transformation Research (Framingham MA), and diisopropylfluorophosphate, benzamidine , phenylmethylsulfonylfluoride and soybean trypsin inhibitor were obtained from Sigma. Fresh bovine brain was obtained froma local slaughterhouse. Connective tissue was dissected away, and the tissue rinsed thoroughly with ice cold distilled water and frozen immediately at -2O'C. One kilogram of frozen tissue was chopped, and then further minced The mince was mixed with 3 1 of ice cold buffer containing 40 mM in a meat grinder. Tris-HCl (pH 7.5), phenylmethylsulfonylfluoride (0.1 mM), soybean trypsin inhibitor (5 mg/l diisopropylfluorophosphate (1.0 mM), leupeptin (5 mg/l), pepstatin (5.0 mg/l), antipain (5.0 mg/l), chymostatin (5.0 mg/l), benzamidine (10.0 mM), DTT (1.0 mM), 2.0 mM EDTA and then homogenized in a Waring blender. The resultant extract was centrifuged at 20,000 x g for 30 minutes and the supernatant was then centrifuged at 100,000 x g for 60 min. The supernatant was diluted into five volumes of 40 mM Tris-HCl (pH 7.5), 0.2 mM DTT and 800 ml of packed DEAE cellulose was then added. The mixture was stirred rapidly for one hour, and then filtered through a coarse scintered-glass funnel. The resultant slurry was washed with 6.0 1 of 40 mM Tris-HCl (pH 7.5) and poured into a 5.0 x 60 cm column. Protein was eluted with a linear gradient made from 2.2 liters each of 40 mM Tris (pH 7.5), 0.2 mM DTT, and 40 mM Tris (pH 7.5), 0.2 mM DTT, 0.45 M NaCl. Samples were dissolved in SDS sample buffer (10 mM 2-mercaptoethanol, 0.1 M Tris-HCl [pH 6.81, 30% glycerol, 2% SDS, 1.0 mM EDTA), heated at 1OO'C for 2 min, and electrophoresed on 12.5% SDS polyacrylamide gels according to Laemmli (3). The electrophoretic transfer was performed according to Towbin (4) at a constant current of 100 mA for 50 min at 4OC. The electrode buffer contained 20% methanol, 0.025 M Tris and 0.13 M glycine (pH 8.5). After electrophoretic transfer the nitrocellulose membrane was rinsed twice with distilled buffer, and incubated with several changes of 60 mM KCl, 5 mM MgC12, and 10 mM imidazole-HCl (pH 6.8). The membrane was incubated in the same buffer containing 1 mCi/liter 45Ca2+ for 15 min, rinsed, dried, and exposed to Kodak XAR-5 X-ray film for 24 h. The 45Ca2+ autoradiography procedure is described in detail in (2). Isoelectric focussing was performed according to O'Farrel (5) using LKH ampholines (pH 3.5-10.0). The chelex competitive calcium binding assay was accomplished according to (6). Calmodulin was assayed according to (7).

Results

and Discussion The

resultant

100,000 fractions

x g supernatant were

analyzed

of by

brain

was

SDS-PAGE

chromatographed and

1139

45Ca2+

on DE52 autoradiography.

(methods) Results

and

the are

Vol. 128, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Molecular Weight (x 10-9

29.0 24.0

14.2 Dye

10

20

30

40

50

60

70

80

100

SO Fraction

Figure

45Ca2+

1.

Autoradiographic

Analysis

Bovine Brain 100,000 x g supernatant 100 eluted by linear NaCl gradient. on 12.5% polyacrylamide, transferred radiography (methods). Electrophoretic cated to the left of the figure.

presented

in

and M,

27.000a.

found

that

ing

the

active

(2).

This

order

to

compare

binding

gel

visualized

calcium

activity

binding peaks

of

45Ca2+

radiographic

Mr

% olecular

weight motility

relative

time

the

was

and

in

in

found

to

150

160

1?0

Chromatographic

180

Fractions.

(1).

M,

to

plots

and M,

1140

weight

radio-

of (determined

(figure

This

identified

calcium 16,000),

bind-

proteins

fractions

autoradiography

molecular

increas

calcium

binding

45Ca2+

of

M,

of

(SDS-PAGE,

by

log

[6])

40,000

detect

since

the

chromatography M,

was

proteins.

chromatography.

autoradiographic

20,000

autoradiography)

calcium

DEAE cellulose

permeation

20,000),

unable

from

gel

the

it

particular

1) with assay

Mr

and

-binding

45Ca2+

binding

permeation

these

45ca2+

(Figure

16,000,

to proteins,

remove

study

by gel

at Mr

visualized.

Ca2+

and

of

45Ca2+

of

SDS-PAGE

autoradiography

(SDS-PAGE,

were

reduced

study

M,230,000

determined

in

previous

of

was

140

shown

completely

calcium

components

We were

bands

present

protein

27,000).

dense

by

chelex

purified

autoradiographic

clearly

resulted

our

our

45ca2+

and

are

binding

(determined

and

determined

less

also

Mr

DEAE Cellulose

non-specific

resolved

pooled

(SDS-PAGE,

vs

wash

to

130

Number

bands

other

procedure

SDS-PAGE

individually

the

due

chromatography

peaks, by

were

proteins

permeation

activity

were

bands

many

120

was chromatographed on DEAE cellulose and fractions ug of each column fraction was subjected to SDS-PAGE to nitrocellulose, and then analysed by 45Ca2+ automobilities of molecular weight standards are indi-

radioactive

addition,

bands

calcium

ing

In

these

Three

1.

nitrocellulose

In

by

Figure

of

110

binding

Mr

38,000

45Ca2+

autothe

calcium

1)

Vol.

128,

8lOCHEMlCALAND8lOPHYSlCAL

No. 3, 1985

Table

Amino

I:

Amino

acida

Acid

Composition

Mr 48,000

Mr

RESEARCH

of

Brain-Stable

Aspartic

acid

Mr %)

16.7

15.5

10.0

1.8

8.1

3.3

acid

4.9

6.5

2.7

4.1

11.3

13.1

18.2

18.7

Proline

3.7

1.8

1.4

3.5

Glycine

9.2

8.3

7.4

5.2

Alanine

10.0

3.0

7.4

6.5

Half-cystinec

1.2

0

0

2.2

Valine

7.4

7.7

4.7

6.5

Methionine

1.3

3.6

6.1

1.9

Isoleucine

5.9

6.5

5.4

3.3

Leucine

10.1

8.3

6.1

10.7

Tyrosine

2.9

1.8

1.4

1.8

Phenylalanine

3.8

7.1

5.4

7.5

Histidine

1.9

1.2

0.7

3.5

Lysine

5.2

8.9

5.4

7.4

Arginine

4.3

3.6

4.1

5.7

Tryptophand

0.7

0.0

0.0

1.3

aValues

represent

bThe

amino

the

as cysteic

ddetermined

after

protein(s)

binding

activity

destroyed

by

SDS-PAGE calcium

of

by (Mr

the

purify

in

methanesulfonic

or

Mr

(Tokuda

et

al.,

assay SDS PAGE),

is

calcium

binding

the

gel

seconds

(13).

presented

(data

binding

acid

protein

of

the

I.

for

these shown)

assay

for

We have

by

45ca2+

exchange

and

in

vacua

these

(14).

calcium was

completely

we concluded

binding of

420,000

peaks

peak

calcium

M,

22h

chromatography

not

composition

table of

110°C

activity

and

in

at

permeation

an inappropriate

amino

hydrolysis.

(12).

activity

preparation)

The

24h

reported

acid

calcium

in

of

oxidation

binding

75,000

(6).

is

420,000

thirty was

the

by

Mr

calcium for

of

chelex

6 subunit performic

the

1OO'C

values

after

75,000

Since

protein

48,000 to

Mr

interpolated

the

autoradiography

HPLC chromatography monitored

the

at

45Ca2+

of acid

peaks.

binding

or

hydrolysis

incubation

and

average

acid composition

'determined

binding

attempts

Mr 27,000

4.3

Serine

CAB-48

20,000

11.6

Threonine

Glutamic

Proteins

16,000b (mol

COMMUNICATIONS

that

proteins. was

The

purified

activity

by was

this

protein,

called

been

unsuccessful

calcium

binding

in activity

peak. Fractions were

individually

demonstrating pooled

calcium and

purified

binding

activity

by both 1141

ion

autoradiography gel

permeation

(Figure chromatography

1)

Vol. 128, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Mr x 10 -: 68-O-

45.036,029-o24,0-

2001-

14.2-

R

DYEFigure

2.

45Ca2+

+

Autoradiograph

of

Purified

Calcium

Binding

Proteins.

Purified calcium binding proteins (10 ng) were analyzed on 12.5% SDS-PAGE transferred to nitrocellulose, and subjected to 45Ca2+ autoradiography (A). In B, the nitrocellulose membrane was stained by amido black. The proteins were a, 16,000 daltons; b, 24,000 daltons; c, 20,000 daltons. The electrophoretic mobilities of molecular weight standards are shown to the left of the figure. isoelectric focussing of the 20,000 dalton protein (0,

4sca2+

on BPLC. fication,

and

tions

to

in

that (data

of

the

coincided sheet

with

reveals

amido

black

M,

60,000

both

non-denaturing protein

has Ca2' The

Mr

60,000;

binding

activity

tentatively

used

2,

to

bands

16,000

bands

when

stained

a single

band

(data

not

consisting

proteins

of

of

with

dalton

the

is

lost

during

the

pres-

positions

nitrocellulose

protein

amido it

not

A) whose

dalton

shown)

purisepara-

revealed

2,

staining

a 60,000

were

proteins

16,000

during

chromatographic

(Figure

by

purified

activity

monitor

of purified

visualized The

binding

binding

radioactive

B).

2+

(Figure

2B.

black.

Since

concluded

that

polypeptides

and

a)

this a 16,000

polypeptide. of

some of

(Table

II)

subunit, of

was

Ca

calcium

proteins

structure

binding

assay

Ssubunit

was

a subunit

identities

by activity

the

Mr

PAGE revealed

labile

as

(Figure and

monitor

Autoradiography

proteins of

to

assay

heat

shown).

bands

used

chelex

potential

binding the

was the

not

Ca2+ with

dalton

addition,

ensure

purification ence

autoradiography

calcineurin after identified

M,

the

as

16,000; is

a heat

separation on the

purified

Ca

calmodulin ref

2+

(Mr

g-10).

stable

binding

proteins

20,000; This

calcium

ref

was

the

binding

7)

have and

first

confirmed

calcineurin

(a subunit,

demonstration

protein

from

the

a subunit

by SDS PAGE.

basis

of

molecular

weight

1142

been

which The

as caligulin,

that

retains M,

the

calcium

27,000 a novel

protein Ca 2+-

Vol.

128,

No. 3, 1985

BIOCHEMICAL

TABLE

II:

Activity

AND

of

BIOPHYSICAL

Purified

Calcium

RESEARCH

Binding

COMMUNICATIONS

Proteins

Calmodulinb activity

CalcineurinC activity

16,000

t

20,000

t

27,000 48,000

aDetermined

by SDS-PAGE

(Figure

2).

bCalmodulin brain cyclic

activity was determined by Ca2' -dependent nucleotide phosphodiesterase (6).

activation

of

bovine

C

Calcineurin of p-nitrophenyl

binding

protein

bovine

brain.

(Table

1).

focussing

activity

was phosphate

determined (7).

originally

described

The identity

of this

The purified and a single

20,000 protein

band was not contaminated

TABLE

dalton

(1)

ref

(calmodulin)

Identification

of

Calcium

100,000

proteins

40,000

Peak

II

75,000 230,000 420,000

III

Peak

(1)

Calcium

b

Mr

48,000

binding

activity

determined

purified

of M, 20,000.

are compared in Table

of

Identity

Mr

27,000

by

protein

a

20,000

Calmodulin

SDS-PAGE. by SDS-PAGE

1143

Caligulin CAB 4ab Calcineurin Unknown

B subun:t,

was destroyed for

the calmodulin

Autoradiography

38,000

a

by isoelectric

x g Supernatant

M r

I

that

protein

Proteins

45ca2t

Gel Permeation Chromatographic Analysis

Peak

proves

binding

Binding

of

by amino acid analysis

This result

binding

x g supematant

was analysed

12), a calcium

calcium

Brain

100,000

has been corroborated

protein

(CBP-18,

Bovine DEAE Cellulose Activity Peak

protein

of the brain

III:

dephosphorylation

(11) in the heat-treated

band was observed.

with

The amino acid compositions

by calmodulin-stimulated

16,000

(11) (15)

1.

Vol.

128,

No. 3, 1985

These proteins

are acidic

The heat stable gulin)

BIOCHEMICAL

calcium

demonatrate

contains

large

of this

protein

proteins binding

high

proteins

ratios

BIOPHYSICAL

and therefore

to distinguish

rich

( Bsubunit

of phenylalanine

amounts of hydrophobic appear

AND

RESEARCH

in glutamic

and aspartic

of calcineurin,

calmodulin,

to tyrosine.

amino acids.

COMMUNICATIONS

By comparison,

intracellular

and caliCAB 48

The amino acid composition

CAB-48 from other

acid.

and M,

calcium

binding

(2),

and chelex

proteins. Using the combined techniques calcium bovine

binding brain

were unable brain

calcium

ing protein

of SDS PAGE, 45Ca2' autoradiography

assay (6) we have identified 100,000 x g supernatant.

to explain, binding

however, proteins

several

These results

why this

approach

such as calregulin

major

calcium

are summarized fails

to identify

(16) vitamin

binding in table several

D-dependent

proteins

of

III.

We

other

known

calcium

bind-

(17) or S-100 (18).

References 1. 2. 3. 4.

Waisman, D.M., Smallwood, J.I., Lafreniere, D., and Rasmussen, H. (1983) Biochem. Biophys. Res. Cormnun. 116, 435-441. Maruyama, IL, Mikawa, T. and Ebashi, S. (1984) J. Biochem. 95, 511-519. Laemmli, U.K. (1970) Nature 227, 680-685. Towbin, Il., Sltaehelin, T., and Gordon, J. (1970) Proc. Natl. Acad. Sci. USA 76, 4350-4354.

5. 6. 7. a. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18.

O'Farrel, P.M. (1975) J. Biol. Chem. 250, 4007-4021. Calcium 6, 89-105. Waisman, D.M., and Rasmussen, Il. (1983rCell Teo, T.S., Wang, T.H., and Wang, J.H. (1973) J. Biol. Chem. 248, 588-595. Wang, J.H., and Desai, R. (1976) Biochem. Biophys. Res. commun. 2, 926-937. Klee, C.B., and Krinks, M.H. (1978) Biochem. 11, 120-126. Sharma, R.K., Desai, R., Waisman, D.M., and Wang, J.H. (1979) J. Biol. Chem. 254, 4276-4282. Waisman, D.M., Muranyi, J., and Ahmed, M. (1983) Febs. Lettr. 164, 80-84. Manalan, A.S., and Klee, C.B. (1984) J. Biol. Chem. 2, 2047-2050. Hirs, C.H.W. (1967) Methods Enzymol. 11, 59-62. T.Y. (1976) J. Biol. Chem. 251, 1936-1940. Simpson, R.J., Neuberger, M.R., and Lit, Pallen, C.J., and Wang, J.H. (1983) J. Biol. Chem. 258, 8550-8553. J. Biol. Chem. 260, 1652-1660. Waisman. D.M., Salimath. B.P., and Anderson, M.J. (1985) Structure Christakos. S., and Norman, A.W. (19801 in Calcium Binding Proteins: and Function. ed. Elsevier, New York, pp. 371-378. Siegal et al. Hidaka, H., Endo, T., and Kata, K. (1983) Methods in Enzymol. 102, 256-261.

1144