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