Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PURIFICATION OF LOW MOLECULAR WEIGHT COPPER PROTEINS FROM COPPER LOADED LIVER J.
R. Riordan
and I.
Gower
Research Institute, The Hospital for Sick Children and Dept. of Clinical Biochemistry, University of Toronto, Toronto, Canada.
Received
July
28,1975
SUMMARY Purification of low molecular weight copper binding proteins from the livers of copper loaded male rats was achieved by sequential ultracentrifugation (186,OOOg, Zh), ultrafiltration (Amicon PM 30), gel filtration (Sephadex G-75) and anion exchange chromatography (DEAE - Biogel A) of soluble tissue extracts. The three major copper-associated polypeptides obtained which had molecular weights of about 7000, 9,000, and 12,000 daltons contained approximately 2.59 atoms of copper per mole. Amino acid analyses indicated a similarity between these proteins and the copper protein 'L-60' isolated earlier from livers of Wilson's disease patients and distinguished them from metallothioneins which have been isolated from animals administered other trace metal ions.
Heavy metals species
in association
described salts
including with
and termed
It
tissue strated.
levels
amount
copper
metallothionein. small content abstract
soluble
that
For example, protein
from
of metallothionein form
that
a protein
in both
(19).
Rajagopalan
different
rats
did
changes
in
have not been demon-
there
very
not exhibit
and colleagues
from metallothionein
and liver
(17,18).
of partially
Evans has demonstrated
or zinc
kidney
effect
of copper
that
first
of cadmium
may not be due to enhanced
loaded
Vallee
in metallothionein,
has suggested
copper
of several
which
has a similar
characterization
proteins this
protein
protein
on administration
copper
but
of this
known to be present
protein
in tissues
Administration
mercury
in some instances,
weight
of protein
whether
also
weight
(l-5).
the amount
clear is
of this
However,
low molecular
a low molecular
increase
is not yet
Although
have been detected
metallothionein
substantially
(6-16).
copper
is
purified a larger
synthesis recently
of a that
the high
a cysteine
have reported appeared
in
on injec-
Vol. 66, No. 2, 1975
tion
of copper
of soluble
proteins
major
thionein loaded
is
from
copper present
liver
Earlier,
(20).
metallothionein. three
BIOCHEMICAL
it
is
Bloomer
copper
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and Sourkes
loaded
rats
The present
comnunication
containing
polypeptides
among the certainly
reports
did
had described not
that
appear
weight
as a minor
copper
a mixture
to contain
on further
can be obtained,
low molecular only
which
(21)
purification
and that proteins
if
metallo-
of copper
component,
METHODS Male Wistar rats (300-4009) after subcutaneous injection for 6 consecutive days with CuSO4 (2 mg Cu++/kg) were sacrificed by decapitation. Portions of the excised livers were used for determination of total tissue copper by a modification of the method of Eden and Green (22). A 20% homogenate of the remainder in 0.05 M Na H2PO4, p H 7.0 was prepared in a Waring blender (5 x 15 set). After centrifugation at 16,300g for 30 min. to remove large particulate material, the supernatant was spun for 2h at 186,000g. This supernatant was passed through an Amicon PM 30 filter, thereby removing material larger than 30,000 daltons, and concentrated by a second ultrafiltration with an Amicon UM 2 filter. The concentrate was applied to a Sephadex G-75 column (2.5 x 90 cm.). 5 ml fractions were monitored for absorbance at 280 and 250 nm and copper concentration (Pye-Unicam SP 1800 Atomic Absorption Spectrometer). Fractions comprising the low molecular weight co per peak were pooled and concentrated either by ultrafiltration (Amicon UM 2 !i or dialysis and lyophilization. This material was applied to a column of DEAE-Biogel A (2.5 x 20 cm) with elution as indicated in the figure legend. Fractions were monitored and copper containing peak fractions were concentrated by either of the methods mentioned above. Protein concentrations were determined by the fluorometric procedure of Bijhlen et al (23) which accurately detected less than 1 pg of protein. This procedure proved a major advantage over other commonly used methods of protein determination since neither the copper binding proteins nor the metallothioneins contain significant amounts of aromatic amino acids. Polyacrylamide gel electrophoresis was performed with discs or slabs of 10% acrylamide in (a) 0.1 M glycine-acetic acid, pH 3.6; (b) 0.1 M Tris-glycine, pH 8.9 or (c) 0.1 M TrisHCL, pH 7.4 containing I%Na dodecyl sulfate. Molecular weights were estimated by interpolation from plots of log molecular weight versus distance migrated in the latter system by the following proteins as standards having the subunit molecular weights indicated: chymotrypsinogen (25,000), hemogl ggin (16,750), Cu++ was prepared lysozyme (14,000), cytochrome C (12,000) and insulin (5,734). (24) by irradiation of natural zinc with Bremsstrahlung from the linear accelerator at the University of Toronto and counted in a well-type gamma spectrometer. Amino acid analyses were performed employing a Technicon TSM analyser after hydrolysis in 6 N HCl in U~CUO for 20h at 1lOoC. RESULTS When the per g. wet
tissue,
of low molecular
livers
of rats
the amount weight
copper
were
loaded
of the metal proteins
with ion
copper
to a level
at the various
was as shown in table
679
of about
stages I.
200 pg
of isolation
The 40% of the
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Distribution
of copper proteins
I
during from
isolation of low molecular copper-loaded liver.
Fraction Total
liver
9400
100.0
5600
60.0
PM 30 concentrate
5250
55.0
PM 30 ultrafiltrate
176
2.0
UM 2 concentrate
69
0.7
UM 2 ultrafiltrate
10
0.1
Sephadex
40
0.4
186,000g
Copper
supernatant
G-75 peak in the homogenate
fractions
total
liver
by atomic
copper
was associated proportion
with
having
a molecular
2% of the
total
total)
copper
0.4% of the
gel
filtration
venously radioactivity
67Cu++
in the
than
the equivalent
fractions
times
more copper
loaded
liver
sacrifice were profile
the
small loaded
with
About copper
proteins
animals
were
the same as those
(or
0.7%
of this obtained
injected
of the listed
total
by intra-
liver
in table
in a preparation material
Of the
l/3 l/2
by an material
daltons. about
the percentages
obtained
the greatest
was retained
molecules
was used as starting
680
By far
of 30,000
UM 2 ultrafilter. with
presumably
have been associated
smaller
When copper
h. before
Fig 1 shows the G-75 elution four
with
by an Amicon
in the various
and in the other
centrifugation
supernatant
greater
was associated
l/2
soluble must
G-75.
(22)
fractions.
therefore,
on Sephadex
with
speed
subcellular
in association
total)
high
and,
was retained
(or
after
particulate remaining
size
chemically
spectrometry.
was removed
of the copper PM 30 ultrafilter
was determined
absorption
which
mainly
Amicon
of the
% of total
cu (119)
homogenate
weight
where
I. about
as in the experi-
Vol. 66, No. 2, 1975
BIOCHEMICAL
20
AND BIOPHYSICAL
40
60
80
FRACTION
100
RESEARCH COMMUNICATIONS
120
NUMBER
Figure 1: Gel filtration of ultrafiltrate (Amicon PM 30) of high speed supernatant from liver homogenate on a Sephadex G-75 column (2.5 x 90 cm) in 1 mM Tris-HCl, pH 8.6. 5 ml fractions were collected and monitored for absorbance at 2 wavelengths and copper content as indicated. The arrows indicate the positions of the void and bed volumes of the column.
ment represented
in table
at 250 and 280 nm. small
amounts
copper
portion that
of the
peak were
content
molecular
weight
animals,
gel
polypeptide 7,000
separately step.
of about
between
with
visibly
(in
fractions
shoulder
latter
part
and only
the
Analysis
of the material
2.5
g atoms
per mole
In several
1 and 5 g atoms (fig
molecular
weights
Over
and the major processed
through
on the
preparations
the
of approximately
the
peak
12,000,
than of the subse-
revealed
basis
of a mean
from
copper-loaded
obtained.
presence
is
the major
portion
the major
per mole were
B) showed
There
at 250 nm is greater
(calculated
of copper
2, gel
of 250 ml.
from
other
and only
and 90 to 100).
absorbance
the
peak of absorbance
non-proteinaceous
75-83 volume
the
comprising
is a large yellow,
at an elution 42 to 69)
electrophoresis
bands
are
present
of 9000 daltons).
values
acrylamide
are
(fractions
exchange
a copper
fractions
The fractions
pooled
anion
Near the bed volume
peak centred
peak
at 280 nm.
quent
These
of copper
a principal
I.
Poly-
of three 9,000
major
and
daltons. Amino
acid
analyses
yielded
the
composition
681
shown in table
II
which
also
Vol.66,No.
BIOCHEMICAL
2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
_*
-lb d
-
A
ti
-me
-
%“n
D
E
F
staining (Coomassie blue) bands after polyacrylamide Figure 2: Protein gel electrophoresis in system (c) of Methods. Samples were prepared by incubation in a boiling water bath for 2 min. in 1% SDS and 40 mM dithioThe following amounts of protein from the chromatography fractions threitol. Gels A and B: 25 pg from fractions 30-41 and indicated were applied: Gels C,D,E: 25, 18, 17 pg from fractions 42-69 of fig 1 respectively. 10-15, 16-40, 41-46 of fig 3 respectively. 1Opg each of chymotrypsinogen, hemoglobin, lysozyme and cytochrome C were applied to gel F.
includes for
for
comparative
purposes
a similar
preparation
from
copper
protein
et al
(25).
rations four
isolated The amount
are very times
proteins
from
less present
The three
major
the
loaded
livers
recently
rat
liver
of a Wilson's
Most notable
that
after
copper
composition
of each of the amino
similar. than
the
acids
loading
with
other
copper-associated
trace
metals
by Evans
as those patient
in the three values
the major
polypeptides
682
as well disease
are the cysteine
of the metallothioneins,
reported
(19)
of a smal by Morel1
different which
prepaare about
low molecular
weight
such as Cd++ and Zn++. of our G-75
copper
peak
BIOCHEMICAL
Vol. 66, No. 2, 1975
Table
Amino
acid
Amino
acid
composition
AND BIOPHYSICAL
II
of low molecular
Cu-loaded
rat
acid
copper
liver
A Aspartic
RESEARCH COMMUNICATIONS
proteins
Liver
from
of Wilson's patieni?
11.4
8.8
Threonine
7.9
7.2
5.8
Serine
7.3
7.6
7.1
12.6
11.9
11.2
Proline
4.0
3.9
5.5
Glycine
10.1
8.8
8.6
Alanine
4.9
6.8
8.2
Valine
4.3
5.1
7.1
Half-cystine
7.5
5.0
4.3
Methionine
3.2
Isoleucine
5.0
4.3
4.9
Leucine
6.2
7.0
6.7
Tyrosine
2.0
1.1
Phenylalanine
3.7
3.1
acid
Lysine
1.5
13.2
Histidine
1.7
Arginine
2.5
13.0
3.0
proteins
obtained
from
B
proteins
obtained
by Evans
a
expressed
b
from
Morel1
et al
10.8 1.5
A
as residues
disease
B
10.2
Glutamic
liver.a
gel
per
filtration et al 100
(25).
683
2.6 step
(19).
(fractions
42-69
of fig.
1)
VoL66,No.
were for
2, 1975
further
resolved
elution
tended
necessary
for
exclusively peak
(fractions
(fig
2, gel
smearing
resolution.
16-39)
the
polypeptides
to cause
polypeptide
D),
largest
weighing
contained
whereas
the
that
very
in two non-dissociating
three
bands
observed
9,000
system
(fig
peak
dalton
(fig
peak
are a result
2, gel
forms systems
of the
Therefore, of disruption
10-15) C),
the
contained broad
copper
and 12,000
daltons
40-46)
seeming
contained
staining
also
of subunit
raises
but gel showed
intensities
seems highly
individual
similarity
same protein
(see Methods)
it
but was
of none of the
Their
used
proteins
7,000
(fractions
contents
together.
gradient
(fractions
weighing
the same relative 2).
salt
- containing
small
copper
may be different
having
The shallow
The copper
electrophoresis
in the dissociating
copper
of the three
they
bands
of the
sharp
that
separated
3).
the polypeptides
polypeptide.
exceeded
A (fig
The first
the possibility
narrowly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on DEAE Biogel
their the
primarily
BIOCHEMICAL
three
as seen
unlikely
that
interactions.
DISCUSSION We have been able
to purify
three
FRACTION
Figure (42-69 with a (mmho)
different
small
proteins
with
which
NUMBER
of pooled gel filtration fractions 3: Anion exchange chromatography of fig 1) on a DEAE Biogel A column (2.5 x 20 cm). Elution was salt gradient from 0 to 0.1 M NaCl which resulted in the conductivity rise shown.
684
the
BIOCHEMICAL
Vol. 66, No. 2, 1975
copper
is
copper.
associated
from
The resolution
been studied
characterization
of these
proteins.
particular
investigators
to avoid
solvent
all
three
as the metallothioneins amino
imately
present from
acid
makes it
animals
content unlikely
of the
As Evans
livers
is
required
that
copper
Wilson's
further
proteins
of have
towards
of the functions we have taken
methods
instead
amounts state
in the
of protein
on very
high
a very
large
speed
respect
metals
pointed
very
the
on copper
this
of such proteins and possibly
that
is livers
here
(26).
L-6D isolated
and co-workers loading
than
it
may also
its
metallothionein
similarity,
others
approx-
of metallothionein
of each of the three
or disprove
the derived
described
by Scheinberg
appearing
from
little
protein
range
drastically
is noteworthy
than
out,
disease
it
contain
proteins
deviation
amount
weight
differ
metallothionein
significant
In this
same molecular
of proteins
of which
characterization
to confirm
pathological
are
the
basis
associated
has already
increased
in this
on the that
to the
Although
isolated
In fact
no trace
with
to be more similar
speculate
step
step
and other
the two groups
liver.
(19)
of a patient
thionein.
proteins
administered
and much less
proteins
understanding
relying
doses which
a further
purification
step,
compositions.
in copper-loaded
liver
final
high
filtration
represents
fractionation
(5,6,9,10)
30% cysteine
name (1)
by gel
a fuller
filtration
fairly
and ultrafiltration.
Although
in total
(8,9,21)
to this
the gel
administered
obtained
and hopefully
before
centrifugation
of rats
components
In addition
care
precipitation
livers
of the
by other
complete
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from the (25)
a metallopurified
is
seems
copper
tempting
be synthesized
to in
(27).
ACKNOWLEDGEMENTS This indebted recent
work was supported
to Dr. findings
G. W. Evans for available
by the Medical making
a preprint
to us.
685
Research
Council
of a manuscript
of Canada. describing
We are his
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Margoshes, M. and Vallee, B.L. (1957) J. Am. Chem. Sot. 79: 4813. K;igi, J.H.R. and Vallee, B.L. (1960) J. Biol. Chem. 235:3460. Ksgi, J.H.R. and Vallee, B.L. (1961) J. Biol. Chem. 234: 2345. i: B.L. (1966) Bioxm. 5: 1768. Pulido, P., Kagi, J.H.R. and Vallee, J.H.R., Himmelhoch, J.R., Whanger, P.D., Bethune, J.r. and 5. Kigi, Vallee, B.L. (1974) 3. Biol. Chem. 249: 3537. 6. Shakh, Z.A. and Lucis, O.J. (1971) Gerientia 27: 1024. 7. Nordberg, G.F., Nordberg, M., Piscator, M. and Esterberg, 0. (1972) Biochem. J. 126: 491. Winge, D.R. and Rajagopalan, K.V. (1972) Archiv. Biochem. Biophys. 153: 755. 9”: Webb, M. (1972) Biochem. Pharmacol. 21: 2751. Buhler, H.O. and Kagi, J.H.R. (1974)FEBS Letters 39: 229. ::: Piotrowski, J.K., Bolanowska, W. and Sapata, A. (lT3) Acta Biochem. Pol. ::
20: 207. 12. Femner, I., Davies, W.T. and Mills, 13. Davies, W.T., Bremner, I. and Mills, 14. Kimura, M., Otaki, W., Yoshiki, S., 15. 16. 17. 18.
C.F. (1973) Biochem. Sot. Trans. 1: 982. C.F. (1973) Biochem. Sot. Trans. i: 985. Suzuki, M., Horiuchi, N. and Suda,f. (1974) Archiv. Biochem. Biophys. 165: 340. Squibb, K.S. and Cousins, R.J. (lm Environ. Physiol. Biochem. 4: 24. Chen, R.W., Whanger, P.D. and Weswig, P.H. (1975) Biochem. Med. 12: 95. B. and Nordberg, G.F. (1974) Environ.Physiol. Nordberg, M., Trojanowska, Biochem. 4: 149. 8. and Sapota, A. (1974) Arch. Toxicol. PiotrowskT, J.K., Trojanowska,
32: 351. G.W., 19. Fans, 20. 21. E: 24. 25. 26. 27.
in press. Winge, D.R.,
Wolenetz, Premakumar,
M.L.
and Grace,
C.I.
(1975)
Nutr.
R. and Rajagopalam,
K.V.
(1974)
Reports Fed.
Int.
Proc.
%~orn~",""; C and Sourkes T L (1973) Biochem Med 8. 78. Eden, A: and'Green, H.H. [19iOj Biochem. J. 34; 12t)2:’ W. and Udenfsend, S. (1973) Archiv. BBhlen, P., Stein, S., Dairman, Biochem. Biophys. 155: 213. McConnell, D.B. and Aspin, N. (1970) Int. J. Marceau, N., Kruck,P.A., Appl. Radiat. Isotop. 21: 667. Morell, A.G., Shapir0,T.R. and Scheinberg, I.H. (1961) in Wilson's J.M. Walshe and J.N. Cumings, Eds. Disease: Some Current Concepts. p. 36. Blackwell, Oxford. Riordan, J.R. and Gower, I. (1975) Biochim. Biophys. Acta. Submitted for publication. V.M. and Sherlock, S. (1968) Smallwood, R.A., Williams, H.A., Rosenoer, Lancet II: 1310.
686
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PURIFICATION OF LOW MOLECULAR WEIGHT COPPER PROTEINS FROM COPPER LOADED LIVER J.
R. Riordan
and I.
Gower
Research Institute, The Hospital for Sick Children and Dept. of Clinical Biochemistry, University of Toronto, Toronto, Canada.
Received
July
28,1975
SUMMARY Purification of low molecular weight copper binding proteins from the livers of copper loaded male rats was achieved by sequential ultracentrifugation (186,OOOg, Zh), ultrafiltration (Amicon PM 30), gel filtration (Sephadex G-75) and anion exchange chromatography (DEAE - Biogel A) of soluble tissue extracts. The three major copper-associated polypeptides obtained which had molecular weights of about 7000, 9,000, and 12,000 daltons contained approximately 2.59 atoms of copper per mole. Amino acid analyses indicated a similarity between these proteins and the copper protein 'L-60' isolated earlier from livers of Wilson's disease patients and distinguished them from metallothioneins which have been isolated from animals administered other trace metal ions.
Heavy metals species
in association
described salts
including with
and termed
It
tissue strated.
levels
amount
copper
metallothionein. small content abstract
soluble
that
For example, protein
from
of metallothionein form
that
a protein
in both
(19).
Rajagopalan
different
rats
did
changes
in
have not been demon-
there
very
not exhibit
and colleagues
from metallothionein
and liver
(17,18).
of partially
Evans has demonstrated
or zinc
kidney
effect
of copper
that
first
of cadmium
may not be due to enhanced
loaded
Vallee
in metallothionein,
has suggested
copper
of several
which
has a similar
characterization
proteins this
protein
protein
on administration
copper
but
of this
known to be present
protein
in tissues
Administration
mercury
in some instances,
weight
of protein
whether
also
weight
(l-5).
the amount
clear is
of this
However,
low molecular
a low molecular
increase
is not yet
Although
have been detected
metallothionein
substantially
(6-16).
copper
is
purified a larger
synthesis recently
of a that
the high
a cysteine
have reported appeared
in
on injec-
Vol. 66, No. 2, 1975
tion
of copper
of soluble
proteins
major
thionein loaded
is
from
copper present
liver
Earlier,
(20).
metallothionein. three
BIOCHEMICAL
it
is
Bloomer
copper
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and Sourkes
loaded
rats
The present
comnunication
containing
polypeptides
among the certainly
reports
did
had described not
that
appear
weight
as a minor
copper
a mixture
to contain
on further
can be obtained,
low molecular only
which
(21)
purification
and that proteins
if
metallo-
of copper
component,
METHODS Male Wistar rats (300-4009) after subcutaneous injection for 6 consecutive days with CuSO4 (2 mg Cu++/kg) were sacrificed by decapitation. Portions of the excised livers were used for determination of total tissue copper by a modification of the method of Eden and Green (22). A 20% homogenate of the remainder in 0.05 M Na H2PO4, p H 7.0 was prepared in a Waring blender (5 x 15 set). After centrifugation at 16,300g for 30 min. to remove large particulate material, the supernatant was spun for 2h at 186,000g. This supernatant was passed through an Amicon PM 30 filter, thereby removing material larger than 30,000 daltons, and concentrated by a second ultrafiltration with an Amicon UM 2 filter. The concentrate was applied to a Sephadex G-75 column (2.5 x 90 cm.). 5 ml fractions were monitored for absorbance at 280 and 250 nm and copper concentration (Pye-Unicam SP 1800 Atomic Absorption Spectrometer). Fractions comprising the low molecular weight co per peak were pooled and concentrated either by ultrafiltration (Amicon UM 2 !i or dialysis and lyophilization. This material was applied to a column of DEAE-Biogel A (2.5 x 20 cm) with elution as indicated in the figure legend. Fractions were monitored and copper containing peak fractions were concentrated by either of the methods mentioned above. Protein concentrations were determined by the fluorometric procedure of Bijhlen et al (23) which accurately detected less than 1 pg of protein. This procedure proved a major advantage over other commonly used methods of protein determination since neither the copper binding proteins nor the metallothioneins contain significant amounts of aromatic amino acids. Polyacrylamide gel electrophoresis was performed with discs or slabs of 10% acrylamide in (a) 0.1 M glycine-acetic acid, pH 3.6; (b) 0.1 M Tris-glycine, pH 8.9 or (c) 0.1 M TrisHCL, pH 7.4 containing I%Na dodecyl sulfate. Molecular weights were estimated by interpolation from plots of log molecular weight versus distance migrated in the latter system by the following proteins as standards having the subunit molecular weights indicated: chymotrypsinogen (25,000), hemogl ggin (16,750), Cu++ was prepared lysozyme (14,000), cytochrome C (12,000) and insulin (5,734). (24) by irradiation of natural zinc with Bremsstrahlung from the linear accelerator at the University of Toronto and counted in a well-type gamma spectrometer. Amino acid analyses were performed employing a Technicon TSM analyser after hydrolysis in 6 N HCl in U~CUO for 20h at 1lOoC. RESULTS When the per g. wet
tissue,
of low molecular
livers
of rats
the amount weight
copper
were
loaded
of the metal proteins
with ion
copper
to a level
at the various
was as shown in table
679
of about
stages I.
200 pg
of isolation
The 40% of the
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Distribution
of copper proteins
I
during from
isolation of low molecular copper-loaded liver.
Fraction Total
liver
9400
100.0
5600
60.0
PM 30 concentrate
5250
55.0
PM 30 ultrafiltrate
176
2.0
UM 2 concentrate
69
0.7
UM 2 ultrafiltrate
10
0.1
Sephadex
40
0.4
186,000g
Copper
supernatant
G-75 peak in the homogenate
fractions
total
liver
by atomic
copper
was associated proportion
with
having
a molecular
2% of the
total
total)
copper
0.4% of the
gel
filtration
venously radioactivity
67Cu++
in the
than
the equivalent
fractions
times
more copper
loaded
liver
sacrifice were profile
the
small loaded
with
About copper
proteins
animals
were
the same as those
(or
0.7%
of this obtained
injected
of the listed
total
by intra-
liver
in table
in a preparation material
Of the
l/3 l/2
by an material
daltons. about
the percentages
obtained
the greatest
was retained
molecules
was used as starting
680
By far
of 30,000
UM 2 ultrafilter. with
presumably
have been associated
smaller
When copper
h. before
Fig 1 shows the G-75 elution four
with
by an Amicon
in the various
and in the other
centrifugation
supernatant
greater
was associated
l/2
soluble must
G-75.
(22)
fractions.
therefore,
on Sephadex
with
speed
subcellular
in association
total)
high
and,
was retained
(or
after
particulate remaining
size
chemically
spectrometry.
was removed
of the copper PM 30 ultrafilter
was determined
absorption
which
mainly
Amicon
of the
% of total
cu (119)
homogenate
weight
where
I. about
as in the experi-
Vol. 66, No. 2, 1975
BIOCHEMICAL
20
AND BIOPHYSICAL
40
60
80
FRACTION
100
RESEARCH COMMUNICATIONS
120
NUMBER
Figure 1: Gel filtration of ultrafiltrate (Amicon PM 30) of high speed supernatant from liver homogenate on a Sephadex G-75 column (2.5 x 90 cm) in 1 mM Tris-HCl, pH 8.6. 5 ml fractions were collected and monitored for absorbance at 2 wavelengths and copper content as indicated. The arrows indicate the positions of the void and bed volumes of the column.
ment represented
in table
at 250 and 280 nm. small
amounts
copper
portion that
of the
peak were
content
molecular
weight
animals,
gel
polypeptide 7,000
separately step.
of about
between
with
visibly
(in
fractions
shoulder
latter
part
and only
the
Analysis
of the material
2.5
g atoms
per mole
In several
1 and 5 g atoms (fig
molecular
weights
Over
and the major processed
through
on the
preparations
the
of approximately
the
peak
12,000,
than of the subse-
revealed
basis
of a mean
from
copper-loaded
obtained.
presence
is
the major
portion
the major
per mole were
B) showed
There
at 250 nm is greater
(calculated
of copper
2, gel
of 250 ml.
from
other
and only
and 90 to 100).
absorbance
the
peak of absorbance
non-proteinaceous
75-83 volume
the
comprising
is a large yellow,
at an elution 42 to 69)
electrophoresis
bands
are
present
of 9000 daltons).
values
acrylamide
are
(fractions
exchange
a copper
fractions
The fractions
pooled
anion
Near the bed volume
peak centred
peak
at 280 nm.
quent
These
of copper
a principal
I.
Poly-
of three 9,000
major
and
daltons. Amino
acid
analyses
yielded
the
composition
681
shown in table
II
which
also
Vol.66,No.
BIOCHEMICAL
2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
_*
-lb d
-
A
ti
-me
-
%“n
D
E
F
staining (Coomassie blue) bands after polyacrylamide Figure 2: Protein gel electrophoresis in system (c) of Methods. Samples were prepared by incubation in a boiling water bath for 2 min. in 1% SDS and 40 mM dithioThe following amounts of protein from the chromatography fractions threitol. Gels A and B: 25 pg from fractions 30-41 and indicated were applied: Gels C,D,E: 25, 18, 17 pg from fractions 42-69 of fig 1 respectively. 10-15, 16-40, 41-46 of fig 3 respectively. 1Opg each of chymotrypsinogen, hemoglobin, lysozyme and cytochrome C were applied to gel F.
includes for
for
comparative
purposes
a similar
preparation
from
copper
protein
et al
(25).
rations four
isolated The amount
are very times
proteins
from
less present
The three
major
the
loaded
livers
recently
rat
liver
of a Wilson's
Most notable
that
after
copper
composition
of each of the amino
similar. than
the
acids
loading
with
other
copper-associated
trace
metals
by Evans
as those patient
in the three values
the major
polypeptides
682
as well disease
are the cysteine
of the metallothioneins,
reported
(19)
of a smal by Morel1
different which
prepaare about
low molecular
weight
such as Cd++ and Zn++. of our G-75
copper
peak
BIOCHEMICAL
Vol. 66, No. 2, 1975
Table
Amino
acid
Amino
acid
composition
AND BIOPHYSICAL
II
of low molecular
Cu-loaded
rat
acid
copper
liver
A Aspartic
RESEARCH COMMUNICATIONS
proteins
Liver
from
of Wilson's patieni?
11.4
8.8
Threonine
7.9
7.2
5.8
Serine
7.3
7.6
7.1
12.6
11.9
11.2
Proline
4.0
3.9
5.5
Glycine
10.1
8.8
8.6
Alanine
4.9
6.8
8.2
Valine
4.3
5.1
7.1
Half-cystine
7.5
5.0
4.3
Methionine
3.2
Isoleucine
5.0
4.3
4.9
Leucine
6.2
7.0
6.7
Tyrosine
2.0
1.1
Phenylalanine
3.7
3.1
acid
Lysine
1.5
13.2
Histidine
1.7
Arginine
2.5
13.0
3.0
proteins
obtained
from
B
proteins
obtained
by Evans
a
expressed
b
from
Morel1
et al
10.8 1.5
A
as residues
disease
B
10.2
Glutamic
liver.a
gel
per
filtration et al 100
(25).
683
2.6 step
(19).
(fractions
42-69
of fig.
1)
VoL66,No.
were for
2, 1975
further
resolved
elution
tended
necessary
for
exclusively peak
(fractions
(fig
2, gel
smearing
resolution.
16-39)
the
polypeptides
to cause
polypeptide
D),
largest
weighing
contained
whereas
the
that
very
in two non-dissociating
three
bands
observed
9,000
system
(fig
peak
dalton
(fig
peak
are a result
2, gel
forms systems
of the
Therefore, of disruption
10-15) C),
the
contained broad
copper
and 12,000
daltons
40-46)
seeming
contained
staining
also
of subunit
raises
but gel showed
intensities
seems highly
individual
similarity
same protein
(see Methods)
it
but was
of none of the
Their
used
proteins
7,000
(fractions
contents
together.
gradient
(fractions
weighing
the same relative 2).
salt
- containing
small
copper
may be different
having
The shallow
The copper
electrophoresis
in the dissociating
copper
of the three
they
bands
of the
sharp
that
separated
3).
the polypeptides
polypeptide.
exceeded
A (fig
The first
the possibility
narrowly
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on DEAE Biogel
their the
primarily
BIOCHEMICAL
three
as seen
unlikely
that
interactions.
DISCUSSION We have been able
to purify
three
FRACTION
Figure (42-69 with a (mmho)
different
small
proteins
with
which
NUMBER
of pooled gel filtration fractions 3: Anion exchange chromatography of fig 1) on a DEAE Biogel A column (2.5 x 20 cm). Elution was salt gradient from 0 to 0.1 M NaCl which resulted in the conductivity rise shown.
684
the
BIOCHEMICAL
Vol. 66, No. 2, 1975
copper
is
copper.
associated
from
The resolution
been studied
characterization
of these
proteins.
particular
investigators
to avoid
solvent
all
three
as the metallothioneins amino
imately
present from
acid
makes it
animals
content unlikely
of the
As Evans
livers
is
required
that
copper
Wilson's
further
proteins
of have
towards
of the functions we have taken
methods
instead
amounts state
in the
of protein
on very
high
a very
large
speed
respect
metals
pointed
very
the
on copper
this
of such proteins and possibly
that
is livers
here
(26).
L-6D isolated
and co-workers loading
than
it
may also
its
metallothionein
similarity,
others
approx-
of metallothionein
of each of the three
or disprove
the derived
described
by Scheinberg
appearing
from
little
protein
range
drastically
is noteworthy
than
out,
disease
it
contain
proteins
deviation
amount
weight
differ
metallothionein
significant
In this
same molecular
of proteins
of which
characterization
to confirm
pathological
are
the
basis
associated
has already
increased
in this
on the that
to the
Although
isolated
In fact
no trace
with
to be more similar
speculate
step
step
and other
the two groups
liver.
(19)
of a patient
thionein.
proteins
administered
and much less
proteins
understanding
relying
doses which
a further
purification
step,
compositions.
in copper-loaded
liver
final
high
filtration
represents
fractionation
(5,6,9,10)
30% cysteine
name (1)
by gel
a fuller
filtration
fairly
and ultrafiltration.
Although
in total
(8,9,21)
to this
the gel
administered
obtained
and hopefully
before
centrifugation
of rats
components
In addition
care
precipitation
livers
of the
by other
complete
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
from the (25)
a metallopurified
is
seems
copper
tempting
be synthesized
to in
(27).
ACKNOWLEDGEMENTS This indebted recent
work was supported
to Dr. findings
G. W. Evans for available
by the Medical making
a preprint
to us.
685
Research
Council
of a manuscript
of Canada. describing
We are his
Vol. 66, No. 2, 1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES Margoshes, M. and Vallee, B.L. (1957) J. Am. Chem. Sot. 79: 4813. K;igi, J.H.R. and Vallee, B.L. (1960) J. Biol. Chem. 235:3460. Ksgi, J.H.R. and Vallee, B.L. (1961) J. Biol. Chem. 234: 2345. i: B.L. (1966) Bioxm. 5: 1768. Pulido, P., Kagi, J.H.R. and Vallee, J.H.R., Himmelhoch, J.R., Whanger, P.D., Bethune, J.r. and 5. Kigi, Vallee, B.L. (1974) 3. Biol. Chem. 249: 3537. 6. Shakh, Z.A. and Lucis, O.J. (1971) Gerientia 27: 1024. 7. Nordberg, G.F., Nordberg, M., Piscator, M. and Esterberg, 0. (1972) Biochem. J. 126: 491. Winge, D.R. and Rajagopalan, K.V. (1972) Archiv. Biochem. Biophys. 153: 755. 9”: Webb, M. (1972) Biochem. Pharmacol. 21: 2751. Buhler, H.O. and Kagi, J.H.R. (1974)FEBS Letters 39: 229. ::: Piotrowski, J.K., Bolanowska, W. and Sapata, A. (lT3) Acta Biochem. Pol. ::
20: 207. 12. Femner, I., Davies, W.T. and Mills, 13. Davies, W.T., Bremner, I. and Mills, 14. Kimura, M., Otaki, W., Yoshiki, S., 15. 16. 17. 18.
C.F. (1973) Biochem. Sot. Trans. 1: 982. C.F. (1973) Biochem. Sot. Trans. i: 985. Suzuki, M., Horiuchi, N. and Suda,f. (1974) Archiv. Biochem. Biophys. 165: 340. Squibb, K.S. and Cousins, R.J. (lm Environ. Physiol. Biochem. 4: 24. Chen, R.W., Whanger, P.D. and Weswig, P.H. (1975) Biochem. Med. 12: 95. B. and Nordberg, G.F. (1974) Environ.Physiol. Nordberg, M., Trojanowska, Biochem. 4: 149. 8. and Sapota, A. (1974) Arch. Toxicol. PiotrowskT, J.K., Trojanowska,
32: 351. G.W., 19. Fans, 20. 21. E: 24. 25. 26. 27.
in press. Winge, D.R.,
Wolenetz, Premakumar,
M.L.
and Grace,
C.I.
(1975)
Nutr.
R. and Rajagopalam,
K.V.
(1974)
Reports Fed.
Int.
Proc.
%~orn~",""; C and Sourkes T L (1973) Biochem Med 8. 78. Eden, A: and'Green, H.H. [19iOj Biochem. J. 34; 12t)2:’ W. and Udenfsend, S. (1973) Archiv. BBhlen, P., Stein, S., Dairman, Biochem. Biophys. 155: 213. McConnell, D.B. and Aspin, N. (1970) Int. J. Marceau, N., Kruck,P.A., Appl. Radiat. Isotop. 21: 667. Morell, A.G., Shapir0,T.R. and Scheinberg, I.H. (1961) in Wilson's J.M. Walshe and J.N. Cumings, Eds. Disease: Some Current Concepts. p. 36. Blackwell, Oxford. Riordan, J.R. and Gower, I. (1975) Biochim. Biophys. Acta. Submitted for publication. V.M. and Sherlock, S. (1968) Smallwood, R.A., Williams, H.A., Rosenoer, Lancet II: 1310.
686
