Vol. 81, No. 2, 1978
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 469-472
March 30,1978
ACTIVE
SUBUNITS
Joe V. Bannister, Dept.
FROM
Angela
of Physiology
Anastasi
February
DISM UTASE
and William
and Biochemistry, Msida
Received
SUPEROXIDE
University
2,1978
When copper-zinc
superoxide
dismutase
is dialyzed
against
the enzyme
dissociates
8 M urea,
are catalytically
Introduction
enzyme
contains
2 atoms
functional
metal
with
a molecular
be obtained
in the presence
bridges
however,
reported
into monomers
from
swordfish
urea
(4).
erythrocyte decided
of about
16,000
that during dissociates
For-man
enzyme to investigate
is active
lauryl
could
and Fridovich wheat
from
(3)
germ
sulphate
have,
dissociates
alone,
whilst
the enzyme
in the presence
had reported
of urea
only
even though
electrophoresis,
in the presence
the effect
to
that no interchain
into monomers
and Fridovich
is the
erythrocytes
weight
from
detergent-gel
and
to be resistant
(1)
Beauchamp
of sodium
is a
33,000
bovine
molecular
that one of the isozymes
liver
from
it has been found (2).
1.15.1.1)
The copper
of 2-mercaptoethanol
in the presence
Since
of zinc.
The enzyme
are present
observed
liver
into monomers
(E.C.
and it has been found
of the protein,
disulphide
swordfish
of approximately
and 2 atoms
studied
Monomers
dismutase
weight
in the enzyme.
dissociation.
we have
superoxide
of copper
has been extensively
the sequence
from
active.
: Copper-zinc
dimeric
of Malta
-Malta
Summ ax-y:
which
H . Bannister
of
that the bovine
of 10 M urea
on the enzyme
from
(51,
we
swordfish
liver.
,M aterials liver
and Methods:
of the swordfish
described
(4).
Copper-zinc (Xiphias
Solutions
superoxide
gladius)
dismutase
was prepared
of the enzyme
469
(30 mg’/ml)
from
the
as previously were
dialyzed
Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 8 1, No. 2, 1978
BIOCHEMICAL
40
50 Fraction
Fig.
1.
A,
indicate
and (c)
Fig.
2.
(A-A)
Fridovich
volume
(b)
(O-O)
protein
15
= 1.8 ml.
chymotrypsinogen
native
concentration
(6)
pH 7.4 containing
Am berlite
. Chromatography AC 54 (LKB buffer.
augmentation
photooxidation monitored
ovalbumin,
Time 10 (Min)
in 8 M urea
Fraction
reaction.
Final
buffer
with
the dialysis
photochemical
oxide
of the assay
enzyme.
Ultrogel with
dismutase
pH 7.4.
of (a)
5
enzyme in the cuvette
mg/ml.
England)
brated
Results
course
was de-ionized
containing
0
c.
0.1 M phosphate
Poole,
buffer,
peaks
cytochrome
Time
was 0.027
urea
elution
monomeric
against
of 30 mg superoxide
0.1 M phosphate
Arrows
02
60
No.
Gel filtration
containing
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
which
takes
of dianisidine
M onobed
dismutase
behaves
AB,
Enzyme
assay advantage
as a dimer
Bromma,
described
by Misra
by riboflavin.
to be unaffected
state
470
equiliby a and
of aerobic
The reaction
is
by 8 M urea.
swordfish
of 32,500
90 x 1.5 cm
Sweden)
rate
The
( B .D . H . Ltd. ,
was measured
of the increased
sensitized
In the native
Resin
at 4OC.
out on a column
activity
recently
at 460 nm and was found
and Discussion:
MB-3
was carried Produkter
8 M urea
molecular
copper-zinc weight
superwith
the
20
BIOCHEMICAL AND BIOF’HYSICAL RESEARCH COMMUNICATIONS
Vol. 81, No. 2, 1978
0 0
12
6
16
Protein
Fig.
3.
native
Reaction
rate
enzyme,
activity
@-A)
in 8 M urea
same
activity
native
cular
and 50% glycerol
enzyme
dialyzed
against
into monomers
weight
of 16,300. the activity
monomer
tended
to show
concentration
lower reaction
lower
These
might
show
cycling
Fielden
--et al. (8) A detailed kinetic superoxide
AC 54 of
that the enzyme patter
a mole-
(in 8 M urea)
was
and the
of both time concentration
than the dimer of the medium
readily
indicated
At low protein
indicate
be able to act independently of superoxide
redox
on Ultrogel
as a function
as similarly
investigations
the dismutation that
activity
lowered
(Fig. ti
and the
3) . This
the fact that the
low activity
was present
in
3).
preliminary
the enzyme
concentration.
. Both the dimer
activity
is due to the viscosity
(Fig.
protein
of the monomer
2 and 3).
(7)
indicate
indicates
dimer
(O-O)
Arrows
The elution
the same
is diffusion-limited
50% glycerol
8 M urea
30
concentration.
Gel filtration
of native
(Figs.
had a slightly activity
(4).
The activity
with
monomer
at low
(Fig.1).
compared
protein
enzyme.
24
(pg/ml)
of protein
monomeric
as bovine
enzyme
dissociates
as a function
concn.
radicals.
of the copper
is not an absolute study
dismutase
of monomeric
that the two active of each other
The results in dim eric necessity
471
enzyme
when also
swordfish
in
catalyzing seem
, as proposed
for the dismutase
and dimeric
is in preparation.
would
sites
activity. liver
to by
Vol. 8 1, No. 2, 1978
BIOCHEMICAL
Acknowledgements and helpful financial
: We thank
comments
:
1.
B.B.,
Keele, Chem., Abernethy,
5.
Irwin
thanks
Fridovich
the Wellcome
for advice Trust
for
Steinman,
J.L.,
C .O.
I.,
(1971)
H.M.
R.L.
J. Bid.
andHill,
(1974)
J. Biol.
and Fridovich,
I.,
(1973)
Biochem
and Bannister,
W.H.
. Biophys.
50-56.
Bannister,
J.V.,
Biochem
. Physiol.,
Forman,
and Fridovich,
7339-7347.
Beauchamp, 317,
J.M.
2875-2880.
249,
Acta, 4.
W.H.B.
McCord,
246,
Chem., 3.
Professor
support.
References
2.
.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
H.J.
Anastasi, 56B,
A.
(1977)
Comp.
235-238.
and Fridovich,
I.,
(1973)
.I. BioI.
Chem.,
248,
2645-2652. 6.
Misra, 181,
H.P.
and Fridovich,
I.,
(1977)
Arch.
Biochem.
Biophys.,
308-312.
7.
Fridovich,
I. , (1974)
8.
Fielden,
E.M . , Roberts,
Mautner, J ., 139,
G.N.,
Rotilio,
Adv.
Enzymol.,
P.B.,
Bray,
41, 35-97. R.C.,
G. and Calabrese,
49-60.
472
Loewe, L.,
D.J.,
(1974)
Biochem.