BIOCHEMICAL
Vol. 86, No. 4, 1979
AND BlOFHYSlCAL
RESEARCH COMMUNICATIONS Pages 1199-1205
February 28, 1979
DIFFELII~,JTIAL EFFECTS OF WE1 ON YEAST 4&D BOVINB COPPER, ZIXC SUPEROXIDE DISMIJTASES, IX HELATIOX +
, F.Bossa
D.BarraO
TO %I-IE EXTENT OF ANALOGY OF PRIMARY STRUC'I'URES +
, F.%rmocchi
',
, F.Martini
A.Rigo*
0
and G.Rotilio 0
+
Isti-tuto
Biologica,
Biologica,
IJniversit&
di Roma e Centro
tuto Received
Universita
di Chimica
December
di
29,
Chimica
di
di Biologia
Fisica,
Istituto
Camerino;
di Chimica * Istide1 C.N.R.;
Molecolare
JJniversitB
di
Venezia
1978
Summarx. Incubation smutase with bi;h$r
in 8X urea (pH 7.4) inactivated yeast Cu, Zn superoxide difi;"t order kinetics (k for the decrease from lOO$ to 167; ; .X for the decrease from 16$ to 0.1): activity = 2.5 ;cj;lip& 5 was fully reversible on dilution with or dialy. The inactivation sis against urea-free buffer. No inactivation was shown to occur in similar experiments with the bovine Cu, Zn enzyme. EPR spectra recorded immediately after addition of 81.; urea showed a more axial line shape and a higher B,, of the copper signal typical of the native enzyme. In the case of the yeast enzyme, this change was more pronounced and further incubation led to a new type of copper signal, typical of the inactivated enzyme. All EPR changes were reversible. Comparative analysis of the amino acid sequence of the two enzymes showed substantial identicontributin. ty of the protein regions g the ligands to the metals and the disulfide bridge. Differential destabilization of active sites by urea should be due to replacements in other protein segments, such as the three C-terminal and some N-terminal residues.
in a recent
report
mers by &1 urea organisms. and
efficiency
class
with
respect
nisms
that
-reactivity
that
features
other
It
is
correlations
distant
parameters
properties,
brought
that
besides
the stability
respect about
Along
dismutases
to reversible by treatment
line
and
properties in partiarrangement
the behavior isolated
,
from
orga-
to make structure-
we have investigated
modifications with
proto-
different
dismutases,
to compare
tree, this
from widely
of the dimeric
interesting
in the phylogenetic
into
the spectral
in Cu, Zn superoxide
therefore
basis.
dissociation
dismutases
of Cu, Zn superoxide
on this
enzymes with
evidence
superoxide
determining
to a Same reagent, are widely
zinc
are conserved
of proteins.
and bovine
site-linked
we have presented
in copper,
[This indicates
the structural
of this
yeast
occurs
catalytic
cular
(1)
814 urea.
the
of some active Tne observed
0006-291X/79/041199-07$01.00/0 1199
Copyright All rights
@ 1979 by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 86, No. 4, 1979
differences
in
the
amino
the
stability
MATERIALS
BIOCHEMICAL
sensitivity
acid
to urea
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were
sequences
, particularly
of the metal
binding
confronted within
with
regions
the degree
of analogy
of crucial
of
importance
to
sites.
AND METHODS.
Yeast (Saccharomyces cerevisiae) superoxide dismutase was prepared by the method of Goscin end Fridovich (Z)., The apoprotein was prepared incubating solutions of the enzyme in 7G$ formic acid at 0" for 2 hours, followed by extensive dialysis against water and lyophilization. Titration of sulfhydryl groups with 5,5'-dithiob&(2-nitrobenzoic acid) was performed according to Cavallini et .al. (3). Reduc tion and allqylation with radioactive iodoacetic acid of the apoprotein (210 mg) in 6.0 I guanidine hydrochloride was performed according to a procedure proved to be effective for the human Cu, Zn superoxide dismutase (4). Digestion with 2.5 mg of Staphylococcus aureus ~8 protease (Miles Biochemicals) was carried out in 0.1 M ammonium bicarbonate at 37% for 24 h. Digestion was stopped by addition of acetic which was completely soluble in this meacid (10% final) and the peptide mixture, dium, was fractionated on a Sephadex 625 column (4x130 cm) equilibrated end eluted with the same solvent. Techniques employed for further purification and subsequent analysis of peptides were as reported in (4). Bovine superoxide dismutase,was purified according to McCord and Fridovioh (5). Urea solutions were deionized before use with AG 501 K8D mixed-bed resin (Bio Rad). Superoxide dismutase activity was assayed according to Rigo et al. (6). ERR measurements were recorded with a Varian E-4 spectrometer at appoximately 9.11 GHz and -150°C.
RESULTS AND DISCUSSION. Effect
of urea
tions xide
(0.1
ted by taking
bovine
-6
M) led
of the native into
strongly
mer (8).
buffer,
( 2 10
typical
refore
on enzyme activity.
M phosphate
dismutase
of that
urea,
treatment
account
supports
the yeast
16% of the initial wer first
order
to immediate enzyme.
effects
yeast
drop
This
that
time
the activity enzyme displayed dependence,
value decay
value
after
that
recorded
a first
in
8M urea
immediately activity order
(k = 2.5 x
min-l
1200
) to a final
that
predic-
(7)
and the-
as active
fall. activity
two days (k = 6.5 x 10 -3 min-') 10 -3
down to 5%
viscosity
the monomer is
value
solu-
Cu, Zn supero-
is approximately
by 8M urea
a further is
or bovine
to buffered
of enzyme activity
decrease
on diffusion
of 8M urea
at 22OC of the enzyme solutions
enzyme retained
had a biphasio
of either
the hypothesis
On incubation
while
pH 7.4)
Addition
as the di-
for
after This
4 deys,the addition inactivation
decrease
down
and a second of 0.1%
of
activity
to slo-
Vol. 86, No. 4, 1979
after
4 days.
solutions,
Hundred-fold
led
tivated EPR
recovery
affected
inactivated
enzyme
up to 5@ of the value
1 min end to a lOO$ recovery by dialysis
yeast
than
spectral
shapes
the g,, regions
of activity
against
of 8M urea
(7)
evidence
buffer
is
gously
to the effects
Sequence
typical
within
8
of the urea-inac-
homologies
primary
chemical
as to allow conditions
we first
zation
further
dialysis
frame
the bovine intenance
enzyme,
The half-cystine
this
of identical
our attention
for
arrangement
to be pin-pointed
reported
which
is
sites
feature
1201
even
out.
Analo-
of the yeast All
dramatic
are
but
effects
difference site
of
explanation (2).
to be found
in Neverin
the
to be as conservative as variant
To unravel
the compactness
the yeast
EPR
had practically
are identical
as to create such structural
and subsequent
which
of the molecule
for
be ruled
of the active
expected
same sites.
in a relatively
in urea
has no apparent
on the existence
a chemical
spectrum
c and cl).
properties
stability
active
viscosity
Therefore
site
The rather
study
which
of the
to be relevant
content
under
of the proteins,
i.e.
ad-
the EPR spectrum
enz,ymes.
differential
stability
was specific,as
buffer.
of the two enzymes,
bonds,
of the dimeric
amenable
and yeast
dismutases
different focussed
urea-free
of the most specific
for
in
incubation
1, curves
the
constant
enzyme).
can not
converted (Fig.
cases
of the
of the active
of signal
against
of bovine
bases
it
in the
the same relative
on the bovine
on the enzyme activity,
new form
splitting
significant-modifications
effects
properties
of disulfide
as to give
while
dismu-
distinctive
In both
on the ERR spectra
to freezing
the formation for
and the hyperfine
of the result
enzyme,
were
b and b').
modifications
the two CU, Zn superoxide the spectroscopic
curves
d shows the effect
to 8M urea
structural
I,
superoxide
were much more pronounced
of specific
after
theless
changes
effect
in much less
an entirely
in sensitivity
changes
(Fig.
The urea
on the bovine
reversible
, and the spectral
Immediate
dismutase
1,
in favor
enzyme into
or yeast
became more axial
(Fig.
attribution
no effect
bovine
at such a concentration
though
were
1).
resulted
enzymes
of either
treatment
increased.
of sucrose
of both
(Fig.
in the bovine line
dition
EPR spectra
by urea
in the two enzymes
is
of the fully
of activity
was obtained
The copper
were
bases
buffer
enzyme.
spectra.
tase
with
recovery
enzyme within
Similar
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dilution
to en abrupt
of the native hours.
BIOCHEMICAL
is
proved,
of the subunit (9,ll)
characteriat least
for
end the ma-
and at the same time
easy manner. enzyme is
3.5-4
residues
per di-
Vol. 86, No. 4, 1979
Fig.
1 - EPR (a): (b) dition piq
mer,
as measured
lue
BIOCHEMICAL
spectra of bovine and ye?tt Cu, Zn superoxide dismutase. bovine enzyme (0.8 x IO M) in 0.1 M phosphate buffer, pH 7.4; and (0): as (a), frozen immediately (b) or 24 hours (c) after-t+ of 81.1urea at ropm temperature;,(d): bovine enzyme (1 x 10 UI) 0.5.M sucrose; (a) , (b) and (c) are the yeast enzyme (1.5 x I) In the seme conditions as (a) (b) and (c). Final concentrations of enzymes were obtained by addition of a few microliters of a coneentrated stock solution of protein to relatively large volumes of either buffer alone or buffer containing 8M urea.
as cysteic
acid
after
of carboxymethylcysteine
(4.1)
iodoacetate
of the apoenzyme
under
with
-SH groups
per
dimer
in 8.0 M urea,
spectively
for
the apo-
under
treatment.
results,
me in 8M urea
(I),
per
feature
subunit,a
From the digest
after
(6.0
indicate
conditions with
S.aureus
acid)
while
with
the established
the presence
psotease
1202
gave 3.9
with hydroand 4.2
borohydride,
no -SH group
omission
dissociation
from higher
of the reduced,
rewas ti-
of the reduction
of anintrasubunit protein
va -
M guanidine
sodium
pracCicr$.l.;;
but with
a similar
and treatment
conditions
common to the homologous with
reduction
reduction
and hoLo-enzyme,
together
clearly
We found
5,5'-dithiobis(2-nitrobenzoic
the same denaturing These
(2,12).
after
denaturing
Titration
trated
oxidation
content
chloride).
11).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
enzy-
disulfide
bond
organisms
(4,3,
carboxymathylated
apoen
Vol. 86, No. 4, 1979
BIOCHEMICAL
me,
two radioactive
Thejr
sequence
chain
of -the bovine
(SA-1
and SA-3)
isolation
is reported
bovine
enzyme is around
His 69,
the bovine
three
the overall
118)
ronment
is
the residues
enzyme were
also
around
the
lytic
sLmilarities
k?hile
this
the N-terminal
region
data
from Petersen
et al.
with
the bovine
cate
an
is
the
two
for
about
46,$
portions that,
beside bond
i.e.
the four
amino acid
residues
of the
zinc
strongly
atom.
protein,
extent
related
,
also that of
44,
fourth
46
ligand
of conservation
to the
the
Conservation
histidines
on the
of
of the
disulfide
we became aware
,
of the envi-
spectroscopic
In fact
(12).
c, is
and cata-
than
contributing
other
a clue
of the yeast
that
this
high
evolution
enough
fragments
to indito that
repor-
substantial
inva-
likely
to be essen -
properties,
such as tho-
ligands
enzymes can be found
are at the subunit
the chain
comparable
above suggest
and catalytic
confirmed
bridge. On the -and the disulfide the differential effects of urea on the active
to explain
of the X-terminal
though
regions
peptides
polypeptide
the polypeptide
of protein
sequen
of the present
fully of
group
terminal
protease
analysis stretch
through
for
of spectroscopic
and bovine
of S.aureus
value,
presented
dismutases
the metal
array
of this
found
the results
the course
and their
Comparison
by a danish
of the amino
during
the whole
of the molecule
lower
of a report
54 residues
of the molecule
of stability
to the maintenance
dues and part
2 encompassing
intrasubunit
2. Such a high
(12).
In conclusion,
that
the
observation
up to now available
to purify
in Cu, Zn superoxide
regions
for
in the
enzyme shows 42$ homology;
extent
hand,
is the
of the first
has been possible
se peptides
tes
is
dismutase
from
tial
performed
accounting
of the bovine
was in preparation
it
riancy
ligands
two metals
work
ted here.
of two more peptides
the corresponding
to be ligands
information
superoxide
of cytochrome
with
conserved,
in fig.
in fig. thus
interest
proved
the determination
ce of yeast
the polypeptide
of the two proteins.
paper
concerning
along
chromatographies
residues,
involved
copper
given
and sequenced.
the sequence
reported
homology
6E;$. Of immediate
and 61 (no sequence 3is
exchange
69 amino acid
out of the four
isolated
by homology
with
chain
78 and Asp 81 are
Ifis
were
fragments.
of polypeptide
subunit:
homology
t ogether
by the ion
include
entire
and SA-4) 2, aligned
enzyme (II),
of the radioactive
half-cystines,
in
(U-2
in fig.
purified
Tne two strectches
the
peptides
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
interface, segment
in
substitutions
such as the last (9).
1203
observed
few C-terminal
siin resi-
b-SOD
y-SOD
b-SOD
b-SOD
Fig.
sA4
2 - Comparison of the primary structure of bovine (b-SOD) and Saccharomyces cerevisiae (g-SOD) superoxide dismutases. Arrows under the yeast enzyme with symbols SR denote peptides derived from digestion of carboxymethyBoxes enclose residues identical lated protein with S.aureus protease. in the two proteins. For the bovine onsyme residues that are ligands of the copper (circles) and the zinc (squares) atoms and the position of the disulfide bond are also indicated.
.
A~-A~-~HR-LYS-~~L~~-VHL-CYS-V~L-LEU-LYS-GLY~GLY-~SP-GLY-PRO-V~L-GLN-GLY-THR-ILE-HIS-PHE-GLU-HLH-LYS-GLY-~SP-THR-VHL-VAL-VHL-THR-
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. larmocchi,F., Venardi,G., Bossa,F., Riga,A. and Rotilio,G. (1978) FEBS Lett., 94, 109-111. 2. Goscin,S.A. and Fridovich,I. (1972) Biochim.Biophys.Acta, 289, 276-283. 3. Cavallini,D., Graziani,M.T. and Orlando,M. (1966) Ital.J.Biochem., 15,268-272. 4. Barra,D., Martini,F., Bossa,F., Rotilio,G., Bannister,J.V. and Bannister,W.H. (1978) Biochem.Biophys.Res.Cormnun., 81, 1195-1200. 5. McCord,J.K and Fridovich,I. (1969) J.Biol.Chem., 244, 6049-6055. 6. Rigo,A., Viglino,P. and Rotilio,G. (1975) Anal.Bioche%, 68, 1-8. 7. Handbook of Chemistry and Physics (Weast,R.C. ed.) 56 Edition 1975-1976, p.D-264, CRC Press, Cleveland. 8. Bannister,J.V., Anastasi,A. and Bannister, W.H. (1978) Biochem.Biophys.Res. comnlun., 81, 469-472. 9. Richardson,J.S., Thomas,K.E., Rubin,B.M. and Richardson,D.C. (1975) Proc.Natl. Aoad.Sci.U.S.A., 72, 1349-1353. 10. Beauchamp,C.O. and Fridovich,I. (1973) Biochim.Biopbys.Acta, 317, 50-64. 11. Steinman,H.K, Naik,V.R., Abernethy,J.L. and Hil1,R.L. (1974) J.Biol.Chem., 249, 7326-7338. 12. Petersen,C., Hasemann,V., Martin,B.f Johansen,J.T., Svendsen,I. and Ottesen,M. (1977) Carlsberg Res.Commun., 42, 391-395.
1205
Vol. 86, No. 4, 1979
AND BlOFHYSlCAL
RESEARCH COMMUNICATIONS Pages 1199-1205
February 28, 1979
DIFFELII~,JTIAL EFFECTS OF WE1 ON YEAST 4&D BOVINB COPPER, ZIXC SUPEROXIDE DISMIJTASES, IX HELATIOX +
, F.Bossa
D.BarraO
TO %I-IE EXTENT OF ANALOGY OF PRIMARY STRUC'I'URES +
, F.%rmocchi
',
, F.Martini
A.Rigo*
0
and G.Rotilio 0
+
Isti-tuto
Biologica,
Biologica,
IJniversit&
di Roma e Centro
tuto Received
Universita
di Chimica
December
di
29,
Chimica
di
di Biologia
Fisica,
Istituto
Camerino;
di Chimica * Istide1 C.N.R.;
Molecolare
JJniversitB
di
Venezia
1978
Summarx. Incubation smutase with bi;h$r
in 8X urea (pH 7.4) inactivated yeast Cu, Zn superoxide difi;"t order kinetics (k for the decrease from lOO$ to 167; ; .X for the decrease from 16$ to 0.1): activity = 2.5 ;cj;lip& 5 was fully reversible on dilution with or dialy. The inactivation sis against urea-free buffer. No inactivation was shown to occur in similar experiments with the bovine Cu, Zn enzyme. EPR spectra recorded immediately after addition of 81.; urea showed a more axial line shape and a higher B,, of the copper signal typical of the native enzyme. In the case of the yeast enzyme, this change was more pronounced and further incubation led to a new type of copper signal, typical of the inactivated enzyme. All EPR changes were reversible. Comparative analysis of the amino acid sequence of the two enzymes showed substantial identicontributin. ty of the protein regions g the ligands to the metals and the disulfide bridge. Differential destabilization of active sites by urea should be due to replacements in other protein segments, such as the three C-terminal and some N-terminal residues.
in a recent
report
mers by &1 urea organisms. and
efficiency
class
with
respect
nisms
that
-reactivity
that
features
other
It
is
correlations
distant
parameters
properties,
brought
that
besides
the stability
respect about
Along
dismutases
to reversible by treatment
line
and
properties in partiarrangement
the behavior isolated
,
from
orga-
to make structure-
we have investigated
modifications with
proto-
different
dismutases,
to compare
tree, this
from widely
of the dimeric
interesting
in the phylogenetic
into
the spectral
in Cu, Zn superoxide
therefore
basis.
dissociation
dismutases
of Cu, Zn superoxide
on this
enzymes with
evidence
superoxide
determining
to a Same reagent, are widely
zinc
are conserved
of proteins.
and bovine
site-linked
we have presented
in copper,
[This indicates
the structural
of this
yeast
occurs
catalytic
cular
(1)
814 urea.
the
of some active Tne observed
0006-291X/79/041199-07$01.00/0 1199
Copyright All rights
@ 1979 by Academic Press, Inc. in any form reserved.
of reproduction
Vol. 86, No. 4, 1979
differences
in
the
amino
the
stability
MATERIALS
BIOCHEMICAL
sensitivity
acid
to urea
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were
sequences
, particularly
of the metal
binding
confronted within
with
regions
the degree
of analogy
of crucial
of
importance
to
sites.
AND METHODS.
Yeast (Saccharomyces cerevisiae) superoxide dismutase was prepared by the method of Goscin end Fridovich (Z)., The apoprotein was prepared incubating solutions of the enzyme in 7G$ formic acid at 0" for 2 hours, followed by extensive dialysis against water and lyophilization. Titration of sulfhydryl groups with 5,5'-dithiob&(2-nitrobenzoic acid) was performed according to Cavallini et .al. (3). Reduc tion and allqylation with radioactive iodoacetic acid of the apoprotein (210 mg) in 6.0 I guanidine hydrochloride was performed according to a procedure proved to be effective for the human Cu, Zn superoxide dismutase (4). Digestion with 2.5 mg of Staphylococcus aureus ~8 protease (Miles Biochemicals) was carried out in 0.1 M ammonium bicarbonate at 37% for 24 h. Digestion was stopped by addition of acetic which was completely soluble in this meacid (10% final) and the peptide mixture, dium, was fractionated on a Sephadex 625 column (4x130 cm) equilibrated end eluted with the same solvent. Techniques employed for further purification and subsequent analysis of peptides were as reported in (4). Bovine superoxide dismutase,was purified according to McCord and Fridovioh (5). Urea solutions were deionized before use with AG 501 K8D mixed-bed resin (Bio Rad). Superoxide dismutase activity was assayed according to Rigo et al. (6). ERR measurements were recorded with a Varian E-4 spectrometer at appoximately 9.11 GHz and -150°C.
RESULTS AND DISCUSSION. Effect
of urea
tions xide
(0.1
ted by taking
bovine
-6
M) led
of the native into
strongly
mer (8).
buffer,
( 2 10
typical
refore
on enzyme activity.
M phosphate
dismutase
of that
urea,
treatment
account
supports
the yeast
16% of the initial wer first
order
to immediate enzyme.
effects
yeast
drop
This
that
time
the activity enzyme displayed dependence,
value decay
value
after
that
recorded
a first
in
8M urea
immediately activity order
(k = 2.5 x
min-l
1200
) to a final
that
predic-
(7)
and the-
as active
fall. activity
two days (k = 6.5 x 10 -3 min-') 10 -3
down to 5%
viscosity
the monomer is
value
solu-
Cu, Zn supero-
is approximately
by 8M urea
a further is
or bovine
to buffered
of enzyme activity
decrease
on diffusion
of 8M urea
at 22OC of the enzyme solutions
enzyme retained
had a biphasio
of either
the hypothesis
On incubation
while
pH 7.4)
Addition
as the di-
for
after This
4 deys,the addition inactivation
decrease
down
and a second of 0.1%
of
activity
to slo-
Vol. 86, No. 4, 1979
after
4 days.
solutions,
Hundred-fold
led
tivated EPR
recovery
affected
inactivated
enzyme
up to 5@ of the value
1 min end to a lOO$ recovery by dialysis
yeast
than
spectral
shapes
the g,, regions
of activity
against
of 8M urea
(7)
evidence
buffer
is
gously
to the effects
Sequence
typical
within
8
of the urea-inac-
homologies
primary
chemical
as to allow conditions
we first
zation
further
dialysis
frame
the bovine intenance
enzyme,
The half-cystine
this
of identical
our attention
for
arrangement
to be pin-pointed
reported
which
is
sites
feature
1201
even
out.
Analo-
of the yeast All
dramatic
are
but
effects
difference site
of
explanation (2).
to be found
in Neverin
the
to be as conservative as variant
To unravel
the compactness
the yeast
EPR
had practically
are identical
as to create such structural
and subsequent
which
of the molecule
for
be ruled
of the active
expected
same sites.
in a relatively
in urea
has no apparent
on the existence
a chemical
spectrum
c and cl).
properties
stability
active
viscosity
Therefore
site
The rather
study
which
of the
to be relevant
content
under
of the proteins,
i.e.
ad-
the EPR spectrum
enz,ymes.
differential
stability
was specific,as
buffer.
of the two enzymes,
bonds,
of the dimeric
amenable
and yeast
dismutases
different focussed
urea-free
of the most specific
for
in
incubation
1, curves
the
constant
enzyme).
can not
converted (Fig.
cases
of the
of the active
of signal
against
of bovine
bases
it
in the
the same relative
on the bovine
on the enzyme activity,
new form
splitting
significant-modifications
effects
properties
of disulfide
as to give
while
dismu-
distinctive
In both
on the ERR spectra
to freezing
the formation for
and the hyperfine
of the result
enzyme,
were
b and b').
modifications
the two CU, Zn superoxide the spectroscopic
curves
d shows the effect
to 8M urea
structural
I,
superoxide
were much more pronounced
of specific
after
theless
changes
effect
in much less
an entirely
in sensitivity
changes
(Fig.
The urea
on the bovine
reversible
, and the spectral
Immediate
dismutase
1,
in favor
enzyme into
or yeast
became more axial
(Fig.
attribution
no effect
bovine
at such a concentration
though
were
1).
resulted
enzymes
of either
treatment
increased.
of sucrose
of both
(Fig.
in the bovine line
dition
EPR spectra
by urea
in the two enzymes
is
of the fully
of activity
was obtained
The copper
were
bases
buffer
enzyme.
spectra.
tase
with
recovery
enzyme within
Similar
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dilution
to en abrupt
of the native hours.
BIOCHEMICAL
is
proved,
of the subunit (9,ll)
characteriat least
for
end the ma-
and at the same time
easy manner. enzyme is
3.5-4
residues
per di-
Vol. 86, No. 4, 1979
Fig.
1 - EPR (a): (b) dition piq
mer,
as measured
lue
BIOCHEMICAL
spectra of bovine and ye?tt Cu, Zn superoxide dismutase. bovine enzyme (0.8 x IO M) in 0.1 M phosphate buffer, pH 7.4; and (0): as (a), frozen immediately (b) or 24 hours (c) after-t+ of 81.1urea at ropm temperature;,(d): bovine enzyme (1 x 10 UI) 0.5.M sucrose; (a) , (b) and (c) are the yeast enzyme (1.5 x I) In the seme conditions as (a) (b) and (c). Final concentrations of enzymes were obtained by addition of a few microliters of a coneentrated stock solution of protein to relatively large volumes of either buffer alone or buffer containing 8M urea.
as cysteic
acid
after
of carboxymethylcysteine
(4.1)
iodoacetate
of the apoenzyme
under
with
-SH groups
per
dimer
in 8.0 M urea,
spectively
for
the apo-
under
treatment.
results,
me in 8M urea
(I),
per
feature
subunit,a
From the digest
after
(6.0
indicate
conditions with
S.aureus
acid)
while
with
the established
the presence
psotease
1202
gave 3.9
with hydroand 4.2
borohydride,
no -SH group
omission
dissociation
from higher
of the reduced,
rewas ti-
of the reduction
of anintrasubunit protein
va -
M guanidine
sodium
pracCicr$.l.;;
but with
a similar
and treatment
conditions
common to the homologous with
reduction
reduction
and hoLo-enzyme,
together
clearly
We found
5,5'-dithiobis(2-nitrobenzoic
the same denaturing These
(2,12).
after
denaturing
Titration
trated
oxidation
content
chloride).
11).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the
enzy-
disulfide
bond
organisms
(4,3,
carboxymathylated
apoen
Vol. 86, No. 4, 1979
BIOCHEMICAL
me,
two radioactive
Thejr
sequence
chain
of -the bovine
(SA-1
and SA-3)
isolation
is reported
bovine
enzyme is around
His 69,
the bovine
three
the overall
118)
ronment
is
the residues
enzyme were
also
around
the
lytic
sLmilarities
k?hile
this
the N-terminal
region
data
from Petersen
et al.
with
the bovine
cate
an
is
the
two
for
about
46,$
portions that,
beside bond
i.e.
the four
amino acid
residues
of the
zinc
strongly
atom.
protein,
extent
related
,
also that of
44,
fourth
46
ligand
of conservation
to the
the
Conservation
histidines
on the
of
of the
disulfide
we became aware
,
of the envi-
spectroscopic
In fact
(12).
c, is
and cata-
than
contributing
other
a clue
of the yeast
that
this
high
evolution
enough
fragments
to indito that
repor-
substantial
inva-
likely
to be essen -
properties,
such as tho-
ligands
enzymes can be found
are at the subunit
the chain
comparable
above suggest
and catalytic
confirmed
bridge. On the -and the disulfide the differential effects of urea on the active
to explain
of the X-terminal
though
regions
peptides
polypeptide
the polypeptide
of protein
sequen
of the present
fully of
group
terminal
protease
analysis stretch
through
for
of spectroscopic
and bovine
of S.aureus
value,
presented
dismutases
the metal
array
of this
found
the results
the course
and their
Comparison
by a danish
of the amino
during
the whole
of the molecule
lower
of a report
54 residues
of the molecule
of stability
to the maintenance
dues and part
2 encompassing
intrasubunit
2. Such a high
(12).
In conclusion,
that
the
observation
up to now available
to purify
in Cu, Zn superoxide
regions
for
in the
enzyme shows 42$ homology;
extent
hand,
is the
of the first
has been possible
se peptides
tes
is
dismutase
from
tial
performed
accounting
of the bovine
was in preparation
it
riancy
ligands
two metals
work
ted here.
of two more peptides
the corresponding
to be ligands
information
superoxide
of cytochrome
with
conserved,
in fig.
in fig. thus
interest
proved
the determination
ce of yeast
the polypeptide
of the two proteins.
paper
concerning
along
chromatographies
residues,
involved
copper
given
and sequenced.
the sequence
reported
homology
6E;$. Of immediate
and 61 (no sequence 3is
exchange
69 amino acid
out of the four
isolated
by homology
with
chain
78 and Asp 81 are
Ifis
were
fragments.
of polypeptide
subunit:
homology
t ogether
by the ion
include
entire
and SA-4) 2, aligned
enzyme (II),
of the radioactive
half-cystines,
in
(U-2
in fig.
purified
Tne two strectches
the
peptides
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
interface, segment
in
substitutions
such as the last (9).
1203
observed
few C-terminal
siin resi-
b-SOD
y-SOD
b-SOD
b-SOD
Fig.
sA4
2 - Comparison of the primary structure of bovine (b-SOD) and Saccharomyces cerevisiae (g-SOD) superoxide dismutases. Arrows under the yeast enzyme with symbols SR denote peptides derived from digestion of carboxymethyBoxes enclose residues identical lated protein with S.aureus protease. in the two proteins. For the bovine onsyme residues that are ligands of the copper (circles) and the zinc (squares) atoms and the position of the disulfide bond are also indicated.
.
A~-A~-~HR-LYS-~~L~~-VHL-CYS-V~L-LEU-LYS-GLY~GLY-~SP-GLY-PRO-V~L-GLN-GLY-THR-ILE-HIS-PHE-GLU-HLH-LYS-GLY-~SP-THR-VHL-VAL-VHL-THR-
Vol. 86, No. 4, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. larmocchi,F., Venardi,G., Bossa,F., Riga,A. and Rotilio,G. (1978) FEBS Lett., 94, 109-111. 2. Goscin,S.A. and Fridovich,I. (1972) Biochim.Biophys.Acta, 289, 276-283. 3. Cavallini,D., Graziani,M.T. and Orlando,M. (1966) Ital.J.Biochem., 15,268-272. 4. Barra,D., Martini,F., Bossa,F., Rotilio,G., Bannister,J.V. and Bannister,W.H. (1978) Biochem.Biophys.Res.Cormnun., 81, 1195-1200. 5. McCord,J.K and Fridovich,I. (1969) J.Biol.Chem., 244, 6049-6055. 6. Rigo,A., Viglino,P. and Rotilio,G. (1975) Anal.Bioche%, 68, 1-8. 7. Handbook of Chemistry and Physics (Weast,R.C. ed.) 56 Edition 1975-1976, p.D-264, CRC Press, Cleveland. 8. Bannister,J.V., Anastasi,A. and Bannister, W.H. (1978) Biochem.Biophys.Res. comnlun., 81, 469-472. 9. Richardson,J.S., Thomas,K.E., Rubin,B.M. and Richardson,D.C. (1975) Proc.Natl. Aoad.Sci.U.S.A., 72, 1349-1353. 10. Beauchamp,C.O. and Fridovich,I. (1973) Biochim.Biopbys.Acta, 317, 50-64. 11. Steinman,H.K, Naik,V.R., Abernethy,J.L. and Hil1,R.L. (1974) J.Biol.Chem., 249, 7326-7338. 12. Petersen,C., Hasemann,V., Martin,B.f Johansen,J.T., Svendsen,I. and Ottesen,M. (1977) Carlsberg Res.Commun., 42, 391-395.
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