Vol. 74, No. 1, 1977
BIOCHEMICAL
GLUTATHIONE PEROXIDASE:
AND BIOPHYSICAL
INHIBITION
RESEARCH COMMUNICATIONS
BY CYANIDE AND RELEASE
OF SELENIUM1 J.
R. Prohaska,
S.-H.
Oh, W. G. Hoekstra,
Departments of Nutritional Sciences Wisconsin, 1270 Linden Drive,
Received
September
and H. E. Ganther
and Biochemistry, Madison, Wisconsin
University 53706
of
27,1976
Summary: Treatment of highly purified ovine erythrocyte glutathione peroxidase withN resulted in loss of enzyme activity and release of selenium from the enzyme. The inactivation by cyanide was time-dependent and the rate was strongly influenced by temperature, concentration of cyanide, oxidation state of the enzyme, and pH. The pH effect could be explained on the basis of the increasing proportion of cyanide ion with increasing pH. Inhibition could be prevented by prior reduction of the purified enzyme with glutathione, dithiothreitol, or dithionite. Oxidation with cumene hydroperoxide was necessary to demonstrate cyanide inhibition of glutathione peroxidase activity in rat liver cytosol. These observations explain why cyanide inhibition of glutathione peroxidase has not been noted previously and provide new approaches for studying the chemical nature of the enzyme-selenium. Glutathione 1.11.1.9)
peroxidase
(GSH-Px)
GSH-Px noted
was discovered
by Mills in contrast
the finding
that
GSH-Px lacked
it
was discovered
(4)
and contained
extensive tissues
to other
4 g-atoms
investigations (reviewed
in
7,8),
heme and various that
the
little
enzyme
A unique (2)
This
peroxidases.
prosthetic
is known
about
insensitivity
for
(5,6).
a variety
the nature
by
groups
selenium
g) of protein from
of
be rationalized
enzyme required
isolated
EC
feature
was its
could
other
(84,000
oxidoreductase,
(1).
investigators
of Se/mole
of the
peroxide
in 1957 by Mills
and by subsequent
to cyanide,
More recently
hydrogen
(glutathione:
(3). activity
Despite
of species of selenium
and in
GSH-Px. In an extension GSH-Px purified the use of papain completely
from
of previous
from
sheep
activated
(9)
with
0 1977 by Academic of reproduction in any
using
proteolytic
one of us (S.-H.
erythrocytes, cyanide
(10)
GSH-Px in a low molecular
1 Research supported University of Wisconsin,
Copyright All rights
studies
weight
caused
Press, Inc. form reserved.
64
0.)
selenium
form.
by the College of Agricultural Madison, and by NIH grants
digestion
It
discovered
of that
to be released was then
found
and Life Sciences, AM-14184 and AM-14881.
that
BIOCHEMICAL
Vol. 74, No. 1, 1977
cyanide this
alone
finding
is readily
would
bring
about
and demonstrates inhibited
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
same effect.
that,
under
The present
the appropriate
paper
describes
conditions,
GSH-Px
by cyanide. MATERIALS AND METHODS
75
Se-labeled GSH-Px was isolated (6) from erythrocytes of an ewe given [75Se] sodium selenite (New England Nuclear, Boston, MA). Radioactivity was determined by means of a well-type gamma scintillation counter and total selenium by a fluorometric procedure (6). The purified enzyme was stored at 4OC as a stock solution (0.04 mg/ml) in 5 mM sodium phosphate, pH 7, containing 10% ethanol. Enzyme activity was measured by a coupled spectrophotometric procedure using glutathione reductase and cumene hydroperoxide (ICN K & K Chemicals, Cleveland, OH) as described elsewhere (11) with the following changes: (1) KCN was omitted; (2) 0.1 M potassium (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonate) (HEPES), pH 7.5, was used in place of pH 7 potassium phosphate buffer; (3) preincubation time with GSH was increased to 10 min. Under these conditions the purified enzyme oxidized 250-300 umoles GSH/min/ng Se. To study the release of selenium from GSH-Px, the enzyme was treated wittl KCN under conditions similar to those used in the initial study using cyanideactivated papain. One ml of a stock solution of [75Se]-GSH-Px (40 ng containing 22,300 cpm) was diluted to 20 ml with water. KCN (65 mg) was added, without neutralization, to give a final concentration of 0.05 M, and the mixture incubated at 40°C for 16 hours. After incubation, the sample was subjected to ultrafiltration on a PM-10 membrane (Amicon Carp). Unless otherwise indicated, studies of GSH-Px inactivation with cyanide and other compounds were carried out at 25OC in stoppered tubes containing 2 x 10s2 M HEPES (pH 7.51, 10e3 M KCN (Mallinkrodt, St. Louis, MO), and 2.5 x 10W8 M GSH-Px (10e7 M Se) and terminated by dilution (200x) into the assay medium. Controls containing KC1 in place of KCN were run simultaneously. RESULTS Treatment
of GSH-Px with
and 85% was recovered ed lo-fold tographed Recovery near
by evaporation on Sephadex of 75 Se off
the position
experiments conditions
in the
the
92% of the 2
PM-10 ultrafiltrate
(Figure
column
1).
selenite
strength.
75
near
We have identified
Earlier experiments had shown that denatured GSH-Px (6) and only 12% of the treatment with 1 N NH40H (9).
6.5
the
the major experiment,
and chroma-
75
at
154 ml.
Se peak eluting subsequent
GSH on G-10 columns selenite
enzyme
was concentrat-
Se peak was located
in this
eluted
2
Se from
The filtrate
.
Although
identified is
75
at room temperature
A major
was 84%.
GSH was not
have shown that of low ionic
released
in an open vessel G-10
for
cyanide
under
and selenocyanate
75Se was not released from heat (65OC) after enzyme 75Se was ultrafiltrable
Vol. 74, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ELUATE
(ml)
75 Se released of ultrafiltrable Figure 1. Sephadex G-10 chromatography from glutathione peroxidaT5 by treatment with cyanide. After ultrafiltration Se released from GSH-Px by cyanide (see text) the and concentration of the sample was chromatographed at 25OC on a 1.8 x 130 cm Sephadex G-10 column (bed volume 330 ml) eluted with 10% ethanol and Q-ml fractions collected. Arrows indicate the elution positions for GSSG (centered at 130 ml) and GSH (155 ml) obtained in a calibration run.
-0 11
\
I
I I TIME
I 3
4
Cl%JRS~
of KCN concentration Figure 2. Effect peroxidase activity. At the various times and assayed for residual enzyme activity. 1 mM KC1 (0) or in 0.1 mM (01, 1 mM (m), or neutralized (pH 7.5) with HCl.
66
on inhibition of glutathione indicated aliquots were removed Incubations were at pH 7.5 in either 10 mM (A) KCN which had been
BIOCHEMICAL
Vol. 74, No. 1, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 3. Effect of temperature on the rate of glutathione peroxidase inactivation by KCN. Reactions were at pH 7.5 with 1 mM KCN (0) or 1 mM KC1 rate constants were obtained from data graphed as in (0 1. Pseudo first-order Figure 2. The inset shows an Arrhenius plot of the data from cyanide-treated enzyme between 19-37OC.
(SeCN-) it
is
the
as two of the products not
implied
cyanide
that
enzyme treated
time
2).
found
whether
that
to 5 mM. when using
of these
cyanide
with
0.1-10
Plotting
the
gave straight
observed
lines;
when cyanide the rate About
log
could
of the
thus
treated
necessarily
inhibit
in large
of inactivation were
percent
an apparent
is present
1.5 hours
the
with
cyanide,
initial
but
product
of
GSH-Px, after
various
time
GSH-Px activity first-order excess.
was proportional necessary
the activity
to inhibit
of the intervals
remaining
kinetic
behavior
is
studies
it
From similar
to KCN concentration 50% of the
vs.
was up
GSH-Px activity
KCN at 1 mM.
of 10 mM did
compounds
is
mM KCN was assayed
NaN3, KOCN, KSCN, KSeCN, KBr, tions
when GSH-Px is
reaction.
To determine
(Figure
either
formed
for
not
at least
KI,
and H-aminopropionitrile
inhibit
GSH-Px when the
2 hours
at pH 7.5 and 25V.
67
at concentra-
enzyme was treated However,
the
with
these
same enzyme
BIOCHEMICAL
Vol. 74, No. 1, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.24
of pH on the rate of glutathione peroxidase inactivation Figure 4. Effect were carried out at 25OC in 0.05 M HEPES (pH 7, 7.5, 8) by 1 mM KCN. Incubations or glycylglycine (pH 8.5, 9, 9.5). Pseudo first-order rate constants were The obtained from data graphed as in Figure 2 and plotted against the final pH. inset shows the relation between these rate constants (pH 7.5 to 9.5) and the concentration of free cyanide ion calculated from the measured pH and using a pK of 9.31 for HCN (13).
preparation
was reversibly
inhibited
by 1 mM Na2S03,
as reported
by others
(12). The effect determined
from
The inset
of temperature the
slopes
shows that
between
19-37OC
apparent
energy
correction
for
pH; a close
correspondence
cyanide
concentration
above
of GSH-Px activity pH 10 and below Table
(AHa)
inactivation
I demonstrates
rate
between
for
of inactivation
between
constants, in
cyanide-treated
plot,
from
which
was calculated,
Figure
increased
of inactivation
with
an after
In the absence
pH 7 and 9.5,
increasing
and the
but
calculated
of KCN no was evident
pH 6. that
GSH-Px was protected
68
against
cyanide
3.
enzyme
of cyanide.
at each pH is evident. was detected
the
of 20 kcal/mole
the rate
rate
2, is presented
in an Arrhenius
in the absence the
first-order
as in Figure
line
of activation
pseudo
of inactivation
gave a straight
4 shows that
loss
of lines
the rates
Figure
ion
on the
inactiva-
Vol. 74, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
I
EFFECT OF PRETREATMENT OF GSH-Px ON INACTIVATION
BY CYANIDEa
% GSH-Px Remaining Pretreatment
Control
None
Cyanide
99
21
105
100
Cumene hydroperoxide
96
22
Dithiothreitol
99
104
Glutathione
Sodium
arsenite
102
21
Sodium
arseniteb
104
30
Sodium
dithionite
96
105
(Na2S204)
aGlutathione peroxidase was preincubated with 2 x 10-4 M of the above compounds for 5 min. prior to the addition of either 10 mM KCN or KC1 (control). Aliquots were assayed for glutathione peroxidase as described in Methods immediately following CN- addition (zero time) compared to 60 min. later. b Sodium
tion
by its
threitol
substrate,
and sodium
hydroperoxide, that
In contrast cytosol
buffer,
pH 6.5,
hydroperoxide
dures;
dithionite
by other
(Na2S204).
protected
nor
GSH-Px was fully to purified (which
at 2 x 10 -3 M.
accelerated
containing
(often
peroxidases)
others
probably
assayed are failed
not
in the
42 hours
presence
69
of activity,
against
phosphate
with
cumene
This
that
crude
suggests
of cyanide
cyanide
dilute
of rat
preincubation
to cyanide
to observe
of loss
of the GSH-Px activity
required
sensitive
cumene
as isolated.
for
10% ethanol)
such as dithio-
substrate,
the rate
inhibition
had been dialyzed
agents
The oxidizing
susceptible
enzyme,
reducing
and the use of 40 mM KCN at pH 9.
enzyme samples and other
tested
GSH, and also
neither
suggesting
liver
arsenite
under
inhibition
to inhibit normal with
catalase assay partially
proce-
Vol. 74, No. 1, 1977
BIOCHEMICAL
purified
the
GSH-Px because
used enzyme that
AND BIOPHYSICAL
reactions
were
was in a nonsusceptible
run
RESEARCH COMMUNICATIONS
in the presence
form
of GSH (1)
or
(2).
DISCUSSION This causes
study
loss
demonstrates
concentration
erature.
inhibition
This
was readily
to 1.5
x 10 -6 M CN- (the
activity assayed
after
from
either
state
recovered
but
(14).
because
is
support
it
the
x-ray
with are
that
states,
photoelectron
contains
in progress
the
spectroscopy
acid
related
derivatives
on the
blood
cells
chemical
it
not
I)
is
to a selenenic
nature
by however, and
with
possible
form
in a more oxidized inhibited
by cyanide
(Table
inhibitors
clearly
in at least
of kinetic
in a
purified
and is not
inhibited
studies
on the basis
of certain
equivalent
GSH-Px is
is presumably (Table
These
It
inacti-
agents;
in highly
Se in GSH-Px can exist
(16).
is
enzyme was diluted
isolated
GSH-Px is
levels;
reducing
of GSH and peroxides,
(15).
as suggested
Se in a form
sulfenic
reduction
cyanide
temp-
1 mM glutathione.
by cyanide
by iodoacetate
likelihood
or oxidation
red
to
The inactivation
GSH-Px with
with
GSH-Px and
by increasing
at pH 7.5,
species).
when GSH-Px is
can be inhibited
inhibited
low
M KCN, which,
by treating
preincubation
or ovine
after
at rather
from
is proportional
accelerated
when cyanide-inactivated
However,
(15);
CN- and is
-4
selenium
of inactivation
reactive
concentrations
bovine
iodoacetate
apparent
a 10 minute
form
at 10
blocked
At physiological reduced
of free
demonstrated
was not
can release
was demonstrated
vation
was completely
cyanide The rate
of enzyme activity.
the calculated
cyanide
that
studies that
(14)
oxidized
and GSH-Px
(81,
since
cyanide
reacts
enzymes
(17,
18).
Further
studies
selenium
I)
two forms
acid
of the
by
in GSH-Px.
REFERENCES 1. 2. 3. 4. 5.
Mills, G. C. (1957) J. Biol. Chem. 229, 189-197. Little, C., and O'Brien, P. J. (1968) Biochem. Biophys. Res. Commun. 31, 145-150. E., Voelter, W., and Wendel, A. (1971) Hoppe-Seyler's Flohe, L., Schaich, Z. Physiol. Chem. 352, 170-180. Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., and Ffoekstra, W. G. (1973) Science 179, 588-590. Flohe, L., Gunzler, W. A., and Schock, H. H. (1973) FEBS Lett. 32, 132-134.
70
Vol. 74, No. 1, 1977
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Oh. S.-H., Ganther, H. E., and Hoekstra, W. G. (1974) Biochemistry 13, 1825-1829. ,, Flohe, L., Gunzler, W. A. and Ladenstein, R. (1976) Glutathione: Metabolism and Function, Arias, I. M., and Jakoby, W. B., eds., pp. 115138, Raven Press, New York. Ganther, H. E. (1975) Chemica Scripta 8A, 79-84. Oh, S.-H. (1975) Ph.D. Thesis, University of Wisconsin, University Microfilms, Inc. Mendel, L. B. and Blood, A. F. (1910) J. Biol. Chem. 8, 177-213. Prohaska, J. R., and Ganther, H. E. (1976) J. Neurochem., in press. Schneider, F., and Flohe, L. (1967) Hoppe-Seyler's Z. Physiol. Chem. 348, 540-552. Weast, R. C., ed. (1967) Handbook of Chemistry and Physics, p. D-91, Chem. Rubber Co., Cleveland. Flohe, L., Loschen, G., Gznzler, W. A., and Eichele, E. (1972) Hoppe-Seyler's Z. Physiol. Chew. 353, 987-999. Flohe, L., and Gunzler, W. A. (1974) Glutathione, pp. 132-145, Academic Press, New York. Wendel, A., Pilz, W., Ladenstein, R., Sawatzki, G., and Weser, U. (1975) Biochim. Biophys. Acta 377, 211-215. Allison, W. S. and Connors, M. J. (1970) Arch. Biochem. Biophys. 136, 383-391. Lin, W. S., Armstrong, D. A. and Gaucher, G. M. (1975) Can. J. Biochem. 53, 298-307.
71
BIOCHEMICAL
GLUTATHIONE PEROXIDASE:
AND BIOPHYSICAL
INHIBITION
RESEARCH COMMUNICATIONS
BY CYANIDE AND RELEASE
OF SELENIUM1 J.
R. Prohaska,
S.-H.
Oh, W. G. Hoekstra,
Departments of Nutritional Sciences Wisconsin, 1270 Linden Drive,
Received
September
and H. E. Ganther
and Biochemistry, Madison, Wisconsin
University 53706
of
27,1976
Summary: Treatment of highly purified ovine erythrocyte glutathione peroxidase withN resulted in loss of enzyme activity and release of selenium from the enzyme. The inactivation by cyanide was time-dependent and the rate was strongly influenced by temperature, concentration of cyanide, oxidation state of the enzyme, and pH. The pH effect could be explained on the basis of the increasing proportion of cyanide ion with increasing pH. Inhibition could be prevented by prior reduction of the purified enzyme with glutathione, dithiothreitol, or dithionite. Oxidation with cumene hydroperoxide was necessary to demonstrate cyanide inhibition of glutathione peroxidase activity in rat liver cytosol. These observations explain why cyanide inhibition of glutathione peroxidase has not been noted previously and provide new approaches for studying the chemical nature of the enzyme-selenium. Glutathione 1.11.1.9)
peroxidase
(GSH-Px)
GSH-Px noted
was discovered
by Mills in contrast
the finding
that
GSH-Px lacked
it
was discovered
(4)
and contained
extensive tissues
to other
4 g-atoms
investigations (reviewed
in
7,8),
heme and various that
the
little
enzyme
A unique (2)
This
peroxidases.
prosthetic
is known
about
insensitivity
for
(5,6).
a variety
the nature
by
groups
selenium
g) of protein from
of
be rationalized
enzyme required
isolated
EC
feature
was its
could
other
(84,000
oxidoreductase,
(1).
investigators
of Se/mole
of the
peroxide
in 1957 by Mills
and by subsequent
to cyanide,
More recently
hydrogen
(glutathione:
(3). activity
Despite
of species of selenium
and in
GSH-Px. In an extension GSH-Px purified the use of papain completely
from
of previous
from
sheep
activated
(9)
with
0 1977 by Academic of reproduction in any
using
proteolytic
one of us (S.-H.
erythrocytes, cyanide
(10)
GSH-Px in a low molecular
1 Research supported University of Wisconsin,
Copyright All rights
studies
weight
caused
Press, Inc. form reserved.
64
0.)
selenium
form.
by the College of Agricultural Madison, and by NIH grants
digestion
It
discovered
of that
to be released was then
found
and Life Sciences, AM-14184 and AM-14881.
that
BIOCHEMICAL
Vol. 74, No. 1, 1977
cyanide this
alone
finding
is readily
would
bring
about
and demonstrates inhibited
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
same effect.
that,
under
The present
the appropriate
paper
describes
conditions,
GSH-Px
by cyanide. MATERIALS AND METHODS
75
Se-labeled GSH-Px was isolated (6) from erythrocytes of an ewe given [75Se] sodium selenite (New England Nuclear, Boston, MA). Radioactivity was determined by means of a well-type gamma scintillation counter and total selenium by a fluorometric procedure (6). The purified enzyme was stored at 4OC as a stock solution (0.04 mg/ml) in 5 mM sodium phosphate, pH 7, containing 10% ethanol. Enzyme activity was measured by a coupled spectrophotometric procedure using glutathione reductase and cumene hydroperoxide (ICN K & K Chemicals, Cleveland, OH) as described elsewhere (11) with the following changes: (1) KCN was omitted; (2) 0.1 M potassium (N-2-hydroxyethylpiperazine-N1-2-ethanesulfonate) (HEPES), pH 7.5, was used in place of pH 7 potassium phosphate buffer; (3) preincubation time with GSH was increased to 10 min. Under these conditions the purified enzyme oxidized 250-300 umoles GSH/min/ng Se. To study the release of selenium from GSH-Px, the enzyme was treated wittl KCN under conditions similar to those used in the initial study using cyanideactivated papain. One ml of a stock solution of [75Se]-GSH-Px (40 ng containing 22,300 cpm) was diluted to 20 ml with water. KCN (65 mg) was added, without neutralization, to give a final concentration of 0.05 M, and the mixture incubated at 40°C for 16 hours. After incubation, the sample was subjected to ultrafiltration on a PM-10 membrane (Amicon Carp). Unless otherwise indicated, studies of GSH-Px inactivation with cyanide and other compounds were carried out at 25OC in stoppered tubes containing 2 x 10s2 M HEPES (pH 7.51, 10e3 M KCN (Mallinkrodt, St. Louis, MO), and 2.5 x 10W8 M GSH-Px (10e7 M Se) and terminated by dilution (200x) into the assay medium. Controls containing KC1 in place of KCN were run simultaneously. RESULTS Treatment
of GSH-Px with
and 85% was recovered ed lo-fold tographed Recovery near
by evaporation on Sephadex of 75 Se off
the position
experiments conditions
in the
the
92% of the 2
PM-10 ultrafiltrate
(Figure
column
1).
selenite
strength.
75
near
We have identified
Earlier experiments had shown that denatured GSH-Px (6) and only 12% of the treatment with 1 N NH40H (9).
6.5
the
the major experiment,
and chroma-
75
at
154 ml.
Se peak eluting subsequent
GSH on G-10 columns selenite
enzyme
was concentrat-
Se peak was located
in this
eluted
2
Se from
The filtrate
.
Although
identified is
75
at room temperature
A major
was 84%.
GSH was not
have shown that of low ionic
released
in an open vessel G-10
for
cyanide
under
and selenocyanate
75Se was not released from heat (65OC) after enzyme 75Se was ultrafiltrable
Vol. 74, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ELUATE
(ml)
75 Se released of ultrafiltrable Figure 1. Sephadex G-10 chromatography from glutathione peroxidaT5 by treatment with cyanide. After ultrafiltration Se released from GSH-Px by cyanide (see text) the and concentration of the sample was chromatographed at 25OC on a 1.8 x 130 cm Sephadex G-10 column (bed volume 330 ml) eluted with 10% ethanol and Q-ml fractions collected. Arrows indicate the elution positions for GSSG (centered at 130 ml) and GSH (155 ml) obtained in a calibration run.
-0 11
\
I
I I TIME
I 3
4
Cl%JRS~
of KCN concentration Figure 2. Effect peroxidase activity. At the various times and assayed for residual enzyme activity. 1 mM KC1 (0) or in 0.1 mM (01, 1 mM (m), or neutralized (pH 7.5) with HCl.
66
on inhibition of glutathione indicated aliquots were removed Incubations were at pH 7.5 in either 10 mM (A) KCN which had been
BIOCHEMICAL
Vol. 74, No. 1, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Figure 3. Effect of temperature on the rate of glutathione peroxidase inactivation by KCN. Reactions were at pH 7.5 with 1 mM KCN (0) or 1 mM KC1 rate constants were obtained from data graphed as in (0 1. Pseudo first-order Figure 2. The inset shows an Arrhenius plot of the data from cyanide-treated enzyme between 19-37OC.
(SeCN-) it
is
the
as two of the products not
implied
cyanide
that
enzyme treated
time
2).
found
whether
that
to 5 mM. when using
of these
cyanide
with
0.1-10
Plotting
the
gave straight
observed
lines;
when cyanide the rate About
log
could
of the
thus
treated
necessarily
inhibit
in large
of inactivation were
percent
an apparent
is present
1.5 hours
the
with
cyanide,
initial
but
product
of
GSH-Px, after
various
time
GSH-Px activity first-order excess.
was proportional necessary
the activity
to inhibit
of the intervals
remaining
kinetic
behavior
is
studies
it
From similar
to KCN concentration 50% of the
vs.
was up
GSH-Px activity
KCN at 1 mM.
of 10 mM did
compounds
is
mM KCN was assayed
NaN3, KOCN, KSCN, KSeCN, KBr, tions
when GSH-Px is
reaction.
To determine
(Figure
either
formed
for
not
at least
KI,
and H-aminopropionitrile
inhibit
GSH-Px when the
2 hours
at pH 7.5 and 25V.
67
at concentra-
enzyme was treated However,
the
with
these
same enzyme
BIOCHEMICAL
Vol. 74, No. 1, 1977
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
0.24
of pH on the rate of glutathione peroxidase inactivation Figure 4. Effect were carried out at 25OC in 0.05 M HEPES (pH 7, 7.5, 8) by 1 mM KCN. Incubations or glycylglycine (pH 8.5, 9, 9.5). Pseudo first-order rate constants were The obtained from data graphed as in Figure 2 and plotted against the final pH. inset shows the relation between these rate constants (pH 7.5 to 9.5) and the concentration of free cyanide ion calculated from the measured pH and using a pK of 9.31 for HCN (13).
preparation
was reversibly
inhibited
by 1 mM Na2S03,
as reported
by others
(12). The effect determined
from
The inset
of temperature the
slopes
shows that
between
19-37OC
apparent
energy
correction
for
pH; a close
correspondence
cyanide
concentration
above
of GSH-Px activity pH 10 and below Table
(AHa)
inactivation
I demonstrates
rate
between
for
of inactivation
between
constants, in
cyanide-treated
plot,
from
which
was calculated,
Figure
increased
of inactivation
with
an after
In the absence
pH 7 and 9.5,
increasing
and the
but
calculated
of KCN no was evident
pH 6. that
GSH-Px was protected
68
against
cyanide
3.
enzyme
of cyanide.
at each pH is evident. was detected
the
of 20 kcal/mole
the rate
rate
2, is presented
in an Arrhenius
in the absence the
first-order
as in Figure
line
of activation
pseudo
of inactivation
gave a straight
4 shows that
loss
of lines
the rates
Figure
ion
on the
inactiva-
Vol. 74, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
I
EFFECT OF PRETREATMENT OF GSH-Px ON INACTIVATION
BY CYANIDEa
% GSH-Px Remaining Pretreatment
Control
None
Cyanide
99
21
105
100
Cumene hydroperoxide
96
22
Dithiothreitol
99
104
Glutathione
Sodium
arsenite
102
21
Sodium
arseniteb
104
30
Sodium
dithionite
96
105
(Na2S204)
aGlutathione peroxidase was preincubated with 2 x 10-4 M of the above compounds for 5 min. prior to the addition of either 10 mM KCN or KC1 (control). Aliquots were assayed for glutathione peroxidase as described in Methods immediately following CN- addition (zero time) compared to 60 min. later. b Sodium
tion
by its
threitol
substrate,
and sodium
hydroperoxide, that
In contrast cytosol
buffer,
pH 6.5,
hydroperoxide
dures;
dithionite
by other
(Na2S204).
protected
nor
GSH-Px was fully to purified (which
at 2 x 10 -3 M.
accelerated
containing
(often
peroxidases)
others
probably
assayed are failed
not
in the
42 hours
presence
69
of activity,
against
phosphate
with
cumene
This
that
crude
suggests
of cyanide
cyanide
dilute
of rat
preincubation
to cyanide
to observe
of loss
of the GSH-Px activity
required
sensitive
cumene
as isolated.
for
10% ethanol)
such as dithio-
substrate,
the rate
inhibition
had been dialyzed
agents
The oxidizing
susceptible
enzyme,
reducing
and the use of 40 mM KCN at pH 9.
enzyme samples and other
tested
GSH, and also
neither
suggesting
liver
arsenite
under
inhibition
to inhibit normal with
catalase assay partially
proce-
Vol. 74, No. 1, 1977
BIOCHEMICAL
purified
the
GSH-Px because
used enzyme that
AND BIOPHYSICAL
reactions
were
was in a nonsusceptible
run
RESEARCH COMMUNICATIONS
in the presence
form
of GSH (1)
or
(2).
DISCUSSION This causes
study
loss
demonstrates
concentration
erature.
inhibition
This
was readily
to 1.5
x 10 -6 M CN- (the
activity assayed
after
from
either
state
recovered
but
(14).
because
is
support
it
the
x-ray
with are
that
states,
photoelectron
contains
in progress
the
spectroscopy
acid
related
derivatives
on the
blood
cells
chemical
it
not
I)
is
to a selenenic
nature
by however, and
with
possible
form
in a more oxidized inhibited
by cyanide
(Table
inhibitors
clearly
in at least
of kinetic
in a
purified
and is not
inhibited
studies
on the basis
of certain
equivalent
GSH-Px is
is presumably (Table
These
It
inacti-
agents;
in highly
Se in GSH-Px can exist
(16).
is
enzyme was diluted
isolated
GSH-Px is
levels;
reducing
of GSH and peroxides,
(15).
as suggested
Se in a form
sulfenic
reduction
cyanide
temp-
1 mM glutathione.
by cyanide
by iodoacetate
likelihood
or oxidation
red
to
The inactivation
GSH-Px with
with
GSH-Px and
by increasing
at pH 7.5,
species).
when GSH-Px is
can be inhibited
inhibited
low
M KCN, which,
by treating
preincubation
or ovine
after
at rather
from
is proportional
accelerated
when cyanide-inactivated
However,
(15);
CN- and is
-4
selenium
of inactivation
reactive
concentrations
bovine
iodoacetate
apparent
a 10 minute
form
at 10
blocked
At physiological reduced
of free
demonstrated
was not
can release
was demonstrated
vation
was completely
cyanide The rate
of enzyme activity.
the calculated
cyanide
that
studies that
(14)
oxidized
and GSH-Px
(81,
since
cyanide
reacts
enzymes
(17,
18).
Further
studies
selenium
I)
two forms
acid
of the
by
in GSH-Px.
REFERENCES 1. 2. 3. 4. 5.
Mills, G. C. (1957) J. Biol. Chem. 229, 189-197. Little, C., and O'Brien, P. J. (1968) Biochem. Biophys. Res. Commun. 31, 145-150. E., Voelter, W., and Wendel, A. (1971) Hoppe-Seyler's Flohe, L., Schaich, Z. Physiol. Chem. 352, 170-180. Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G., and Ffoekstra, W. G. (1973) Science 179, 588-590. Flohe, L., Gunzler, W. A., and Schock, H. H. (1973) FEBS Lett. 32, 132-134.
70
Vol. 74, No. 1, 1977
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Oh. S.-H., Ganther, H. E., and Hoekstra, W. G. (1974) Biochemistry 13, 1825-1829. ,, Flohe, L., Gunzler, W. A. and Ladenstein, R. (1976) Glutathione: Metabolism and Function, Arias, I. M., and Jakoby, W. B., eds., pp. 115138, Raven Press, New York. Ganther, H. E. (1975) Chemica Scripta 8A, 79-84. Oh, S.-H. (1975) Ph.D. Thesis, University of Wisconsin, University Microfilms, Inc. Mendel, L. B. and Blood, A. F. (1910) J. Biol. Chem. 8, 177-213. Prohaska, J. R., and Ganther, H. E. (1976) J. Neurochem., in press. Schneider, F., and Flohe, L. (1967) Hoppe-Seyler's Z. Physiol. Chem. 348, 540-552. Weast, R. C., ed. (1967) Handbook of Chemistry and Physics, p. D-91, Chem. Rubber Co., Cleveland. Flohe, L., Loschen, G., Gznzler, W. A., and Eichele, E. (1972) Hoppe-Seyler's Z. Physiol. Chew. 353, 987-999. Flohe, L., and Gunzler, W. A. (1974) Glutathione, pp. 132-145, Academic Press, New York. Wendel, A., Pilz, W., Ladenstein, R., Sawatzki, G., and Weser, U. (1975) Biochim. Biophys. Acta 377, 211-215. Allison, W. S. and Connors, M. J. (1970) Arch. Biochem. Biophys. 136, 383-391. Lin, W. S., Armstrong, D. A. and Gaucher, G. M. (1975) Can. J. Biochem. 53, 298-307.
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