- Email: [email protected]
Invivo inactivation of γ-aminobutyric acid-α-ketoglutarate transaminase by 4-amino-5-fluoropentanoic acid
Vol. 102, No. 1,1981 September
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages 520-523
16, 1981
--IN VIVO INACTIVATION OF y-AMINOBUTYRIC ACID-a-KETOGLUTARATE TRANSAMINASE RY 4-AMINO-5-FLLJOROPENTANOIC ACID Richard
8. Silverman,* Mark A. Levy,*, A. Jabbar ?Iuztar,* and James D. Hirsch* *Department of Chemistry, Northwestern University, Evanston, Illinois 60201 *Department
Received
August
of Biological P.,O. Box 5110,
Research, G.D. Chicago, Illinois
Searle and Co., 60680
6,1981
SUMMARY Intraperitoneal injection into mice of varying concentrations of (S)-4-amino-5-fluoropentanoic acid c(S)-AFPA) produces a dose-dependent irreversible decrease in brain Y-aminobutyric acid-a-ketoglutaric acid aminotransferase (E.C. 2.6.1.19) activity. Concomitant with this inactivation is an increase in whole brain Y-aminobutyric acid (GABA) levels. Four hours after a dose of 100 mg/kg body weight of (S)-AFPA to mice, endogenous brain GABA concentrations increase to 16 times that of the untreated animals and the enzyme activity decreases to 20% that of the controls. The binding of (S)-AFPA to GABA receptors was more than three orders of magnitude poorer than for GABA itself. INTRODUCTION (S)-4-Amino-5-fluoropentanoic brain
GABA-T --in vitro
(S)-AFPA
is
through
included
process
this
category
include
(5),
4-aminohex-5-enoic GABA-T
transmitter would for that
brain
is
(61,
which Other
isogabaculine
enzyme
brain
(8),
by the
it
inhibition (8-12).
inactivate
(41,
for
the
(2). effect
compounds
inhibition that
acid
GABA, y-aminobutyric aminotransferase acid.
inhibitory
G 1981 by Academrc Press, Inc. of reproducrion in any form reserved.
520
acid
neuro-
that
(S)-AFPA
report
shown
we demonstrate
and produces
exaggerated
acid; GABA-T, Y-aminobutyric acid-a(EC 2.6.1.19); (S)-AFPA, (S)-4-amino-5-
0006-291X/81/170520-04$01.00/0
into
(7).
of GABA-T as has been
of GABA-T --in vivo
fit
4-aminohex-5-ynoic
was expected
In this
pig
Thus,
GABA levels.
Abbreviations: ketoglutaric fluoropentanoic
Copyrighr All rights
inactivators
and ethanolamine-O-sulfate
inactivators inactivator
to irreversibly
mode of action
enzyme.
catabolic
GABA content
a potent
target
(3),
the major
mechanism-based
(Sl-AFPA
brain
gabaculine
GABA in the mammalian
elevate other
of GABA-T
of the
acid is
was shown
by a mechanism-based
in a class
a catalytic
Since
(1)
acid
BIOCHEMICAL
Vol. 102, No. 1,198l
MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNtCATIONS
AND METHODS
(S)-AFPA was prepared as previously reported (13). All substrates and cofactors were purchased from Sigma Chemical Co. Male CD/CR Ham-ICR mice weighing 25-30 g were bought Erom the Charles River Breeding Labs (Portage, MI). Groups of three mice were injected intraperitoneally with different concentration's of (S)-AFPA in 0.2 mL of saline; control animals were injected with an equal volume of saline. Brain GABA levels were determined 4 hours after drug treatment by the radioreceptor assay method of Bernasconi et al. (14). Animals were killed by microwave irradiation (General Medical Engineering Corp., Peabody, MA) to prevent postmortem changes in GABA concentrations (14). Residual GABA-T activity, 4 hours after drug treatment, was determined in the brains of animals killed by decapitation. The brains were excised within one minute, frozen on Dry ice, and stored at -80°C until processed for analysis by a modified version of the method of Jung et al. (10). Bach brain was hand homogenized at 4'C in 10 volumes (v/m) of a buffer consisting of 0.\3% Triton X-100, 0.1 mM glutathione (reduced), 0.1 mM pyridoxal phosphate, 1.0 mM EDTA, 1 mM a-ketoglutarate, 20% (v/v) glycerol and 20 mM potassium phosphate adjusted to pH 6.8 with acetic acid. The homogenates were frozen at -80°C overnight, thawed, and The slightly turbid supernatants were centrifuged at 2500 x g for 20 min. assayed for GABA-T ac.tivity at 25'C using the enzyme-coupled, succinic semialdehyde dehydrogenase assay as previously described (2), modified only in that the potassium pyrophosphate concentration was increased to 100 mM. Each brain was assayed in triplicate and the values were averaged. Samples of the supernatants were dialyzed against the homogenization buffer to determine the reversibility of inhibition. G4BA receptor binding was carried out the by the method of Bernasconi et al. (14). RESULTS AND DISCUSSION Four into
hours
mice,
in whole
brain
results
This
indicates
inactivates
compares whole
of mice
increase
with
in the brain
same dose;
(10)
(S)-AFPA
of different
of GABA-T activity was observed
crosses
concomitant
(Figure).
enzyme activity
doses
did
of (S)-AFPA
with
an increase
Analogous
not
return
to the -in
upon dialysis.
the blood-brain
barrier
and irreversibly
to other
GABA-T inactivators
GABA-T.
treatment
quite
favorably
brain
concentrations
GABA levels
twice
8 hours.
80% by (S)-AFPA,
92%
and 45% by 4-aminohex-5-enoic
that
acid
Under by
and 7 times (10)
these
(9), (11).
521
after
(S)-AFPA
an
(9)
at the
by gabaculine higher
than
brain
drug
caused
that
and 4-aminohex-S-enoic
same conditions,
gabaculine acid
produced
in its
Four hours
body weight,
was 2.5
of 4-aminohex-5-ynoic after
of GABA.
100 mg inactivator/kg
the GABA concentration
same doses
tivated
loss
the --in vivo
that
to raise
respectively
injection
GABA concentration cl),
(S)-AFPA
the
intraperitoneal
a dose-dependent
vitro
ability
after
induced acid
(11)
GABA-T was inac-
90% by 4-aminohex-5-ynoic
acid
by
Vol. 102, No. 1,198l
BIOCHEMICAL
I 60
0
I 100
mg Figure
Dose-Dependent Increase in
AND
BIOPHYSICAL
I 150
AFPA
I 200
I kg
Decrease in GABA Concentration.
RESEARCH
I 260
COMMUNICATIONS
I 300.
Mouse
GABA-T
Activity
by AFPA Concomitant
with
ao
Mice were injected intraperitoneally with various doses of (S)-APPA. After 4 hours, the brain GABA levels and QUA-T activity were determined as described in Materials and Methods. The GABA concentration for 6 control mice was 175 f 10 !Jg GABA/g braio (wet weight) and the GABA-T activity was 24 t 2 ~rsol NADPH / h / g brain. ‘Ihe GABA-T activity is expressed as a % of the controls. Results represent the mean f SEM.
IC50
Binding
of
values
(the
[3H]-muscimol
These
vivo alanine
to
--in
(14))
at
results
indicate
GABA-T inhibition transaminase.
(S)-4-amino-5-chloropentanoic
the
is
in
(S)-AFPA
and
unlikely
that
that
(S)-AFPA
preliminary there glutamate
--In
vivo
was
resulting
sites.
inactivator; brain
receptor
receptor
Furthermore,
of
GABA
for it
effects
vivo.
the
concentration
Therefore,
GABA-ergic
specific
drug
binding
respectively.
GABA-T
(S)-AFPA
GABA
results
acid
GABA-T (2,13),
(S)-AFPA.
522
poor;
inhibition
were
to
will
but
to
M and
little
also a lesser
(S)-AFPA
no --in
vitro
lowered extent
or
a
-in and
by than
of is
transaminase, is
M
inhibitor
that or
3.0~10’~ any
irreversible
aspartate activity
specific
exhibit
indicate be
the of
8.6~10’~
a potent,
decarboxylase, brain
50%
(S)-AFPA
is
appears
relatively
with
Vol. 102, No. 1,198l
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNlCATlONS
ACKNOWLEDGMENTS We are grateful and to the National
to Paul Institutes
Sumner
for
determining
of Health
(Grant
the mouse brain NS 15703)
for
financial
GABA levels support.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Silverman, R.B. and Levy, M.A. (1980) Biochem. Biophys. Res. Commun. 95, 250-255. Xlverman, R.B. and Levy, M.A. (1981) Biochemistry 2, 1197-1203. Rando, R.R. and Bangerter, F.W. (1976) J. Am. Chem. Sot. 9Z& 6762-6764. Metcalf, B.W. and Jung, M.J. (1979) Molecular Pharmacol. 16, 539-545. Jung, M.J. and Metcalf, B.W. (1975) Biochem. Biophys. Res. Commun. 67, 301-306. zppert, B., Metcalf, B.W., Jung, M.J., and Casara, P. (1977) Eur. J. Biochem. I'+, 441-445. FLetcher, A. and Fowler, L.J. (1980) Biochem. Pharmacol. 29, 1451-1454. Balazs, R., Machiyama, Y., Hammond, B.J., Julian, T., andRichter, D. (1970) Biochem. J. 116, 445-467. F.W. (1977) Biochem. Biophys. Res. Rando, R.R., and Bangerter, Commun. 76, 1276-1281. Jung, M.J., Lippert, B., Metcalf, B.W., Schechter, P.J., Bblhlen, P., and Sjoerdsma, A. (1977) J. Neurochem. 28, 717-723. Jung, M.J., Lippert, B., Metcalf, B.W., *hIen, P., and Schechter, P.J. (1977) J. Neurochem. 2, 797-802. Leach, M.J. and Walker, J.M.G. (1977) Biochem. Pharmacol. 2, 1569-1572. Silverman, R.B. and Levy, M.A. (1980) J. Org. Chem. 45, 815-818. Bernasconi, R., Bittiger, H., Reid, J., and Martin, P. (1980) J. Neurochem. 2, 614-618.
523
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages 520-523
16, 1981
--IN VIVO INACTIVATION OF y-AMINOBUTYRIC ACID-a-KETOGLUTARATE TRANSAMINASE RY 4-AMINO-5-FLLJOROPENTANOIC ACID Richard
8. Silverman,* Mark A. Levy,*, A. Jabbar ?Iuztar,* and James D. Hirsch* *Department of Chemistry, Northwestern University, Evanston, Illinois 60201 *Department
Received
August
of Biological P.,O. Box 5110,
Research, G.D. Chicago, Illinois
Searle and Co., 60680
6,1981
SUMMARY Intraperitoneal injection into mice of varying concentrations of (S)-4-amino-5-fluoropentanoic acid c(S)-AFPA) produces a dose-dependent irreversible decrease in brain Y-aminobutyric acid-a-ketoglutaric acid aminotransferase (E.C. 2.6.1.19) activity. Concomitant with this inactivation is an increase in whole brain Y-aminobutyric acid (GABA) levels. Four hours after a dose of 100 mg/kg body weight of (S)-AFPA to mice, endogenous brain GABA concentrations increase to 16 times that of the untreated animals and the enzyme activity decreases to 20% that of the controls. The binding of (S)-AFPA to GABA receptors was more than three orders of magnitude poorer than for GABA itself. INTRODUCTION (S)-4-Amino-5-fluoropentanoic brain
GABA-T --in vitro
(S)-AFPA
is
through
included
process
this
category
include
(5),
4-aminohex-5-enoic GABA-T
transmitter would for that
brain
is
(61,
which Other
isogabaculine
enzyme
brain
(8),
by the
it
inhibition (8-12).
inactivate
(41,
for
the
(2). effect
compounds
inhibition that
acid
GABA, y-aminobutyric aminotransferase acid.
inhibitory
G 1981 by Academrc Press, Inc. of reproducrion in any form reserved.
520
acid
neuro-
that
(S)-AFPA
report
shown
we demonstrate
and produces
exaggerated
acid; GABA-T, Y-aminobutyric acid-a(EC 2.6.1.19); (S)-AFPA, (S)-4-amino-5-
0006-291X/81/170520-04$01.00/0
into
(7).
of GABA-T as has been
of GABA-T --in vivo
fit
4-aminohex-5-ynoic
was expected
In this
pig
Thus,
GABA levels.
Abbreviations: ketoglutaric fluoropentanoic
Copyrighr All rights
inactivators
and ethanolamine-O-sulfate
inactivators inactivator
to irreversibly
mode of action
enzyme.
catabolic
GABA content
a potent
target
(3),
the major
mechanism-based
(Sl-AFPA
brain
gabaculine
GABA in the mammalian
elevate other
of GABA-T
of the
acid is
was shown
by a mechanism-based
in a class
a catalytic
Since
(1)
acid
BIOCHEMICAL
Vol. 102, No. 1,198l
MATERIALS
AND
BIOPHYSICAL
RESEARCH
COMMUNtCATIONS
AND METHODS
(S)-AFPA was prepared as previously reported (13). All substrates and cofactors were purchased from Sigma Chemical Co. Male CD/CR Ham-ICR mice weighing 25-30 g were bought Erom the Charles River Breeding Labs (Portage, MI). Groups of three mice were injected intraperitoneally with different concentration's of (S)-AFPA in 0.2 mL of saline; control animals were injected with an equal volume of saline. Brain GABA levels were determined 4 hours after drug treatment by the radioreceptor assay method of Bernasconi et al. (14). Animals were killed by microwave irradiation (General Medical Engineering Corp., Peabody, MA) to prevent postmortem changes in GABA concentrations (14). Residual GABA-T activity, 4 hours after drug treatment, was determined in the brains of animals killed by decapitation. The brains were excised within one minute, frozen on Dry ice, and stored at -80°C until processed for analysis by a modified version of the method of Jung et al. (10). Bach brain was hand homogenized at 4'C in 10 volumes (v/m) of a buffer consisting of 0.\3% Triton X-100, 0.1 mM glutathione (reduced), 0.1 mM pyridoxal phosphate, 1.0 mM EDTA, 1 mM a-ketoglutarate, 20% (v/v) glycerol and 20 mM potassium phosphate adjusted to pH 6.8 with acetic acid. The homogenates were frozen at -80°C overnight, thawed, and The slightly turbid supernatants were centrifuged at 2500 x g for 20 min. assayed for GABA-T ac.tivity at 25'C using the enzyme-coupled, succinic semialdehyde dehydrogenase assay as previously described (2), modified only in that the potassium pyrophosphate concentration was increased to 100 mM. Each brain was assayed in triplicate and the values were averaged. Samples of the supernatants were dialyzed against the homogenization buffer to determine the reversibility of inhibition. G4BA receptor binding was carried out the by the method of Bernasconi et al. (14). RESULTS AND DISCUSSION Four into
hours
mice,
in whole
brain
results
This
indicates
inactivates
compares whole
of mice
increase
with
in the brain
same dose;
(10)
(S)-AFPA
of different
of GABA-T activity was observed
crosses
concomitant
(Figure).
enzyme activity
doses
did
of (S)-AFPA
with
an increase
Analogous
not
return
to the -in
upon dialysis.
the blood-brain
barrier
and irreversibly
to other
GABA-T inactivators
GABA-T.
treatment
quite
favorably
brain
concentrations
GABA levels
twice
8 hours.
80% by (S)-AFPA,
92%
and 45% by 4-aminohex-5-enoic
that
acid
Under by
and 7 times (10)
these
(9), (11).
521
after
(S)-AFPA
an
(9)
at the
by gabaculine higher
than
brain
drug
caused
that
and 4-aminohex-S-enoic
same conditions,
gabaculine acid
produced
in its
Four hours
body weight,
was 2.5
of 4-aminohex-5-ynoic after
of GABA.
100 mg inactivator/kg
the GABA concentration
same doses
tivated
loss
the --in vivo
that
to raise
respectively
injection
GABA concentration cl),
(S)-AFPA
the
intraperitoneal
a dose-dependent
vitro
ability
after
induced acid
(11)
GABA-T was inac-
90% by 4-aminohex-5-ynoic
acid
by
Vol. 102, No. 1,198l
BIOCHEMICAL
I 60
0
I 100
mg Figure
Dose-Dependent Increase in
AND
BIOPHYSICAL
I 150
AFPA
I 200
I kg
Decrease in GABA Concentration.
RESEARCH
I 260
COMMUNICATIONS
I 300.
Mouse
GABA-T
Activity
by AFPA Concomitant
with
ao
Mice were injected intraperitoneally with various doses of (S)-APPA. After 4 hours, the brain GABA levels and QUA-T activity were determined as described in Materials and Methods. The GABA concentration for 6 control mice was 175 f 10 !Jg GABA/g braio (wet weight) and the GABA-T activity was 24 t 2 ~rsol NADPH / h / g brain. ‘Ihe GABA-T activity is expressed as a % of the controls. Results represent the mean f SEM.
IC50
Binding
of
values
(the
[3H]-muscimol
These
vivo alanine
to
--in
(14))
at
results
indicate
GABA-T inhibition transaminase.
(S)-4-amino-5-chloropentanoic
the
is
in
(S)-AFPA
and
unlikely
that
that
(S)-AFPA
preliminary there glutamate
--In
vivo
was
resulting
sites.
inactivator; brain
receptor
receptor
Furthermore,
of
GABA
for it
effects
vivo.
the
concentration
Therefore,
GABA-ergic
specific
drug
binding
respectively.
GABA-T
(S)-AFPA
GABA
results
acid
GABA-T (2,13),
(S)-AFPA.
522
poor;
inhibition
were
to
will
but
to
M and
little
also a lesser
(S)-AFPA
no --in
vitro
lowered extent
or
a
-in and
by than
of is
transaminase, is
M
inhibitor
that or
3.0~10’~ any
irreversible
aspartate activity
specific
exhibit
indicate be
the of
8.6~10’~
a potent,
decarboxylase, brain
50%
(S)-AFPA
is
appears
relatively
with
Vol. 102, No. 1,198l
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNlCATlONS
ACKNOWLEDGMENTS We are grateful and to the National
to Paul Institutes
Sumner
for
determining
of Health
(Grant
the mouse brain NS 15703)
for
financial
GABA levels support.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Silverman, R.B. and Levy, M.A. (1980) Biochem. Biophys. Res. Commun. 95, 250-255. Xlverman, R.B. and Levy, M.A. (1981) Biochemistry 2, 1197-1203. Rando, R.R. and Bangerter, F.W. (1976) J. Am. Chem. Sot. 9Z& 6762-6764. Metcalf, B.W. and Jung, M.J. (1979) Molecular Pharmacol. 16, 539-545. Jung, M.J. and Metcalf, B.W. (1975) Biochem. Biophys. Res. Commun. 67, 301-306. zppert, B., Metcalf, B.W., Jung, M.J., and Casara, P. (1977) Eur. J. Biochem. I'+, 441-445. FLetcher, A. and Fowler, L.J. (1980) Biochem. Pharmacol. 29, 1451-1454. Balazs, R., Machiyama, Y., Hammond, B.J., Julian, T., andRichter, D. (1970) Biochem. J. 116, 445-467. F.W. (1977) Biochem. Biophys. Res. Rando, R.R., and Bangerter, Commun. 76, 1276-1281. Jung, M.J., Lippert, B., Metcalf, B.W., Schechter, P.J., Bblhlen, P., and Sjoerdsma, A. (1977) J. Neurochem. 28, 717-723. Jung, M.J., Lippert, B., Metcalf, B.W., *hIen, P., and Schechter, P.J. (1977) J. Neurochem. 2, 797-802. Leach, M.J. and Walker, J.M.G. (1977) Biochem. Pharmacol. 2, 1569-1572. Silverman, R.B. and Levy, M.A. (1980) J. Org. Chem. 45, 815-818. Bernasconi, R., Bittiger, H., Reid, J., and Martin, P. (1980) J. Neurochem. 2, 614-618.
523