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Isoenzymicity in mouse liver guanine deaminase demonstrable under substrate stress
Vol. 70, No. 2, 1976
BIOCHEMICAL
ISOENZYMICITY
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IN MOUSE LIVER GUANINE DEAMINASE DEMONSTRABLE UNDER SUBSTRATE STRESS , K. Sree Kumar**
A. Sitaramayya*
Department of Biochemistry, Lucknow, U.P., India
Received
March
and P.S.
Lucknow
Krishnan***
University,
15,1976
SUMMARY: Two isoenzymes of guanine deaminase could be demonstrated sliver of mice subjected to guanine stress while the salinetreated controls showed only one. The one appearing under stress was a regulatory protein showing a sigmoidal substrate saturation curve, but was not influenced by GTP, allantoin or Mg2+
rat
Guanine
daminase
was mostly
localised
reported the
earlier
least
and liver
(2).
the
isoenzymicity
was lacking
that
of rat
communication
guanine
and mouse under
we report
the one present
in the untreated
affected
response
to
*Present
address
**Present
address
***Present
address
deaminase
contained
demonstrable (I).
the other activity
substrate
deaminase
Partially by GTP,
tissues
(3).
was inducible
stress
(2).
in brain
In the present
different
substrate
This
from
mice,
and in addition
inducible concentration,
isoenzyme
or Mg 2+ .
480
to showed
but was not
: Centre de Neurochimie C.N.R.S., 11, rue Humann, 67085 STRASBOURG, Prance : Department of Pharmacology, USC School of Medicine, 2025 Zonal Avenue, Los Angeles, Calif. 90033, U.S.A. : Department of Botany, Calicut University Calicut, Kerala State, India
Copyright 0 1976 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
We have
in the guanine-stressed
animals.
increasing
by GTP, allantoin
We have
was shown to be unaffected
the observation,
of guanine
(I).
mouse liver
in the mouse liver
the enzyme from
of an isoenzyme
a sigmoidal
x g supernatant studied
Also
of mouse and
and brain
tissues
or Mg2+ unlike
demonstrated
15,000
liver
of the four
enzyme from mouse liver
allantoin also
that
tissues
in the
in the
enzyme activity
in the other purified
activity
Vol. 70, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS : Adult albino male mice were divided into three groups of 24 animals each. The first group was injected with normal saline and the second with guanine throughout the experimental period. The third group received guanine during the first 5 days followed by a mixture of guanine and ethionine thereafter. Guanine and ethionine were suspensions in normal saline and the dosage 120 and 70 mg per kg. body weight per day respectively. The injections were intraperitoneal and were repeated every 24 h. In experiments reported earlier (2), the mice were sacrificed after one injection while in the present experiments they were sacrificed in batches of 8 from each group after receiving 5, 10 and 15 injections. The livers were extracted and homogenized in 0.25 M sucrose solution. The enzyme assays and the kinetic studies were conducted as descrjked earlier (1, 3). The influence of GTP (at 50 PM), allantoin (both at 1 mM) was tested by including them in the assay system. or Mg The homogenate was centrifuged for 20 min. at 15,000 x g and the supernatant obtained served as the starting material for the partial purification of the enzyme. Briefly, the enzyme activity precipitating between 35-55 % ammonium sulphate saturation was dialysed against water for 6 h and the contents of the dialysis bag were centrifuged. The supernatant was adjusted to 20 mM phosphate, pH 7.0 and loaded on a DEAE-cellulose column (1 x 10 cm) equilibrated with 20 mM phosphate, pH 7.0. The column was washed with the same buffer and eluted stepwise with 65 ml each of 50 mM, 85 mM, 125 mM and 200 mM phosphate buffer, pH 7.0. Five ml fractions were collected and analysed for enzyme activity and protein content. The protein content in the DUE-cellulose column eluates was determined spectrophotometrically (4) and in all other fractions by the method of Lowry et al (5). RESULTS AND DISCUSSION We have mouse liver
reported
has increased
decreased
slightly
4 days
(2).
rates,
77 % after
to 45-50
kinetic from
properties the normal
a comparison, animals deaminase column
of stress
animals
a partial
activity
from
be eluted
purified
purification in order
synthesized
from
under
saline-
under
saline-treated with
control
enzyme in untreated
the enzyme activity partially
could
of the
slightly
showed
was attempted
of guanine
125 mM phosphate
level
but
for
higher
induction
IO and 15 days activity.
of the enzyme
to observe
whether
substrate
stress
animals
and ethionine-
the same conditions.
animals
481
in
the administration
we found
animals
of the enzyme
was also
injection
activity
and 53 % and 50 % after
experiment
pattern
deaminase
% on prolonging
Ethionine-treated
stressed
guanine
experiment
5 days
In the present the
that
by 65 % on a single
In the present
respectively.
from
earlier
loaded (A enzyme)
(1).
the altered
To afford treated Guaninc
on a DEAE-cellulose with
no more of
BIOCHEMICAL
Vol. 70, No. 2, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
F . 5 Y i? I.OP
Fraction
Fig.
1 : Elution pattern (0) and protein column.
activity That
getting agrees
enzyme from
eluted
with
of liver supernatant (@) of guanine-stressed
on increasing
the earlier
the untreated
guanine-treated
animals
was an additional
enzymic
activity
for from
it
increased
loaded
the
stressed
ethionine-treated shoulder
of activity
that
analysis
the
from
buffer.
(B enzyme)
A typical
animals
was shown in Fig.
animals
also
exhibited
in the 14 % of the for
5 days
animals
pattern
eluates
showed
small
stressed
of the enzyme
but
in spite
no increase
the
there
1. The enzyme preparation
a very
in 200 mM phosphate
for
from
elution
of the
column,
stressed
preparations
to 200 mM.
from
appearing
peak accounted
from animals
to 19 % and 32 % for
of the homogenates
behaviour
the DEAE-cellulose
This
on the column
IO and 15 days respectively.
of the buffer
But when the preparation
peak of enzyme activity
of 200 mM phosphate
deaminase activity from DEAE-cellulose
chromatographic
(I).
was eluted
guanine mice
the molarity
reported animals
eluates
while
no.
from
definite of the
fact!
in enzyme
activity. The 1\ enzyme from Michaelis-Mentes
response
all
three
groups
to increasing
482
of animals substrate
showed
concentration
classical as reported
BIOCHEMICAL
Vol. 70, No. 2, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
>
[Guanin&@A)
Fig.
for
2 : Reaction guanine stressed
rate-substrate concentration deaminase isoenzymes A (0) mice.
the enzyme in untreated
treated
animals
guanine
deaminase
both
the
deaminase
like
demonstrable
is
mouse brain normal
Substrate the concentration
significant
enhanced
rat
animals
mouse liver
under
concentrations.
It
response
(Fig.
liver
were
the -B enzyme of guanine2).
and brain
But unlike
the
or mouse brain,
animals
and the A enzyme in
unaffected
by GTE, allantoin
. Obviously
elevating
from
while
in the guanine-treated
or ethionine-treated 2+
(I),
a sigmoidal
isoenzymes
isoenzymes
saline0rMg
showed
animals
relationship for liver and g (0) from guanine-
under
that substrate
also and rat
has two molecular brain
conditions stress
while reveals
and liver.
Only
the g enzyme this
of the g isoenzymc the concentration
species
the A enzyme is
isoenzymicity to demonstrable
of the regulatory
of guanine is
in very
low
clearly
by
levels. isoenzyme
is
stress.
ACKNOWLEDGEMENTS A.S. is grateful to the Council of Scientific and industrial Research and K.S.K. to the University grants Commission, New Dehli, for the award of fello>?shipp.This department is indebted to the Rockefeller Foundation for generous grants.
483
Vol. 70, No. 2; 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1. Sree Kumar, K., Sitaramayya, A., and Krishnan, P.S. (1972) Biochem. J. 128, 1079-1088. 2. Sitaramayya, A., Shahid Ali, Sree Kumar, K. and Krishnan, P.S. (1974) Biochem. J. 2, 143-146. 3. Sree Kumar, K., Sitaramayya, A. and Krishnan, P.S. (1973) Biochem. 3. 131, 683-687. 4. Kalckar, H.M. (1947) J. Biol. Chem. 167, 461-475. 5. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.
484
BIOCHEMICAL
ISOENZYMICITY
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
IN MOUSE LIVER GUANINE DEAMINASE DEMONSTRABLE UNDER SUBSTRATE STRESS , K. Sree Kumar**
A. Sitaramayya*
Department of Biochemistry, Lucknow, U.P., India
Received
March
and P.S.
Lucknow
Krishnan***
University,
15,1976
SUMMARY: Two isoenzymes of guanine deaminase could be demonstrated sliver of mice subjected to guanine stress while the salinetreated controls showed only one. The one appearing under stress was a regulatory protein showing a sigmoidal substrate saturation curve, but was not influenced by GTP, allantoin or Mg2+
rat
Guanine
daminase
was mostly
localised
reported the
earlier
least
and liver
(2).
the
isoenzymicity
was lacking
that
of rat
communication
guanine
and mouse under
we report
the one present
in the untreated
affected
response
to
*Present
address
**Present
address
***Present
address
deaminase
contained
demonstrable (I).
the other activity
substrate
deaminase
Partially by GTP,
tissues
(3).
was inducible
stress
(2).
in brain
In the present
different
substrate
This
from
mice,
and in addition
inducible concentration,
isoenzyme
or Mg 2+ .
480
to showed
but was not
: Centre de Neurochimie C.N.R.S., 11, rue Humann, 67085 STRASBOURG, Prance : Department of Pharmacology, USC School of Medicine, 2025 Zonal Avenue, Los Angeles, Calif. 90033, U.S.A. : Department of Botany, Calicut University Calicut, Kerala State, India
Copyright 0 1976 by Academic Press, Inc. Ail rights of reproduction in any form reserved.
We have
in the guanine-stressed
animals.
increasing
by GTP, allantoin
We have
was shown to be unaffected
the observation,
of guanine
(I).
mouse liver
in the mouse liver
the enzyme from
of an isoenzyme
a sigmoidal
x g supernatant studied
Also
of mouse and
and brain
tissues
or Mg2+ unlike
demonstrated
15,000
liver
of the four
enzyme from mouse liver
allantoin also
that
tissues
in the
in the
enzyme activity
in the other purified
activity
Vol. 70, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS : Adult albino male mice were divided into three groups of 24 animals each. The first group was injected with normal saline and the second with guanine throughout the experimental period. The third group received guanine during the first 5 days followed by a mixture of guanine and ethionine thereafter. Guanine and ethionine were suspensions in normal saline and the dosage 120 and 70 mg per kg. body weight per day respectively. The injections were intraperitoneal and were repeated every 24 h. In experiments reported earlier (2), the mice were sacrificed after one injection while in the present experiments they were sacrificed in batches of 8 from each group after receiving 5, 10 and 15 injections. The livers were extracted and homogenized in 0.25 M sucrose solution. The enzyme assays and the kinetic studies were conducted as descrjked earlier (1, 3). The influence of GTP (at 50 PM), allantoin (both at 1 mM) was tested by including them in the assay system. or Mg The homogenate was centrifuged for 20 min. at 15,000 x g and the supernatant obtained served as the starting material for the partial purification of the enzyme. Briefly, the enzyme activity precipitating between 35-55 % ammonium sulphate saturation was dialysed against water for 6 h and the contents of the dialysis bag were centrifuged. The supernatant was adjusted to 20 mM phosphate, pH 7.0 and loaded on a DEAE-cellulose column (1 x 10 cm) equilibrated with 20 mM phosphate, pH 7.0. The column was washed with the same buffer and eluted stepwise with 65 ml each of 50 mM, 85 mM, 125 mM and 200 mM phosphate buffer, pH 7.0. Five ml fractions were collected and analysed for enzyme activity and protein content. The protein content in the DUE-cellulose column eluates was determined spectrophotometrically (4) and in all other fractions by the method of Lowry et al (5). RESULTS AND DISCUSSION We have mouse liver
reported
has increased
decreased
slightly
4 days
(2).
rates,
77 % after
to 45-50
kinetic from
properties the normal
a comparison, animals deaminase column
of stress
animals
a partial
activity
from
be eluted
purified
purification in order
synthesized
from
under
saline-
under
saline-treated with
control
enzyme in untreated
the enzyme activity partially
could
of the
slightly
showed
was attempted
of guanine
125 mM phosphate
level
but
for
higher
induction
IO and 15 days activity.
of the enzyme
to observe
whether
substrate
stress
animals
and ethionine-
the same conditions.
animals
481
in
the administration
we found
animals
of the enzyme
was also
injection
activity
and 53 % and 50 % after
experiment
pattern
deaminase
% on prolonging
Ethionine-treated
stressed
guanine
experiment
5 days
In the present the
that
by 65 % on a single
In the present
respectively.
from
earlier
loaded (A enzyme)
(1).
the altered
To afford treated Guaninc
on a DEAE-cellulose with
no more of
BIOCHEMICAL
Vol. 70, No. 2, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.0
F . 5 Y i? I.OP
Fraction
Fig.
1 : Elution pattern (0) and protein column.
activity That
getting agrees
enzyme from
eluted
with
of liver supernatant (@) of guanine-stressed
on increasing
the earlier
the untreated
guanine-treated
animals
was an additional
enzymic
activity
for from
it
increased
loaded
the
stressed
ethionine-treated shoulder
of activity
that
analysis
the
from
buffer.
(B enzyme)
A typical
animals
was shown in Fig.
animals
also
exhibited
in the 14 % of the for
5 days
animals
pattern
eluates
showed
small
stressed
of the enzyme
but
in spite
no increase
the
there
1. The enzyme preparation
a very
in 200 mM phosphate
for
from
elution
of the
column,
stressed
preparations
to 200 mM.
from
appearing
peak accounted
from animals
to 19 % and 32 % for
of the homogenates
behaviour
the DEAE-cellulose
This
on the column
IO and 15 days respectively.
of the buffer
But when the preparation
peak of enzyme activity
of 200 mM phosphate
deaminase activity from DEAE-cellulose
chromatographic
(I).
was eluted
guanine mice
the molarity
reported animals
eluates
while
no.
from
definite of the
fact!
in enzyme
activity. The 1\ enzyme from Michaelis-Mentes
response
all
three
groups
to increasing
482
of animals substrate
showed
concentration
classical as reported
BIOCHEMICAL
Vol. 70, No. 2, 1976
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
>
[Guanin&@A)
Fig.
for
2 : Reaction guanine stressed
rate-substrate concentration deaminase isoenzymes A (0) mice.
the enzyme in untreated
treated
animals
guanine
deaminase
both
the
deaminase
like
demonstrable
is
mouse brain normal
Substrate the concentration
significant
enhanced
rat
animals
mouse liver
under
concentrations.
It
response
(Fig.
liver
were
the -B enzyme of guanine2).
and brain
But unlike
the
or mouse brain,
animals
and the A enzyme in
unaffected
by GTE, allantoin
. Obviously
elevating
from
while
in the guanine-treated
or ethionine-treated 2+
(I),
a sigmoidal
isoenzymes
isoenzymes
saline0rMg
showed
animals
relationship for liver and g (0) from guanine-
under
that substrate
also and rat
has two molecular brain
conditions stress
while reveals
and liver.
Only
the g enzyme this
of the g isoenzymc the concentration
species
the A enzyme is
isoenzymicity to demonstrable
of the regulatory
of guanine is
in very
low
clearly
by
levels. isoenzyme
is
stress.
ACKNOWLEDGEMENTS A.S. is grateful to the Council of Scientific and industrial Research and K.S.K. to the University grants Commission, New Dehli, for the award of fello>?shipp.This department is indebted to the Rockefeller Foundation for generous grants.
483
Vol. 70, No. 2; 1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
REFERENCES 1. Sree Kumar, K., Sitaramayya, A., and Krishnan, P.S. (1972) Biochem. J. 128, 1079-1088. 2. Sitaramayya, A., Shahid Ali, Sree Kumar, K. and Krishnan, P.S. (1974) Biochem. J. 2, 143-146. 3. Sree Kumar, K., Sitaramayya, A. and Krishnan, P.S. (1973) Biochem. 3. 131, 683-687. 4. Kalckar, H.M. (1947) J. Biol. Chem. 167, 461-475. 5. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.
484