- Email: [email protected]
On the effect of adenosine 3′,5′ cyclophosphate on the kinase of UDPG: α-1,4-glucan α-4 glucosyl transferase
Vol.
23, No. 3, 1966
BIOCHEMICAL
AND
BIOPHYSICAL
ON THE EFFECT OF ADENOS;INE 3',5' OF UDPG:W1,4-GLUCAN Fr.
and J.
Department of Biochemistry, University of Minnesota,
Received
March28,
Adenosine converts
form
for
(D) (Friedman 1964).
This
the cyclophosphate The present
information
3',5' 0.25
(1965).
cyclophosphate or 0.4
reaction
*This work Institute Service.
kinase
independent into
1963;
stimulate
(I)
form
nor
the mechanism assesses
the kinase
neither
6-phosphate
and Lamer, from
dependent
1964; Appleman
phosphorylase
the sensitivity
of action
which
of UDPG:Ol-1,4-glucan
the glucose
can be distinguished However,
paper
to
Rosell-Perez
1965).
I kinase
b kinase
of the kinase
of the cyclophosphate
the sensitivity
was tested
To 0.1 ml of
a
range
in principle
solution
(concentrations
ml of a solution
2.5 or 5 mM caffeine, kinase
known
and offers
are
some
on the mechanism.
The transferase and Larner
is
(EC 2.4.1.11)
and Larner,
and Iarner,
known.
6-phosphate
transferase
(Friedman
Iarner
College of Medical Sciences Minneapolis, Minnesota
cyclophosphate
the glucose
-et al.,
TRANSFERASE"
1966
3',5'
a-4-glucosyl
COMMUNICATIONS
CYCLOPHOSPHATE ON THE KINASE
a-4-GLUCOSYL **
Huijing
RESEARCH
containing
was stopped
was supported of Arthritis
by adding
containing
as indicated enzyme,
and 50 mM mercaptoethanol.
as described ATP, MgCl
in Figures
and adenosine 2 1 and 2) was added
8 mM glycerophosphate After
by Friedman
7 min at 30'
0.1 ml EDTA, 100 mM, and 0.05
pH 7.8, C, the ml aliquots
in part by a grant (No. AM 09071) from the National and Metabolic Diseases of the Il. S. Public Health
**On leave of absence from the Department of Biochemistry of the University recipient of a fellowship of the Netherlands of Amsterdam, The Netherlands, Organization for the Advancement of Pure Research (Z.W.0.)
259
Vol. 23, No. 3, 1966
were
then
glucose
BIOCHEMICAL
transferred
to tubes
6-phosphate)
Villar-Palasi
for
vity
or where
procedures
ferase
activity
was determined
transferase
I kinase
into
comparable
activity
did
from
UDPG into
before
the reaction
change
was determined. in acti-
incubation,
i.e.,
to controls
where
was started.
In some tubes,
in the presence
by
as a decrease
kinase
compared
(without
15 min at 30' C
glycogen
glycogen
not
for
is expressed
rates.
mixture
as described
incubated
the 7 minute
EDTA was added
gave
I activity
were
I kinase
I during
in cpm incorporated
was added,
total
The tubes
transferase
of the transferase
decrease
trol
of the
0.1 ml of the test
of transferase
of C14 glucose
and the incorporation The activity
containing
assay
-et a1.,(1966).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
of glucose
appreciably
total
the
no ATP Both
(I+D)
6-phosphate.
during
as a
con
transThe
transferase
test.
[ATP] IOmM [Mg2’] 5mM
E
304k3005 01 I
2
3
bdenosine Fig.
1 Transferase I kinase 3',5' cyclophosphate.
L 4
5
6
3’, 5’ cyclophosphote] activity
at varying
7
8
9
IO
x 10e7M concentrations
of adenosine
0.4 ml of 51,000 x g supernatant containing 2.5 mM caffeine was added to 0.1 ml of a solution containing ATP and MgC12, final concentrations 10 mM and 5 mM and cyclophosphate as indicated. The transferase I kinase reaction was allowed to proceed for 7 min, the transferase I activity was assayed for 15 min.
260
Vol.
23,
No.
As nized then
3, 1966
BIOCHEMICAL
source
a
of both
in 3 volumes for
for
3 hours
x g and the precipitate
50 mM mercaptoethanol.
Fig.
mately
3',5' 6 x 10
stimulation terms
by the cyclic
is
achieved
and a total
sensitivity that
in the
tration
of ader,osine
Robison
et al.,
ferase
which
is
1965,
Huijing,
cogen
concentration
sion
of transferase
3',5'
for
I form,
of the heart
(Posner
al. -et -J
varying
Mg
I (Huijing,
Butcher
2 shows the activiry 2+
concentrations.
et -- al 2
is
causes
heart,
half
max-
of the cyclic is
found.
This
surprising
in view
increases
the concen-
et al.,
1965;
196.5; Robison
results
of
of the trans-
a decrease
decrease
I kinase
et al.,
in the glyin a conver-
under
system
of skeletal
muscle
these
conditions
of skeletal
muscle
is
and the
same
and liver
1965).
of the transferase Both
in
1966).
transferase
kinase
maximal
a 20% increase
on the fraction
and this
at approxi
the
of rat
and Mayer,
3', 5' cyclophosphate
of the phosphorylase 1964;
effect
by
nucleotide.
Lhich
epinephrine
However,
as that
conditions
(Hammermeister
(Williams
super-
caused obtained
concentration
I kinase
cyclophosphatti
of the
assay
epinephrine,
in the
adenosine
Figure
organ
has no direct
D into
is
of the same magnitude
1965),
1966).
activation
cyclic
an identical
transferase
intact
The sensitivity heart
heart
for
To the clear
activity
of 10% homogenate
stimulation
of the rat
the fact
with
x g.
from
pH 7.8
was incubated
represents
of added
x g supernatant
was obtained
I kinase
these
nucleotide
in the absence
a 12,000
activation
maximal
Under
was cen-
of 2.5 or 5 mM.
in transferase
M cyclophosphate.
supernatant that
45 min at 51,000
Half
was homoge-
of 8 mM glycerophosphate,
ml
to a concentration
cyclophosphate. -8
of the activity
nucleotide
for
the increase
produced
Using imal
was added
The resulting
The enzyme solution
C and centrifuged
1 shows
adenosine
in 5
muscle
5 mM EDTA; 100 mM KF and
at 78,000
containing
x g.
COMMUNICATIONS
rabbit
pH 7.8 containing
was homogenized
caffeine
kinase
12,000
muscle
natant,
RESEARCH
2.0 min at
10 g fresh
45 min at 30'
BIOPHYSICAL
and its
transferase
50 mM Tris-HCl,
centrifuged
trifuged
AND
curves
261
with
I kinase
and without
as a function adenosine
3',5'
of
Vol.
23, No.
3, 1966
BIOCHEMICAL
AND
BIOPHYSICAL
8
Pig.
2
Transferase
I kinase
IO
activity
RESEARCH
14
12
at varying
COMMUNICATIONS
16
I8
concentrations
20
of M&12.
0.25 ml of 51,000 x g supernatant containing 5 mM caffeine was added to 0.1 ml ATP (final concentration 8 mM) and adenosine 3',5' cyclophosphate where indicated. The transferase I kinase reaction was I activity was assayed allowed to proceed for 7 min, the transferase for 15 min. cyclophosphate
are
cyclophosphate
changes
ditions causes
At low Mg
sigmoidal. the rate
the cyclophosphate
the cyclophosphate
from zero
may exert
a 100% stimulation.
concentrations to a finite
a regulatory
high
At
under
2+
Mg
conditions
2+
adenosine
role.
Mg
2+
there
itself
these
At 4 mM M&12
concentrations
where
Under
value.
3',5' con-
it
is no effect
has maximally
of
stimulated
the kinase. On the basis it
appears
serves form tions
that
the dual
of kinetic these role
analysis
phenomena
of Mg2+.
affinity
Adenosine
of the Mg
2+
can be explained
of activating
of the Mg ATP complex.
for
to be dealt
the kinase,
The activation, 3',5'
in a future
by the
possibility
and being however,
cyclophosphate
the activating
with
site
may
act
that
a substrate
requires
publication
high
to increase
in the concentrathe
on the enzyme.
References Appleman, Comm .,
H.M., l4,
Belocopitow, 550 (1964).
E. and Torres,
262
H.H.,
Biochem.
Bi.ophys.
Mg
Res.
2i
Vol.
23, No.
3, 1966
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Butcher, R.W., Ho, R.J., Meng, H.C. and Sutherland, E.W., J, Biol. Chem., 240, 4515 (1965). Friedman, D.L. and Larner, J., Biochemistry, 3 669 (1963). Friedman, D.L. and Larner, J,., Biochemistry, & 2261 (1965). Hammermeister, K.E., Yunis, A.A. and Krebs, E.G., J. Biol. Chem., 240, 986 (1965). Huijing, F., Fed. Proc., G 449 (1966). Posner, J.B., Hammermeister, K., Bratvold, G.E. and Krebs, E.G., Biochemistry, 1, 1040 (1964). Robison, G.A., Butcher, R.W., @ye, I., Morgan, H.E. and Sutherland, E.W., Mol. Pharmacol., & 168 (1965). Rosell-Perez, M. and Larner, J., Biochemistry, 2, 75 (1964). Villar-Palasi, C., Rosell-Perez, M., Hizukuri, S., Huijing, F. and Larner, J. in Methods in Enzymology (Ed. S.P. Colowick and N.O. Kaplan) Vol. VIII (64) (1966). Williams, B.J. and Mayer, S.E:., Fed. Proc., 2& 151 (1965).
263
23, No. 3, 1966
BIOCHEMICAL
AND
BIOPHYSICAL
ON THE EFFECT OF ADENOS;INE 3',5' OF UDPG:W1,4-GLUCAN Fr.
and J.
Department of Biochemistry, University of Minnesota,
Received
March28,
Adenosine converts
form
for
(D) (Friedman 1964).
This
the cyclophosphate The present
information
3',5' 0.25
(1965).
cyclophosphate or 0.4
reaction
*This work Institute Service.
kinase
independent into
1963;
stimulate
(I)
form
nor
the mechanism assesses
the kinase
neither
6-phosphate
and Lamer, from
dependent
1964; Appleman
phosphorylase
the sensitivity
of action
which
of UDPG:Ol-1,4-glucan
the glucose
can be distinguished However,
paper
to
Rosell-Perez
1965).
I kinase
b kinase
of the kinase
of the cyclophosphate
the sensitivity
was tested
To 0.1 ml of
a
range
in principle
solution
(concentrations
ml of a solution
2.5 or 5 mM caffeine, kinase
known
and offers
are
some
on the mechanism.
The transferase and Larner
is
(EC 2.4.1.11)
and Larner,
and Iarner,
known.
6-phosphate
transferase
(Friedman
Iarner
College of Medical Sciences Minneapolis, Minnesota
cyclophosphate
the glucose
-et al.,
TRANSFERASE"
1966
3',5'
a-4-glucosyl
COMMUNICATIONS
CYCLOPHOSPHATE ON THE KINASE
a-4-GLUCOSYL **
Huijing
RESEARCH
containing
was stopped
was supported of Arthritis
by adding
containing
as indicated enzyme,
and 50 mM mercaptoethanol.
as described ATP, MgCl
in Figures
and adenosine 2 1 and 2) was added
8 mM glycerophosphate After
by Friedman
7 min at 30'
0.1 ml EDTA, 100 mM, and 0.05
pH 7.8, C, the ml aliquots
in part by a grant (No. AM 09071) from the National and Metabolic Diseases of the Il. S. Public Health
**On leave of absence from the Department of Biochemistry of the University recipient of a fellowship of the Netherlands of Amsterdam, The Netherlands, Organization for the Advancement of Pure Research (Z.W.0.)
259
Vol. 23, No. 3, 1966
were
then
glucose
BIOCHEMICAL
transferred
to tubes
6-phosphate)
Villar-Palasi
for
vity
or where
procedures
ferase
activity
was determined
transferase
I kinase
into
comparable
activity
did
from
UDPG into
before
the reaction
change
was determined. in acti-
incubation,
i.e.,
to controls
where
was started.
In some tubes,
in the presence
by
as a decrease
kinase
compared
(without
15 min at 30' C
glycogen
glycogen
not
for
is expressed
rates.
mixture
as described
incubated
the 7 minute
EDTA was added
gave
I activity
were
I kinase
I during
in cpm incorporated
was added,
total
The tubes
transferase
of the transferase
decrease
trol
of the
0.1 ml of the test
of transferase
of C14 glucose
and the incorporation The activity
containing
assay
-et a1.,(1966).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
of glucose
appreciably
total
the
no ATP Both
(I+D)
6-phosphate.
during
as a
con
transThe
transferase
test.
[ATP] IOmM [Mg2’] 5mM
E
304k3005 01 I
2
3
bdenosine Fig.
1 Transferase I kinase 3',5' cyclophosphate.
L 4
5
6
3’, 5’ cyclophosphote] activity
at varying
7
8
9
IO
x 10e7M concentrations
of adenosine
0.4 ml of 51,000 x g supernatant containing 2.5 mM caffeine was added to 0.1 ml of a solution containing ATP and MgC12, final concentrations 10 mM and 5 mM and cyclophosphate as indicated. The transferase I kinase reaction was allowed to proceed for 7 min, the transferase I activity was assayed for 15 min.
260
Vol.
23,
No.
As nized then
3, 1966
BIOCHEMICAL
source
a
of both
in 3 volumes for
for
3 hours
x g and the precipitate
50 mM mercaptoethanol.
Fig.
mately
3',5' 6 x 10
stimulation terms
by the cyclic
is
achieved
and a total
sensitivity that
in the
tration
of ader,osine
Robison
et al.,
ferase
which
is
1965,
Huijing,
cogen
concentration
sion
of transferase
3',5'
for
I form,
of the heart
(Posner
al. -et -J
varying
Mg
I (Huijing,
Butcher
2 shows the activiry 2+
concentrations.
et -- al 2
is
causes
heart,
half
max-
of the cyclic is
found.
This
surprising
in view
increases
the concen-
et al.,
1965;
196.5; Robison
results
of
of the trans-
a decrease
decrease
I kinase
et al.,
in the glyin a conver-
under
system
of skeletal
muscle
these
conditions
of skeletal
muscle
is
and the
same
and liver
1965).
of the transferase Both
in
1966).
transferase
kinase
maximal
a 20% increase
on the fraction
and this
at approxi
the
of rat
and Mayer,
3', 5' cyclophosphate
of the phosphorylase 1964;
effect
by
nucleotide.
Lhich
epinephrine
However,
as that
conditions
(Hammermeister
(Williams
super-
caused obtained
concentration
I kinase
cyclophosphatti
of the
assay
epinephrine,
in the
adenosine
Figure
organ
has no direct
D into
is
of the same magnitude
1965),
1966).
activation
cyclic
an identical
transferase
intact
The sensitivity heart
heart
for
To the clear
activity
of 10% homogenate
stimulation
of the rat
the fact
with
x g.
from
pH 7.8
was incubated
represents
of added
x g supernatant
was obtained
I kinase
these
nucleotide
in the absence
a 12,000
activation
maximal
Under
was cen-
of 2.5 or 5 mM.
in transferase
M cyclophosphate.
supernatant that
45 min at 51,000
Half
was homoge-
of 8 mM glycerophosphate,
ml
to a concentration
cyclophosphate. -8
of the activity
nucleotide
for
the increase
produced
Using imal
was added
The resulting
The enzyme solution
C and centrifuged
1 shows
adenosine
in 5
muscle
5 mM EDTA; 100 mM KF and
at 78,000
containing
x g.
COMMUNICATIONS
rabbit
pH 7.8 containing
was homogenized
caffeine
kinase
12,000
muscle
natant,
RESEARCH
2.0 min at
10 g fresh
45 min at 30'
BIOPHYSICAL
and its
transferase
50 mM Tris-HCl,
centrifuged
trifuged
AND
curves
261
with
I kinase
and without
as a function adenosine
3',5'
of
Vol.
23, No.
3, 1966
BIOCHEMICAL
AND
BIOPHYSICAL
8
Pig.
2
Transferase
I kinase
IO
activity
RESEARCH
14
12
at varying
COMMUNICATIONS
16
I8
concentrations
20
of M&12.
0.25 ml of 51,000 x g supernatant containing 5 mM caffeine was added to 0.1 ml ATP (final concentration 8 mM) and adenosine 3',5' cyclophosphate where indicated. The transferase I kinase reaction was I activity was assayed allowed to proceed for 7 min, the transferase for 15 min. cyclophosphate
are
cyclophosphate
changes
ditions causes
At low Mg
sigmoidal. the rate
the cyclophosphate
the cyclophosphate
from zero
may exert
a 100% stimulation.
concentrations to a finite
a regulatory
high
At
under
2+
Mg
conditions
2+
adenosine
role.
Mg
2+
there
itself
these
At 4 mM M&12
concentrations
where
Under
value.
3',5' con-
it
is no effect
has maximally
of
stimulated
the kinase. On the basis it
appears
serves form tions
that
the dual
of kinetic these role
analysis
phenomena
of Mg2+.
affinity
Adenosine
of the Mg
2+
can be explained
of activating
of the Mg ATP complex.
for
to be dealt
the kinase,
The activation, 3',5'
in a future
by the
possibility
and being however,
cyclophosphate
the activating
with
site
may
act
that
a substrate
requires
publication
high
to increase
in the concentrathe
on the enzyme.
References Appleman, Comm .,
H.M., l4,
Belocopitow, 550 (1964).
E. and Torres,
262
H.H.,
Biochem.
Bi.ophys.
Mg
Res.
2i
Vol.
23, No.
3, 1966
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Butcher, R.W., Ho, R.J., Meng, H.C. and Sutherland, E.W., J, Biol. Chem., 240, 4515 (1965). Friedman, D.L. and Larner, J., Biochemistry, 3 669 (1963). Friedman, D.L. and Larner, J,., Biochemistry, & 2261 (1965). Hammermeister, K.E., Yunis, A.A. and Krebs, E.G., J. Biol. Chem., 240, 986 (1965). Huijing, F., Fed. Proc., G 449 (1966). Posner, J.B., Hammermeister, K., Bratvold, G.E. and Krebs, E.G., Biochemistry, 1, 1040 (1964). Robison, G.A., Butcher, R.W., @ye, I., Morgan, H.E. and Sutherland, E.W., Mol. Pharmacol., & 168 (1965). Rosell-Perez, M. and Larner, J., Biochemistry, 2, 75 (1964). Villar-Palasi, C., Rosell-Perez, M., Hizukuri, S., Huijing, F. and Larner, J. in Methods in Enzymology (Ed. S.P. Colowick and N.O. Kaplan) Vol. VIII (64) (1966). Williams, B.J. and Mayer, S.E:., Fed. Proc., 2& 151 (1965).
263