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Nucleotide activation of phosphorylase b in the presence and absence of salmine
Vol. 30, No. 4, 1968
BIOCHEMICAL AND 8lOPHYSlCAL
RESEARCH COMMUNICATIONS
NUCLECTIDE ACTIVATION OF PHOSPHORILASE _b IN THE PRESENCEANDABSENCEOFSAIMINE by &vid
M. Mott and Allan L. Bieber
Department of Chemistry, Arizona State University, Tempe, Arizona
85281
ReceivedJanuary 15, 1968 Phosphorylase 12is activated et al, 1953).
to its maximumvelocity
Similar concentrations
however IMP or less than saturating
of IMP activate concentrations
of phosphorylase 1 by structural
the enzyme to about lO$,
Of AMP completely activate
the enzyme in the presence of salmine (Krebs, 1954). the activation
by 1x10-% AMP (Cori
The following
work on
analogues of AMP is part of a
study designed to explore the chemical nature of the nucleotide binding site and clarify
the action of salmi.ne on activation.
nucleotide activation markedly altered
indicate
Preliminary
a high degree of specificity
in the presence of salmine.
studies
of
for AMP which is
Nucleotide activation
of
phosphorylase 2 in the presence of salmine has manifested the critical tance of the 5'-phosphate and less rigid
impor-
requirements for the pyrimidine
and
imidazole rings of the purine base. Materials
and Methods
Phosphorylase 2 was isolated from rabbit of Fischer and Krebs (1958). glycerophosphate,
Third crystals
5x10m4MEM'A buffer prior
Enzymatic activity
skeletal
muscle by the method
were dissolved in a pH 6.8, O.&M to AMP removal by Norite treatment.
was measured according to the method of Illingsworth
and Cori (1953) with the following
changes. The enzymatic reaction
in the previously
using 0.1s glycogen.
for activation
described buffer
were g.4t0.2x10-4M in the final
The compoundsexamined
reaction media. All activity
measurementswere made against a blank containing substrate,
363
was run
enzyme and salmine
BIOCHEMICAL
Vol. 30, No. 4, 1968
(where applicable)
AND BlOPHYSlCAL
in order to correct
for residual phosphorylase activity
As noted by Cori -et aL(1943),
under these conditions. under the conditions
described above does not yield
For this reason, all
assay procedures were identical
anslogues and enzyme concentrations analogues to activate activity,
calculated
The additional
purification
except for structural of structural
of phosphorylase b. and inosine 5'-phosphofluoridate of Alberta.
adenine 5'-monophosphate (iso-AMP) was a gift
purity.
order kinetics.
from the moles of inorganic phosphate released in five
of Dr. Alexander JJampton,University
available
zero or first
the enzyme is expressed as the percent of AMP mediated
Inosine 5'-chloromethylphosphonate
of Illinois.
assay of phosphorylase
employed. Tne ability
minutes, with the sameconcentration
gifts
RESEARCH COMMUNICATIONS
were
3-&D-ribofuranosyl-
from Dr. Nelson Leonard, University
nucleotides were commercial samples of highest
m\e to limited
availability
of someof the compoundsfurther
was not attempted unless indicated.
Results and Discussion The percent activation
data for the structural
phosphorylase b is reproducible sistent
analogues of AMP on
to within f 5 percent activity
data was obtained for different
enzyme preparations
units.
Con-
and was reproducible
over a four fold range of enzyme concentration. In view of the enhanced activation the presence of salmine, structural for t&e data in Table I.
of phosphorylase 2 by AMP and IMP in
analogues of these nucleotides were used
The failure
of adenostie to activate
phosphorylase 2
demonstrates the importance of tine phosphate group on AMP. An examination of the results site
which
in Table I indicates a high degree of specificity binds the phosphate moiety.
for those analogues which failed
of the enzyme
Fksmination of the structural
to activate
formula
the enzyme suggests the possible
importance of a hydrogen on the 5'-phosphate group w'hich could readily
hydrogen
bond to the enzyme. The chemical nature of the enzyme-adenine ring interaction to active
which leads
phosphorylase 2 in the presence and absence of salmine is not readily
364
Vol. 30, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
apparent from the data of Table II.
A number of general characteristics,
however,
Table I STRUcmTRALANALOGUES OFAMP ANDIMP Modification
on the Ribose 5-phosphate Moietya
$ activity without salmine
compound adenosine 5'-monophosphate (control) adenosine 5'-phosphoramidate adenoeine 5'-acetate
$ activity with salmine
100
132
traceb
83
0
0
adenylic acid, 3'(2')
mixed isomer
0
0"
adenosine 3',5'-cyclic
phosphate
0
0
0
0
adenosine 5'-triphosphate
w-e
OC
trace
89
inosine 5t-phosphofluoridate
0
0
inosine 5t-chloromethylphosphonate
0
0
adenosine inosine 5'-monophosphate (control)
a Ribose-5'-phosphate failed to activate without salmine (Krebs, 1954).
phosphorylase 2 with or
b Values from 5 to 12 percent. ' E. G. Krebs (1954). are suggested.
In the absence of salmine, little
any of the analogue compoundsexcept iso-AMP. this nucleotide activate
activation
Specificity
data for modifications
is sensitive
and complete deletion
to alteration (uridine
is observed for
In the presence of salmine
is again unique, showing even greater ability
phosphorylase.
the activation
activity
than AMPto
at the AMPbinding site is apparent from of the purine ring structure.
Ehzyme
(8-azaguanosine 5'-phosphate and iso-AMY)
5'-monophosphate and cytidine
5'-monophosphate)
of the imidazole ring. Until
the mechanismby which salmine alters the high specificity
365
for
8lOCHEMlCAL
Vol. 30, No. 4, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table II OF AMP
STRUCTURALANAU3GUB
Modifications
on the Adenine Ring
$ activity without salmine
compound adenosine 5'-monophosphate (control) 5-B-D-ribofuranosyladenine
100
5'-monophosphate
87
151
trace
94
inosine 5'-monophosphate'
trace
69
guanosine 5'-monophosphatea
trace
61
uridine
5'-monophosphate'
trace
41
8-azaguanosine gs-monophosphate
trace
39
cytidine
trace
25
0
0
0
0
6-mercaptopurtie
riboside
5'-phosphate
5'-monophosphatea
6-azauridine
5'-monophosphate
xanthosine 5'-monophosphate
a Purified nucleotide
by Dowex 1 column chromatography.
activators
of phosphorylase b is elucidated,
the chemistry of activation ments to activate
the significance
in the presence of salmine relative
phosphorylase JJ in its absence will
enzyme are not affected en activating stigating
ability
a different
by salmine.
nucleotide
structures
currently
of salmine on the dissociation
rather than in-
Investigation
of
constants of various analogues is
in progress to determine if there is a parallel
specifications
the native
mechanism. Ifrebs (1954) reported a decrease
of the I$ values for IMP and AMP in the presence of salmine. the effect
It
This suggests that salmine is enhancing
inherent to certain activation
to the require-
remain obscure.
should be noted that only those compoundswhich do not activate
of
of the nucleotides for binding and their
vation.
366
between structural requirements for acti-
Vol. 30, No. 4, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgements: The authors wish to thank Dr. Nelson Leonard and Dr. Alexander Hamptonfor their viously
generosity
indicated.
in supplying the compoundspre-
This work was supported by American Cancer
Society Grant BP-383Aand by a special graduate fellowship grant from the Arizona Division of the American Cancer Society. References Cori, C. F., Cori, G. T., and Green, A. A., J. Biol. C&em., 151:;59 (1943). Escher, E. H., and Krebs, E. G., J. Biol. Chem., 231:65 (lgm. Illingsworth, B., and Cori, G. T., Biochem. Prepar~ons, J:l (1953). Krebs, E. G., Biochim. Biophys. Acta, s:'jOg (1954).
367
BIOCHEMICAL AND 8lOPHYSlCAL
RESEARCH COMMUNICATIONS
NUCLECTIDE ACTIVATION OF PHOSPHORILASE _b IN THE PRESENCEANDABSENCEOFSAIMINE by &vid
M. Mott and Allan L. Bieber
Department of Chemistry, Arizona State University, Tempe, Arizona
85281
ReceivedJanuary 15, 1968 Phosphorylase 12is activated et al, 1953).
to its maximumvelocity
Similar concentrations
however IMP or less than saturating
of IMP activate concentrations
of phosphorylase 1 by structural
the enzyme to about lO$,
Of AMP completely activate
the enzyme in the presence of salmine (Krebs, 1954). the activation
by 1x10-% AMP (Cori
The following
work on
analogues of AMP is part of a
study designed to explore the chemical nature of the nucleotide binding site and clarify
the action of salmi.ne on activation.
nucleotide activation markedly altered
indicate
Preliminary
a high degree of specificity
in the presence of salmine.
studies
of
for AMP which is
Nucleotide activation
of
phosphorylase 2 in the presence of salmine has manifested the critical tance of the 5'-phosphate and less rigid
impor-
requirements for the pyrimidine
and
imidazole rings of the purine base. Materials
and Methods
Phosphorylase 2 was isolated from rabbit of Fischer and Krebs (1958). glycerophosphate,
Third crystals
5x10m4MEM'A buffer prior
Enzymatic activity
skeletal
muscle by the method
were dissolved in a pH 6.8, O.&M to AMP removal by Norite treatment.
was measured according to the method of Illingsworth
and Cori (1953) with the following
changes. The enzymatic reaction
in the previously
using 0.1s glycogen.
for activation
described buffer
were g.4t0.2x10-4M in the final
The compoundsexamined
reaction media. All activity
measurementswere made against a blank containing substrate,
363
was run
enzyme and salmine
BIOCHEMICAL
Vol. 30, No. 4, 1968
(where applicable)
AND BlOPHYSlCAL
in order to correct
for residual phosphorylase activity
As noted by Cori -et aL(1943),
under these conditions. under the conditions
described above does not yield
For this reason, all
assay procedures were identical
anslogues and enzyme concentrations analogues to activate activity,
calculated
The additional
purification
except for structural of structural
of phosphorylase b. and inosine 5'-phosphofluoridate of Alberta.
adenine 5'-monophosphate (iso-AMP) was a gift
purity.
order kinetics.
from the moles of inorganic phosphate released in five
of Dr. Alexander JJampton,University
available
zero or first
the enzyme is expressed as the percent of AMP mediated
Inosine 5'-chloromethylphosphonate
of Illinois.
assay of phosphorylase
employed. Tne ability
minutes, with the sameconcentration
gifts
RESEARCH COMMUNICATIONS
were
3-&D-ribofuranosyl-
from Dr. Nelson Leonard, University
nucleotides were commercial samples of highest
m\e to limited
availability
of someof the compoundsfurther
was not attempted unless indicated.
Results and Discussion The percent activation
data for the structural
phosphorylase b is reproducible sistent
analogues of AMP on
to within f 5 percent activity
data was obtained for different
enzyme preparations
units.
Con-
and was reproducible
over a four fold range of enzyme concentration. In view of the enhanced activation the presence of salmine, structural for t&e data in Table I.
of phosphorylase 2 by AMP and IMP in
analogues of these nucleotides were used
The failure
of adenostie to activate
phosphorylase 2
demonstrates the importance of tine phosphate group on AMP. An examination of the results site
which
in Table I indicates a high degree of specificity binds the phosphate moiety.
for those analogues which failed
of the enzyme
Fksmination of the structural
to activate
formula
the enzyme suggests the possible
importance of a hydrogen on the 5'-phosphate group w'hich could readily
hydrogen
bond to the enzyme. The chemical nature of the enzyme-adenine ring interaction to active
which leads
phosphorylase 2 in the presence and absence of salmine is not readily
364
Vol. 30, No. 4, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
apparent from the data of Table II.
A number of general characteristics,
however,
Table I STRUcmTRALANALOGUES OFAMP ANDIMP Modification
on the Ribose 5-phosphate Moietya
$ activity without salmine
compound adenosine 5'-monophosphate (control) adenosine 5'-phosphoramidate adenoeine 5'-acetate
$ activity with salmine
100
132
traceb
83
0
0
adenylic acid, 3'(2')
mixed isomer
0
0"
adenosine 3',5'-cyclic
phosphate
0
0
0
0
adenosine 5'-triphosphate
w-e
OC
trace
89
inosine 5t-phosphofluoridate
0
0
inosine 5t-chloromethylphosphonate
0
0
adenosine inosine 5'-monophosphate (control)
a Ribose-5'-phosphate failed to activate without salmine (Krebs, 1954).
phosphorylase 2 with or
b Values from 5 to 12 percent. ' E. G. Krebs (1954). are suggested.
In the absence of salmine, little
any of the analogue compoundsexcept iso-AMP. this nucleotide activate
activation
Specificity
data for modifications
is sensitive
and complete deletion
to alteration (uridine
is observed for
In the presence of salmine
is again unique, showing even greater ability
phosphorylase.
the activation
activity
than AMPto
at the AMPbinding site is apparent from of the purine ring structure.
Ehzyme
(8-azaguanosine 5'-phosphate and iso-AMY)
5'-monophosphate and cytidine
5'-monophosphate)
of the imidazole ring. Until
the mechanismby which salmine alters the high specificity
365
for
8lOCHEMlCAL
Vol. 30, No. 4, 1968
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table II OF AMP
STRUCTURALANAU3GUB
Modifications
on the Adenine Ring
$ activity without salmine
compound adenosine 5'-monophosphate (control) 5-B-D-ribofuranosyladenine
100
5'-monophosphate
87
151
trace
94
inosine 5'-monophosphate'
trace
69
guanosine 5'-monophosphatea
trace
61
uridine
5'-monophosphate'
trace
41
8-azaguanosine gs-monophosphate
trace
39
cytidine
trace
25
0
0
0
0
6-mercaptopurtie
riboside
5'-phosphate
5'-monophosphatea
6-azauridine
5'-monophosphate
xanthosine 5'-monophosphate
a Purified nucleotide
by Dowex 1 column chromatography.
activators
of phosphorylase b is elucidated,
the chemistry of activation ments to activate
the significance
in the presence of salmine relative
phosphorylase JJ in its absence will
enzyme are not affected en activating stigating
ability
a different
by salmine.
nucleotide
structures
currently
of salmine on the dissociation
rather than in-
Investigation
of
constants of various analogues is
in progress to determine if there is a parallel
specifications
the native
mechanism. Ifrebs (1954) reported a decrease
of the I$ values for IMP and AMP in the presence of salmine. the effect
It
This suggests that salmine is enhancing
inherent to certain activation
to the require-
remain obscure.
should be noted that only those compoundswhich do not activate
of
of the nucleotides for binding and their
vation.
366
between structural requirements for acti-
Vol. 30, No. 4, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Acknowledgements: The authors wish to thank Dr. Nelson Leonard and Dr. Alexander Hamptonfor their viously
generosity
indicated.
in supplying the compoundspre-
This work was supported by American Cancer
Society Grant BP-383Aand by a special graduate fellowship grant from the Arizona Division of the American Cancer Society. References Cori, C. F., Cori, G. T., and Green, A. A., J. Biol. C&em., 151:;59 (1943). Escher, E. H., and Krebs, E. G., J. Biol. Chem., 231:65 (lgm. Illingsworth, B., and Cori, G. T., Biochem. Prepar~ons, J:l (1953). Krebs, E. G., Biochim. Biophys. Acta, s:'jOg (1954).
367