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THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 234, No. 11, November 1959
Printed in U.S. A.
Factors Affecting the Activity of Muscle Phosphorylase b Kinase
EDWIN G. KREBS, DONALD J. GRAVES,* AND EDMOND H.
FISCHER
From the Department of Biochemistry, University of Washington, Seattle, Washington
(Received for publication, February 13, 1959)
Resting muscle contains a higher proportion of phosphorylase
b than of phosphorylase a as determined by analysis of extracts
(1). Under the influence of epinephrine (2, 3) or with muscle
contraction (4), the amount of phosphorylase a increases. Two
enzymes which arc involved in the interconversion of the two
forms of phosphorylase have been described. One of these,
phosphorylase 6 kinasc, catalyzes the phosphorylation of phos-
phorylase b by idenosine triphosphate to give phosphorylase a
(5-7); the other enzyme, phosphorylase phosphatase,l catalyzes
the hydrolytic cleavage of phosphate from phosphorylase a to
give phosphorylase b (9). It appears reasonable to assume that
the relative activities of these two enzymes would be important
factors in determining the proportion of phosphorylase a and b
in muscle (4, 11).
It has been found that phosphorylase b kinase exists in fresh
rabbit muscle extracts in a form that is inactive at pH 7.0 or
below. The enzyme becomes highly active in this region when
incubated for a short period of time with Ca++ ions. As it has
been suggested that the release of bound Ca++ may be a link
between excitation of muscle and contraction (12, 13) or that
Ca++ may be mobilized and undergo transfer from one site to
another in connection with muscle contraction (14, 15), it is
possible that the present effect is related to an important control
process. Activation of phosphorylase 6 kinase would result in
the production of phosphorylase a, which in turn catalyzes the
first step in glycogenolysis.
The outstanding investigations of Sutherland et al. (16-18)
have shown that the increased formation of active liver phos-
phorylase observed in the presence of epinephrine is medi-
ated by adenosine 3’,5’-phosphoric acid formed from adeno-
sine triphosphate. They also have observed the formation of
this substance in other tissues including skeletal muscle. No
explanation has been given, however, as to how this compound
acts in shifting the balance between the active and inactive
forms of phosphorylase. In the present investigation it is shown
that in muscle extract the effect of adenosine 3’,5’-phosphoric
acid is in the activation of phosphorylase b kinase. In the activa-
tion process, which is apparent when the kinase is assayed below
its pH optimum, Mg++ ions and adenosine triphosphate are
required for the cyclic nucleotide effect. A preliminary report
of this work has been given (19).
* Predoctoral Fellow of the National Institute of Arthritis and
Metabolic Diseases, United States Public Health Service.
1 This enzyme was originallv called PR enzvme at the time of
its discovery- (8). On the bas& of its function”(g) the name phos-
phorylase phosphatase, as originally applied to the analogous liver
enzyme by Wosilait and Sutherland (lo), is used here.
METHODS
fMateriaZsCrystalline rabbit muscle phosphorylase b was
isolated as described previously (20). Phosphorylase a and
P32-labeled phosphorylase a were made from phosphorylase b
with ATP and phosphorylase b kinase (7). Samples of crystal-
line cyclic 3’, 5’-AMP2 were kindly furnished by Dr. David
Lipkin and Dr. Earl W. Sutherland. The glycogen used in the
assay of phosphorylase activities was obtained from Krishell
Laboratories, Inc., and was freed from traces of AMP3 and other
impurities by passage through Dowex l-X10,200 to 400 mesh, in
the OH- form and Dowex 50-X4,100 to 200 mesh, in the H+
form. The effectiveness of this treatment was ascertained by
spectrophotometric analysis of hydrolyzed glycogen solutions
before and after the procedure.
Muscle E&acts-White female rabbits were anesthetized
deeply with sodium pentobarbital solution and the blood was
drained from the jugular veins. The muscles from the lower
extremities and back were removed immediately, packed in
crushed ice, and then passed through an ordinary meat grinder
in the cold room. Portions of 100 g of the ground muscle were
mixed with 200 ml amounts of cold distilled water and homog-
enized for 1 minute in a Waring Blendor. The homogenate was
then centrifuged at 4,000 x g for 30 minutes and the supernatant
solution was collected after filtering through glass wool to remove
traces of lipid material. The extracts were kept at 0” and used
within 3 hours. pH adjustments of the extract to neutrality
were made by careful addition of 1
N
NaOH.
Acid Precipitation-The pH of the extracts was lowered to 5.7
to 5.8 by the addition of 1
N
acetic acid. The precipitate which
formed was collected by centrifugation at 10,000 X g and
partially dissolved4 in sufficient 0.08
M
sodium glycerophosphate
to give a final volume approximately & that of the extract used,
and the pH was adjusted to 7.0 by careful addition of 1
N
NaOH.
This fraction was stored at -20”. On some occasions the solu-
tion was made 0.002
M
with respect to EDTA before freezing
and storage.
2 The abbreviations used are : cyclic 3’,5’-AMP, adenosine 3’, 5’-
phosphoric acid; EDTA, ethylenediaminetetraacetic acid; Tris,
tris(hydroxymethyl)aminomethane.
3 In a previous publication (7) the activity of phosphorylase a,
as measured in the absence of AMP, was erroneously found to be
equal to the activity of this form of the enzyme measured in the
presence of AMP. This was due to traces of AMP in the glycogen
used in the reaction mixture for the activity test. The slight
activity reported for charcoal-treated samples of crystalline phos-
phorylase b (20), presumably determined in the absence of AMP,
was also due to this contaminant.
4 Most of the protein appears to go into solution although the
final mixture is still turbid; it can be clarified by centrifugation
at 25,000 X 9 without loss of phosphorylase b kinase.
2867
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2868 Phosphorylase b Kinase
Vol. 234, No. 11
r
6.0 7.0 8.0 9.0
PH
FIG.
1. Phosphorylase b kinase activity of muscle extracts;
effects of Ca++ and cvclic 3’.5’-AMP. Phosnhorvlase b kinase
activities were determined at a final dilution of1 to”l,320 of muscle
extract at the pH values indicated. The usual assay system for
measurement of initial reaction rates (see “Methods”) was em-
ployed. A sodium glycerophosphate-glycine buffer instead of a
sodium glycerophosphate-Tris buffer was used at pH 9.1. In
Curve A the extract was diluted and assayed without treatment.
In Curves B and C the extract was adjusted to pH 7.0 and incubated
for 15 minutes at 30” with either 1 X 10e4 M cyclic 3’, 5’.AMP or 3
X lo+
M
calcium acetate, respectively, before dilution and assay.
A control experiment in which extract was adjusted to pH 7.0 and
incubated without additions gave a curve identical to Curve A.
Phosphorylase b Kinase Activities-Phosphorylase
b
kinase
activities were determined by a modification of the method de-
scribed previously (6). Reaction mixtures are made up as
follows: 0.1 ml of 0.25
M
Tris-0.25
M
sodium glycerophosphate
buffer, 0.1 ml of Hz0 or additions, 0.2 ml of phosphorylase
b
solution in 0.015
M
neutral cysteine, 0.1 ml of kinase solution
diluted in cold 0.015
M
neutral cysteine, and 0.1 ml of 6 X 10v2
M
Mg(Ac)r1.8 X 1O-2
M
ATP solution to start the reaction, which
is carried out at 30”. The pH of the buffer and of the Mg-ATP
solution are adjusted to give whatever final pH is desired. At 5
minutes an aliquot is removed and diluted in 0.04
M
sodium
glycerophosphate-0.03
M
cysteine buffer containing 0.001
M
EDTA, pH 6.8, for assay of phosphorylase activity according
to the method of Illingworth and Cori (21). A unit of phos-
phorylase b kinase activity is defined as before (6), i.e. that
amount of enzyme that gives rise to the formation of 100 units
of phosphorylase activity (measured without AMP) per ml of
kinase reaction mixture in 5 minutes at 30”. The amount of
phosphorylase b in the reaction mixture corresponds to about
10,000 units (6 mg) and assays are carried out at sufficient dilu-
tion of the kinase so that no more than + of the phosphorylase
b
will be converted within 5 minutes. In addition to the measure-
ment of initial reaction rates, as described in the above assay
system, the entire time course of the phosphorylase b to a reac-
tion was followed with similar reaction mixtures. In this case
aliquots were removed for phosphorylase assay at additional
times beyond 5 minutes.
Phosphorylase Phosphatase Activities-Phosphorylase phos-
phatase activities were determined essentially as described by
Keller and Cori (22) except that the phosphorylase a concentra-
tion was approximately thirty times greater than in their assay.
This required an intermediate dilution before the assay for the
phosphorylase.
RESULTS
Phosphorylase b Kinase Activity of Muscle Extracts-Phos-
phorylase b kinase of fresh rabbit muscle extracts is inactive
when tested at pH 7 or below (Curve A of Fig. 1). Above this
pH the enzyme is active, reaching its maximum around pH 8.5.
This behavior is markedly different from that obtained with the
preparations of purified phosphorylase b kinase described pre-
viously (6), which had their optimum in the same region but
were more than half maximally active at pH 7. Since the kinase
activity determinations are carried out at more than a lOOO-fold
dilution of the extract, it is unlikely that nonspecific effects due
to salts or other components in the extract would affect the pH
optimum curve appreciably and thus account for the observed
difference.
Activation
of
Phosphorylase b Kinase by Ca++--When muscle
extracts are incubated with added Ca++ ions for a short period of
time, a striking activation5 of the kinase results. This activation
is made manifest when the kinase activities are determined at
pH values below the optimum range (Curve C of Fig. 1). The
activation process is rapid with a maximum activity being
reached after 3 to 5 minutes of incubation at 30”; the level of
activity remains constant when the incubation times are pro-
longed to 45 minutes. No reversal of the activation is seen when
a a-fold molar excess of EDTA over the Ca++ ions is added after
activation has occurred. This concentration of EDTA added
before the addition of the Ca+* ions prevents the activation.
Other metals studied including Zn++, Mg++, Cu++, Fe++, Mn++,
and Ni++ have not been found to be effective in activating the
kinase of muscle.
Fractionation
of
“pH 7-inactive” Kinase
from
Muscle Extract-
When the pH of fresh muscle extract is lowered to 5.7 to 5.8 (see
“Methods”), the precipitate which forms contains the phos-
phorylase b kinase. If this precipitate is dissolved in sodium
glycerophosphate buffer, pH 7, and assayed the same day, it can
be shown that the kinase is present in essentially the same “pH
7-inactive” form as in the original extract (Curve A of Fig. 2).
Slight activation may have occurred, however, since there is
now detectable activity at pH 7 and lower. On incubation with
Ca++ the enzyme is again markedly activated (Curve B of Fig. 2);
here there appears to be some increase in activity even at high
pH values near the optimum which was not seen to any marked
degree in the extracts. If the acid precipitate fraction is stored
without FDTA, it is slowly6 transformed to the activated type
of enzyme. Storage with EDTA holds the enzyme in its “pH 7-
inactive” state. Once the acid precipitate fraction has been
activated with Ca++, it is not converted back to the ‘<pH 7-
inactive” form by dialysis against repeated changes of neutral
2 x 1O-3
M
EDTA. The Ca++-activated and -dialyzed kinase
does not lose activity when added back to the nonactivated acid
precipitate fraction.
Dud Role of Ca++-In the experiments described thus far, the
effect of Ca++ on the kinase has been studied by adding the metal
to the muscle extracts, incubating for a short period, and then
assaying at a high dilution of the treated extract. Under these
5 Phosphorylase b kinase which is inactive at pH 7.0 and below
will be referred to as “pH 7-inactive” enzyme. A change in the
kinase so that its activitv is increased at nH 7 will be referred to
”
as “activation.”
6This fraction is stored in the frozen state in plastic tubes.
After thawing and use on 2 or 3 successive days, the enzyme will
have become activated.
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November 1959 E. G. Krebs, D. J. Graves, and E. H. Fischer 2869
6.0 7.0 8.0
PH 9.0
FIG. 2. Phosphorylase b kinase activity of acid precipitate
fraction before and after activation with Ca++. Curve
A:
acid
precipitate fraction control. Kinase activities were carried out
at a 1 to 24,000 final dilution of the fraction at the pH values in-
dicated. Curve B: the acid-precipitate fraction was incubated
with 5 X 10e3
M
Ca++ for 15 minutes at 30”. Kinase activities were
carried out as in
A.
pH 7.0 pH 7.5 pH 8.3
FIG. 3. Effect of Ca* in the kinase test system with muscle
extract as source of enzyme. Fresh muscle extract was assayed
at a final dilution of 1 to 1,200. [Ca++] = 1 X 10W3 M in the assay
reaction mixture where indicated. The pH of the assay is shown
beneath the columns.
conditions Ca++ is present at very low concentrations (of the
order of lo+’ M) during the kinase reaction itself. When Ca++
is introduced directly into the kinase reaction mixture an interest-
ing dual role for this metal appears (Fig. 3). Now Ca++ acts
either as an activator or as an inhibitor, depending upon the pH.
Below pH 8 the first of these effects prevails; above this point
the enzyme is inhibited. The explanation for this complicated
picture lies in the superposition of two separate and opposite
actions of this metal. First, as shown above, Ca++ causes a
change in the “pH 7-inactive” kinase present in muscle extracts,
2 4 6 16
[Ca++] X IO3
FIG. 4. Inhibition of phosphorylase
b
kinase by Ca+. The
concentration of Mg++ was 1 X 10m2
M
in the kinase assay reaction
mixture, [ATP] = 3 X W8 M, pH = 7.8. Source of kinase was
activated acid-precipitate fraction diluted 1 to 5,000 in reaction
mixture.
so that the enzyme becomes active at a lower pH:
“pH 7.inactive” phosphorylase
6
kinase Ca++
“activated” phosphorylase
b
kinase (1)
The second action is an inhibition of the phosphorylase b kinase
reaction, which proceeds according to Equation 2:
2 Phosphorylase
b
+ 4 ATP Mg++
phosphorylase a + 4 ADP (2)
This inhibition occurs over the entire pH range of activity of the
kinase, but below pH 8.0 the effect of forming active kinase as
in Equation 1 predominates.
Inhibition
of
Phosphorylase b Kinuse Reaction by Ca++--With
the use of an acid-precipitated fraction activated with 5 x 1O-3 M
Ca++ and then diluted so that the amount of this metal carried
over into the assay reaction mixture would be of the order of
10-r M, the inhibitory properties of Ca++ in the phosphorylase b
to a reaction itself were studied. Fig. 4 shows the effect of
increasing concentrations of Ca++ on the kinase activity; in this
experiment with 1 X 10m2 M Mg++ the reaction is inhibited 50%
at 2 x 1O-3 M Ca++. The inhibition is noncompetitive with
respect to ATP7 (Fig. 5)) but is competitive with respect to Mg++
(Fig. 6). The value of
K,
for Mg++ is 1.9 x 1O-3 M and the
value of
K;
for Ca++ is 3.0 X 10e4 M under the conditions of the
experiment illustrated in Fig. 6.
E$ect
of
Ca++ on Phosphorylase b Kinase Reaction in Presence
of
EDTA-It
appeared to be of interest to study the interrela-
tionship that might exist between Mg++ and Ca++ in the phos-
7 The anomalous plot shows inhibition by high ATP concentra-
tions which is especially marked in the presence of Ca++.
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2870 Phosphorylase b Kinase Vol. 234, No. 11
6 12 18 24 30
j/[*TP] x lo-*
FIG. 5. Effect of varying ATP on the inhibition of phosphoryl-
ase
b
kinase by Ca++. Conditions as in experiment of Fig. 4.
ATP concentration as shown. [Ca++] = 1.60 X 10-s
M
in
A.
80
60
I , I
4 8
x 10-2
12
FIG. 6. Effect of varying Mg++ on the inhibition of phosphoryl-
ase
b
kinase by Ca++. Conditions as in legend of Fig. 4 except
that the source of kinase was a different activated acid precipitate
fraction assayed at a 1 to 14,000 dilution. @a++] = 1.43 X 1OW
M in
A.
phorylase b kinase reaction in the presence of an agent that
chelated these metals. In the experiment of Fig. 7 increasing
amounts of EDTA are added to the assay reaction mixture in
the presence (Curve A) and absence of Ca++ (Curve B). The
source of kinase is again the activated and diluted acid precipitate
fraction. It can be seen that without Ca++ the extent of inhibi-
tion increases with increasing EDTA until it becomes essentially
complete at the point where the concentration of the chelating
agent and Mg++ are equal (1.4 X 10e2 M). The reaction with
Ca++ (Curve A) is inhibited relative to that without Ca++ (Curve
B) at the lower EDTA concentrations, but the reverse is true
at the higher EDTA levels. These results can be understood
on the basis of the ability of Ca ++ to displace the more loosely
bound Mg++ from EDTA.
Whether this model system with EDTA is comparable to
anything existing in muscle or in muscle extracts is not known.
Muscle contains substances (phosphates, nucleotides, proteins,
and so forth) which can serve as chelating agents for metals, and
under certain circumstances the displacement of one metal by
another could serve as a regulatory mechanism.
Effect of Cyclic 3’ ,5’-AMP on Phosphorylase b Kinase Activity-
When fresh muscle extracts are incubated with cyclic 3’) 5’-AMP,
their phosphorylase b kinase activity is increased; this is espe-
cially noticeable when the kinase assays are carried out at pH
values below the optimum for this enzyme (Curve B of Fig. 1).
In this latter respect the activation of the kinase in the extract
by the cyclic nucleotide resembles the action of Ca++ ions,
except that the extent of activation is not as great as with the
metal. In contrast to the effect of Ca++ ions, the activation of
the kinase by cyclic 3’) 5’-AMP appears to be transient as is
shown in the experiment of Table I.
The activation of phosphorylase b kinase by cyclic 3’) 5’-AMP
requires the presence of a labile factor in the muscle extract.
This is evident from the experiment of Table I, where it can be
seen that readdition of cyclic 3’,5’-AMP after incubation of the
extract for 60 minutes fails to reactivate the kinase appreciably.
Evidence that the factor lost from the extract during the incuba-
tion period might be ATP is seen by the effectiveness of this
substance when it is added along with cyclic 3’,5’-AMP; more-
over, in separate experiments it was shown that sufficient ATPase
activity exists in this type of extract to account readily for the
destruction of endogenous ATP during an incubation period of
60 minutes at 30”. The experiment of Table I shows that cyclic
3’,5’-AMP is also lost from the extract during the incubation,
since addition of ATP alone at 60 minutes has no effect. The
requirement for ATP to enable cyclic 3’) 5’-AMP to act can also
FIG. 7. Effect of Ca++ on phosphorylase
b
kinase in presence of
EDTA. Conditions as described in legend of Fig. 4 except [Mg++]
= 1.4 X 10-Z M. Curve A: with 1 X 1O-3 M Ca++ in the kinase
reaction mixture. Curve
B:
without Ca++.
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November 1959 E. G. Krebs, D. J. Graves, and E. H. Fischer 2871
be shown when an initial dilution of fresh muscle extract is made
before its incubation with activating components (Table II).
Now, even though the extract contains endogenous ATP, the
dilution lowers its concentration to a level where cyclic 3’,5’-
AMP alone is not effective.
Time Course of Phosphorylase b to a Reaction
with “pH 7-
inactive”
Kinase-It can be seen in the experiment of Table II
that incubation of diluted muscle extract with ATP and Mg++
ions caused some activation of phosphorylase 6 kinase in the
absence of added cyclic 3’, 5’-AMP. This effect was more
marked in the incubation carried out at pH 8.4 than in the experi-
ment at pH 7.0. Since ATP and Mg++ ions are both compo-
nents of the phosphorylase b to a reaction itself, this reaction
was studied beyond the 5-minute period used in the measurement,
of initial reaction rates in the kinase assay.8 An experiment at
various pH’s with dilute muscle extract as a source of “pH 7-
inactive” kinase, is shown in Fig. 8. At the two lower pH values
the reactions are clearly autocatalytic with zero initial reaction
rates. With the particular extract used in the experiment of
Fig. 8 some phosphorylase a formation was evident at 5 minutes
at pH 6.8; this amounted to 6% of that found at pH 8.6. More
commonly, as shown with the extracts used in Fig. 1 and Tables
I and II, essentially no phosphorylase a was formed at pH 7.0
or lower in 5 minutes, the period of time employed in the usual
kinase assay (see “Methods”). As the pH is increased the
requirement for activation becomes less apparent, until at pH
8.2 the enzyme seems to be fully active initially.
In another experiment at pH 6.8 (Fig. 9), cyclic 3’,5’-AMP
is included in the phosphorylase b kinase reaction mixture
(Curve B). Its presence does not abolish the lag period, but
accelerates the activation process and results in the attainment
of a rate which is equal to that shown in Curve C, where the
extract has been activated by preincubation with Ca++ ions.
In the control reaction (Curve A), without added cyclic 3’, 5’-
AMP the rate is still increasing slowly at 20 minutes. Protein-
free filtrates of reaction mixtures similar to that in Curve B at
15 minutes have not been found to contain anything that will
abolish the lag phase of the kinase reaction. Heating, addition
of perchloric acid, and addition of organic solvents have been
used as methods for deproteinization.
The specificity of cyclic 3’) 5’-AMP for its role in the activation
of phosphorylase b kinase has been tested with other nucleotides
alone or in combination with ATP by preincubating these sub-
stances with “pH 7-inactive” kinase in the presence of Mg++.
No other nucleotide was found to be effective. Those tested
include: cyclic 2’,3’-AMP, 2’-AMP, 3’-AMP, 5’-AMP, ADP,
ATP, GMP, GDP, GTP, UMP, UDP, UTP, CMP, CDP, CTP,
IMP, IDP, and ITP.
Localization of Effects of Cyclic S’,S’-AMP to Activation of
Phosphorylase b
K&use-The problem in localizing an effect that
shifts the balance between the phosphorylated and the nonphos-
phorylated forms of phosphorylase has been discussed by Rall
and Sutherland (17). This is especially difficult when extracts
or crude fractions containing both phosphorylase kinase and
phosphorylase phosphatase are studied under conditions in which
8 The earlier study (6) showing the entire course of the phos-
phorylase
b
to a reaction was carried out with an acid precipitate
fraction as the source of phosphorylase
b
kinase. As this fraction
had been stored without EDTA, it was probably of the “acti-
vated” type (see footnote 6).
TABLE I
Requirements
for
activation
of
phosphorylase
b kinase in muscle
extracts by
adenosine
3’,5’-phosphoric acid
Phosphorylase
b
kinase activities were determined at a final
dilution of 1 to 900 of muscle extract. Incubation of the extract
was carried out at 30” at pH 7.0. Cyclic 3’,5’-AMP was added to
a final concentration of 1 X 1w4
M
and ATP to a final concentra-
tion of 2 X IO+
M
where shown. The mixture was divided into
3 portions at 60 minutes.
Treatment of extract
No additions
Incubation for 10 minutes after addition
of cyclic 3’,5’-AMP
Incubation continued for 30 minutes
Incubation continued for 60 minutes
Incubation continued for 71 minutes (cy-
clic 3’,5’-AMP readded at 61 minutes)
Incubation continued for 71 minutes (ATP
readded at 61 minutes)
Incubation continued for 71 minutes (cy-
clic 3’,5’-AMP and ATP added at 61
minutes)
No additions. Extract incubated for 71
minutes
TABLE
II
Activation
of phosphorylase
b kinase in diluted
muscle extract
at
diferent pH
values
Complete incubation mixtures for the activation of the kinase
were made up with the following components: 0.2 ml of 0.125 M
Tris-0.125
M
sodium glycerophosphate buffer, 0.1 ml of 5 X 10m4
M
cyclic 3’,5’-AMP, 0.1 ml of 5 X 10m2
M
Mg(Ac)z-1.5 X 10m2
M
ATP,
and 0.1 ml of fresh muscle extract diluted 1 to 30. Incubations
were carried out for 15 minutes at 30” and aliquots were removed
for assay of phosphorylase b kinase at pH 6.8.
Phosphorylase
b
kinase
activity at
PH 7.0
pH 8.6
units/ml
200
3,100
0
0
400
300
3,400
100
units/ml
12,400
12,400
13,100
11,200
12,900
ImitS/d*
U?dS/d” W&S/Vd’
7.0 100 200 1,200
8.4 0 0 2,100
* Calculated back to original muscle extract.
U?ZitS/Td*
3,700
4,100
both reactions can occur at demonstrable rates. In the present
work, however, phosphorylase kinase assays are carried out at
dilutions IO-fold higher than the maximum dilution at which any
phosphorylase phosphatase activity can be detected in the usual
phosphorylase phosphatase activity test.
To eliminate completely the possibility that some special con-
dition might exist in the kinase reaction that would permit
phosphorylase phosphatase to act, a control experiment was set
up in which a trace of P32-labeled phosphorylase a was included
in the kinase reaction mixture. Reactions were carried out under
these conditions with fresh muscle extract, extract activated by
Ca++ ions, and extract activated by cyclic 3’,5’-AMP (Table
III). In every instance all the radioactive phosphorus could
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Phosphorglase b Kinase
Vol. 234, No. 11
MINUTES
FIG.
8. Time course of the phosphorylase
b
to a reaction with
“pH 7-inactive” kinase at different pH values. Fresh muscle
extract at a final dilution of 1 to 1,000 in the kinase reaction mix-
ture was used. The pH value for each reaction mixture is shown
beside the curves. The ordinate gives units of phosphorylase a
per ml of kinase reaction mixture.
5 IO 15 20
MINUTES
FIG.
9. Course of the phosphorylase
6
to a, reaction in the
presence of cyclic 3’,5’-AMP. Fresh muscle extract at a final
dilution of 1 to 700 in the kinase reaction mixture was used. In
Curve A no additions to the reaction mixture were made. In
Curve
B
the reaction mixture contained 1.5 X 10e4
M
cyclic 3’,5’-
AMP. In Curve C the reaction mixture was identical to that of
Curve A except that the extract was preincubated with 5 X 10W3
M
calcium acetate before dilution and use. The pH of the reac-
tion mixtures was 6.8 in all instances.
be recovered in a protein-bound form, showing that no phos-
phatase action had occurred. Since the ATP used in the kinase
reactions was not labeled, any phosphatase action followed by
rephosphorylation would lead to a loss of the bound isotopic
phosphorus. It is concluded that the activation effects reported
in this study are not due to inhibition of phosphorylase phos-
TABLE
III
Absence
of
phosphorylase phosphatase activity during
phosphorylase
kinase reaction
The kinase reaction mixtures at pH 7.5 contained 0.1 mg of
crystalline P32-labeled phosphorylase a in addition to the regular
components. At 5 minutes and at 60 minutes aliquots were re-
moved and the proteins were precipitated with 5% trichloroacetic
acid, washed, dissolved in formic acid, plated, and counted as
described previously (7). Phosphorylase a determinations were
also made on separate aliquots removed at 5 minutes. Theoreti-
cal c.p.m. on samples = 350, assuming no phosphatase action.
Protein-bound radioactive phosphorus at
Treatment of extract
5 minutes 60 minutes
C.~.rn. c.p.m.
None 340 400
Activation* with 5
X
lo+
M
cal- 398
338
cium acetate
Activation* with 1 X low4
M cy-
clic 3’,5’-AMP
325 417
* The extract showed 13,000 kinase units per ml before activa-
tion and 26,000 units per ml after activation with either Ca++
or
cyclic 3’,5’-AMP.
phatase. Control experiments have also been carried out show-
ing that the ATP level in the kinase assay is not rate limiting,
so that a secondary effect due to a variation in activity of con-
taminating ATPase can be ruled out as an explanation for the
role of cyclic 3’, 5’-AMP.
DISCUSSION
The results reported in this paper are not readily explained
on the basis of the information available. It appears certain
that phosphorylase
b
kinase can be extracted from muscle in a
form that is not fully active. Seemingly diverse methods are
available for the activation of the enzyme. These include:
(a) incubation with Ca++, which can be carried out in the crude
extract or with a partially purified preparation of the “pH 7-
inactive” kinase; and
(b)
incubation with ATP and Mg++ ions.
The latter type of activation is accelerated by addition of cyclic
3’, 5’-AMP, a substance which has no effect by itself. The
studies involving the time course of the phosphorylase
b
to a
reaction at different pH values, with “pH 7-inactive” kinase,
suggest that the kinase may be completely inactive initially and
appears to be fully active at the higher pH values only because
here the activation by ATP is essentially instantaneous. It has
not been excluded that ATP may be giving rise to some cyclic
3’,5’-AMP in addition to acting in conjunction with this sub-
stance in the activation process.
A highly speculative hypothesis to advance in relation to the
observed phenomena is that phosphorylase
b
kinase itself exists
in phosphorylated and dephosphorylated forms, which are active
and inactive in a manner analogous to phosphorylases a and
b.
According to this hypothesis an enzyme system consisting of
another kinase and a phosphatase might be involved in the
activation and inactivation of phosphorylase
b
kinase. The
effects of Ca++, pH, ATP, and cyclic 3’,5’-AMP on the state
of kinase activity could involve either the activating or inactivat-
ing enzymic reactions.
It is possible that phosphorylase
b
kinase is extracted from
by guest, on July 13, 2011www.jbc.orgDownloaded from
November 1959
E. G. Krebs, D. J. Graves, and E. H. Fischer 2873
muscle in the form of an organized complex9 involving other Acknowledgment-The authors wish to acknowledge the excel-
proteins by which the kinase is inhibited. Activation by Ca++ lent technical assistance of Mrs. Marion E. Lord.
might occur as the result of disruption of such a complex. The
action of ATP and cyclic 3’, 5’-AMP could also involve some
modification of this complex. It is of interest that the acid
precipitate fraction of rabbit muscle, which can be isolated
containing all the phosphorylase
b
kinase in its inactive form,
also contains large amounts of phosphorylase
bLo
and most of the
phosphorylase phosphatase (22) of muscle. This fraction
exhibits all of the behavior of the original extract in relation to
the various types of activation seen (illustrated only with Ca++
in this paper). When the kinase of the fraction is activated by
ATP and Mg++, with or without added cyclic 3’, 5’-AMP, all
the endogenous phosphorylase 6 in the fraction is converted to
phosphorylase a.
The phosphorylase
b
to a reaction appears to be sensitive to
a variety of possible control mechanisms including variation of
pH, changes in Mg++ or ATP concentrations (6), inhibition by
Ca++, and perhaps most important, the activation and inactiva-
tion of phosphorylase
b
kinase reported here. Experiments are
being undertaken to determine the state of activity of the kinase
in relation to muscle contraction.
SUMMARY
1. Phosphorylase
b
kinase can be extracted from rabbit muscle
in a form which shows no activity at pH 7.0 or lower.
2. The kinase which is inactive at pH 7.0 or below is activated
by a short period of incubation with Ca++ ions. This type of
activation has not been found to be reversible.
3. The inactive kinase is activated by preincubation with
adenosine triphosphate in the presence of Mg++ ions. This
activation is enhanced in the presence of added adenosine 3’, 5’-
phosphoric acid.
4. In the phosphorylase
b
to a reaction itself Ca++ is a competi-
tive inhibitor with respect to Mg++.
9 Such a complex could not be large in size, since the “pH 7-
inactive” kinase remains in the supernatant solution after cen-
trifugation of muscle extracts at 100,000 X g.
lo Unpublished results in this laboratory.
1.
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