PHOSPHORYLASE ACTIVITY OF SKELETAL

MUSCLE EXTRACTS*

BY EDWIN G. KREBS AND EDMOND H. FISCHER

(From the Department of Biochemistry, University of Washington,
Seattle, Washington)

(Received for publication, January 17, 1955)

Cori, Cori, and Green found that muscle phosphorylase exists in two
forms, which they designated as phosphorylase a and phosphorylase b
(l-5). Phosphorylase a exhibits 60 to 70 per cent of its maximal activity
in the absence of AMP,l whereas phosphorylase b is inactive unless this
nucleotide is present. Extracts of resting muscle were found to contain
on the average 92 per cent phosphorylase a, but muscle stimulated to fa-
tigue yielded mainly phosphorylase b (5). The hypothesis that the a
form is converted to the b form in vivo during muscle contraction was ad-
vanced (5).
The present study was initiated to determine whether environmental
temperature affected the phosphorylase content of skeletal muscle. Sig-
nificant temperature effects were not noted; however, a surprising result
was the finding that the muscle extracts contained mainly phosphorylase
b rather than phosphorylase a. This was so, even though the animals
were handled in ways to minimize muscle contraction. Experiments were
conducted to rule out the possibility that a conversion of phosphorylase a
to b occurred in vitro after extraction of the enzyme.
Methods
Animals-White female rabbits weighing from 2.5 to 4.5 kilos were used.
No restrictions on rations2 were imposed. Some of the rabbits were kept
in a room3 regulated at 5” and used at intervals as indicated. One rabbit
was kept at 31”. The rabbits maintained at 24-28’ were kept in the ordi-
nary animal quarters, which were subject to small temperature variations.
Preparation of Muscle Extracts
Method I-The procedure used was essentially the same as that de-
scribed by Green and Cori in their method for the isolation of crystalline
* Supported by the Initiative 171 Research Fund of the State of Washington and
by a research grant (G-2399) from the National Institutes of Health, Public Health
Service.
1 AMP = adenosine-5’.phosphate; EDTA = ethylenediaminetetraacetate.
2 Rabbit Rreeder Paks, Albers Milling Company.
3 The authors wish to thank Dr. Loren D. Carlson for making available the special
animal quarters.
113

This is an Open Access article under the CC BY license.

114 MUSCLE PHOSPHORYLASE

phosphorylase a (1, 6). Rabbits were anesthetized deeply by intravenous

injection of Nembutal, care being taken to insure that no evident muscle
contraction occurred during this process. The animals were bled from the
jugular veins, and the back and hind leg muscles were excised immediately.
During the excision, it was impossible to avoid some stimulation and a
slight degree of muscle twitching was always present, despite the efforts
made to keep these contractions minimal. The total lapse of time be-
tween injection of the anesthetic and completion of the removal of the
muscles was between 10 and 15 minutes. The muscles were ground im-
mediately in the cold room, extracted twice with cold distilled water,4 and
filtered through cheese-cloth. The extracts were centrifuged at 0” for 15
minutes at 2000 X g and filtered rapidly through a small wad of cotton.
Activities of the extracts were determined immediately at this point unless
noted otherwise.
Method %--The rabbit was anesthetized with Nembutal, then bled, and
the eviscerated carcass was cooled to 5” according to the procedure of
Dounce et al. (7) for obtaining muscle in a manner that minimizes con-
tractions. No twitching was observed during excision of the muscle.
The excised muscle was treated as described in Method 1 for the prepara-
tion of the extract.
Method S-The rabbit was anesthetized with Nembutal and then in-
jected intraperitoneally with 900 mg. of MgSO4 per kilo as described by
DuBois et al. (8). The eviscerated carcass was chilled in ice before re-
moval of the muscle. As in Method 2, no twitching was apparent during
excision of the muscle. The remainder of the procedure is as described
in Method 1.
Method &--The procedure is the same as Method 1 except that neutral
0.001 M EDTA solution was used instead of water in extracting the ground
muscle.
Method 5-The rabbits were injected intravenously with approximately
1 mg. of strychnine per kilo. Death occurred after severe convulsions of
1 to 3 minutes duration. The animals were bled from the jugular veins
and handled as described in Method 1.
Preparation of Crystalline Phosphorylase a-The enzyme was prepared
according to the procedure of Green and Cori (1, 6).
Phosphorylase Activity Determinations-Activity measurements on mus-
cle extracts were made in the presence and absence of AMP at pH 6.0 ac-
cording to the procedure of Illingworth and Cori (6). Preincubation of
the enzyme in cysteine-glycerophosphate buffer was carried out for 8 min-
utes at 30” before the reaction was started. Preincubation of the enzyme
4 Each extraction is made with an amount of water equal to the original weight
of the ground muscle.
 E. G. KREBS AND E. H. FISCHER 115

in the presence of glycogen (6) was found to be without effect on activity

measurements in crude extracts and was not done routinely. Units of
phosphorylase activity were corrected to pH 6.8, as has been described,
and are given per ml. of original extract.

Results
Phosphorylase Activity and Temperature-Rabbits adapted to different
environmental temperatures were used for a study of the total phosphoryl-
ase content5 of muscle extracts. Table I gives the results obtained with
animals kept at 5” and a control group maintained at 24-28“. The aver-
age total phosphorylase activity is approximately the same for the two
groups. It was reported by Illingworth and Cori (6) that the phosphor-
ylase content of muscle drops to low values when the room temperature is
30” or higher. A single rabbit, No. 24 of Table I, kept at 31”, did not
show this, but a large series is obviously needed, since the individual var-
iation in activity can be very great (1, 5).
Phosphorylase Activity and Rabbit Size-With the weight of muscle as
an index of the size of the animals, the data of Table I show that there is
an increase in total phosphorylase activity per ml. of extract with increas-
ing rabbit size. This is equivalent to an increase of activity per unit
weight of muscle, since the volume of extracting fluid always bears a con-
stant ratio to the amount of tissue. The correlation between the two var-
iables is significant with a probability of less than 2 per cent that the result
was due to chance. If Rabbit 13 is excluded, this probability is less than
1 per cent.
Relative Amounts of Phosphorylase a and Phosphorylase b in Muscle Ex-
tracts-Most of the extracts from rabbits in either series of Table I showed
low activities when tested in the absence of added AMP, indicating that
the phosphorylase was predominantly in the b form. For example, the
room temperature series (Rabbits 1 to 15) showed on the average only 32
per cent phosphorylase a, which is much lower than the 92 per cent re-
ported by Cori (5) for a comparable series. The difference actually is
more pronounced than these figures would indicate, since phosphorylase a
present in the crude extracts gives a higher ratio ((activity without AMP)/
(activity with AMP)) than the 0.65 used in the calculation of phosphoryl-
ase a concentration; this was pointed out by Cori (5) and was attributed
to traces of AMP in the crude extracts. The average percentage of total
phosphorylase in the a form was slightly lower (24 per cent) in the series
at 5” of Table I, but this is not significant statistically.
Since evidence had been presented (5) that muscular contraction causes
6 Activity determined in the presence of added AMP (1 X 10e3 M) is taken as total
phosphorylase activity (6).
116 MUSCLE PHOSPHORYLASE

TABLE I
Phosphorylase Activity of Muscle Extracts
Extracts prepared by Method 1. Activity determinations carried out as de-
scribed under “Methods.” The figures in parentheses are the number of days the
rabbits were kept at the temperature indicated. The weight of back and hind leg
muscle obtained in each case is given in the second column. The temperatures at
which the animals were kept are as follows: Rabbits 1 to 15,24-28”; Rabbits 16 to 23,
5”; Rabbit 24, 31”.
7 -

Rabbit No. Muscle

I Activity of extract
Phosphorylase a
+ AMP* - AMP

gm. units ger ml. units ger ml. per centt

1 325 1260 130 16
2 330 770 380 76
3 340 1320 300 34
4 370 1270 360 44
5 390 1000 130 20
6 395 750 70 15
7 400 840 110 21
8 415 1100 100 14
9 480 1820 20 2
10 515 1470 1310 lOO$
11 600 1200 1060 1001
12 605 1690 310 28
13 650 760 20 4
14 650 2360 20 1
15 685 1420 70 5

Average. .. 1270 293 32

16 (18) 200 1240 130 16

17 (21) 400 1040 0 0
18 (26) 410 1130 90 12
19 (60) 660 1180 230 19
20 (105) 670 1870 60 5
21 (54) 650 1680 130 12
22 (63) 600 1760 600 52
23 (11) 375 1070 550 79

Average. . 1370 220

-
24 (27) 465 1560 80

* Added in the activity assay system.

t Calculated on the basis that phosphorylase a is 65 per cent active without AMP
(1).
$ Activity without AMP > theoretical for phosphorylase a.
 E. G. KREBS AND E. H. FISCHER 117

conversion of phosphorylase a to b, it appeared important to obtain mus-

cle in as completely a resting condition as possible. Methods that have
been shown to preserve muscle adenosine triphosphate (7, 8) were used;
however, again phosphorylase was found mainly in the b form. In one
experiment, extract prepared by Method 2 was found to contain 810 units
per ml. of total phosphorylase,4 of which all was phosphorylase b. In an-
other experiment, extract prepared by Method 3 was found to contain 800
units per ml., of which 87 per cent was phosphorylase b.
Ruling Out Formation of Phosphorylase b in Vitro-Extracts obtained
before or immediately after filtration through cheese-cloth were tested
and found to contain the same amount of phosphorylase b as was present
after the centrifugation step (see Method 1).
The possibility was considered that the concentration of PR enzyme6
was unusually high in the crude extracts and that phosphorylase a was be-
ing converted to phosphorylase b in the handling of the extract or in the
process of carrying out an activity test. Several types of evidence made
this appear extremely unlikely: (1) When preincubation of the diluted ex-
tract in cysteine-glycerophosphate buffer was omitted entirely, essentially
the same relative phosphorylase b to a ratios were obtained as with the
preincubation, although slightly lower total phosphorylase activities were
found. In these experiments fresh cold extract was diluted rapidly in
cold buffer and immediately mixed with substrate for the activity test.
(2) Crystalline phosphorylase a was added to the fresh crude extract, and
its activity, as tested without added AMP, was completely recoverable un-
der the usual conditions. (3) Fluoride was added to the fresh crude mus-
cle extracts in a concentration of 0.1 M. This did not alter the relative
amounts of phosphorylase b and a found. It has been shown that I+ at
this concentration effectively inhibits the PR enzyme (9).
Extraction of Muscle with EDTA-It was found that muscle extracts
containing only phosphorylase b could serve perfectly well for the isolation
of crystalline phosphorylase a by the method of Green and Cori (1). For
example, extracts of Rabbits 13, 14, and 17 of Table I were used for the
isolation of crystalline phosphorylase a, which was obtained in good yields.
These observations pointed to a conversion of phosphorylase b to a in vitro,
which is the subject of the accompanying paper (10). It was determined
that, the reaction of phosphorylase b to a required a divalent metal ion;
hence it appeared that extraction of muscle with EDTA solution (Method
4) rather than with water might give a more exact picture concerning the
state of phosphorylase in viva, since the effect of chance contamination by
metals and a conversion of phosphorylase b to a in vitro would be avoided.
6 The enzyme catalyzing a transformation of phosphorylase a to b in vitro is called
PR enzyme (2, 4).
118 MUSCLE PHOSPHORYLASE

Table II presents the results obtained when the muscle from rabbits
was divided into equal portions, one of which was extracted with water
and the other with EDTA. It can be seen that in each case the EDTA
extracts contained phosphorylase b almost exclusively. Separate experi-
ments carried out with crystalline phosphorylase a and purified PR en-
zyme have shown no accelerating effect of added EDTA on PR enzyme
action; thus these results cannot be interpreted as being due to an activa-
tion of PR enzyme. A larger series of animals is being studied by the
EDTA extraction method.

TABLE II
Phosphorylase Activity of EDTA and Water Extracts of Rabbit Muscle
The muscle from each rabbit was divided into equal portions. Extractions by
Method 1 (HzO) and Method 4 (EDTA) were used.

Activity of water extract Activity of EDTA extract

Experiment No. -.
+ AMP - AMP + AMP - AMP
_.---
units per ml. units per ml. units per ml. units per ml.

1* 1270 360 1150 60

2 1420 70 1065 10
3 1560 80 1540 40
41 1850 190 1500 10

Average. 1525 175 1314 30

* Experiment 1, Rabbit 4 of Table I; Experiment 2, Rabbit 15 of Table I; Experi-

ment 3, Rabbit 24 of Table I.
t Rabbit killed with strychnine (Method 5).

DISCUSSION

It is difficult to determine with certainty the relative amounts of phos-

phorylase a and b present in muscle tissue in a given state of physiological
activity, i.e. resting or fatigued muscle. According to the concept pre-
sented by Cori (5) and Sutherland (9, 11) the two forms of phosphorylase
are interconvertible in the intact tissue, the particular balance existing at
any time being determined by hormonal and other factors. In the type
of assay employed in the present study, the muscle must first be removed
from the anesthetized animal, ground, and extracted before any measure-
ments are made. Complete control of the transformations that might
tend to distort the ratio of phosphorylase a to phosphorylase b that existed
in viva cannot be obtained until the extraction step has been reached. In
the extract itself phosphorylase a can change to phosphorylase b in the re-
 E. G. KREBS AND E. H. FISCHER 119

action catalyzed by PR enzyme (2), but this reaction is relatively slow and
ample time is available for enzyme assay before marked changes occur.
A more rapid reaction is the conversion of phosphorylase b to phosphoryl-
ase a (lo), which takes place if any of several common divalent cations are
introduced into the extract. When the extraction of muscle is carried
out with water, it is entirely possible that some conversion of phosphoryl-
ase b to a may occur before assays can be made; hence values such as
those of Table I may indicate too much phosphorylase CL. The use of
EDTA would appear to be the most reliable method in approaching this
problem.
The determination of whether resting skeletal muscle contains phos-
phorylase predominantly in the a or b form is of considerable physiological
significance. Phosphorylase b has been interpreted as being relatively in-
active in viva, since it requires AMP in concentrations7 that probably do
not exist in muscle (5, 14). If resting muscle contains mainly phosphoryl-
ase b, as the present study would appear to indicate, then pronounced ac-
tivation of the phosphorylase reaction under various conditions is possible.
This is in keeping with Sutherland’s concept that epinephrine increases
glycogenolysis by causing a conversion of phosphorylase b to phosphoryl-
ase a (9, 11).

The authors wish to acknowledge the work done in connection with this
problem by Mr. William 0. Rieke and Mr. James Kauth of the Univer-
sity of Washington School of Medicine and also the technical assistance of
Mr. Charles Beaudreau and Mrs. Jeanne Dills.
SUMMARY

1. Muscle extracts of rabbits adapted to 5’ showed the same total phos-

phorylase content as extracts from animals kept at 24-28’.
2. There was a significant correlation between the size of rabbits and the
phosphorylase activity per given amount of muscle.
3. Extracts of resting muscle were found to contain phosphorylase pre-
dominantly in the form of phosphorylase b. Controls were conducted to
rule out the possibility that phosphorylase b was formed through PR ac-
tion during excision of the muscle or in the handling of the extracts.
BIBLIOGRAPHY

1. Green, A. A., and Cori, G. T., J. Bid. Chem., 151, 21 (1943).

2. Cori, G. T., and Green, A. A., J. Bio.?. Chem., 161,31 (1943).

7 The concentration of AMP required for half maximal activation of phosphorylase

b is 5 X 10-c M (12); however, the sensitivity of the enzyme to this nucleotide is in-
creased in the presence of positively charged peptides such as protamine or synthetic
polylysine (13).
120 MUSCLE PHOSPHORYLASE

3. Green, A. A., J. Biol. Chem., 168, 315 (1945).

4. Cori, G. T., and Cori, C. F., J. Biol. Chem., 168, 321 (1945).
5. Cori, G. T., J. Biol. Chem., 158,333 (1945).
6. Illingworth, B., and Cori, G. T., Biochem. Preparations, 3, 1 (1953).
7. Dounce, A. L., Rothstein, A., Beyer, G. T., Meier, R., and Freer, R. M., J. Biol.
Chem., 174, 361 (1948).
8. DuBois, K. P., Albaum, H. G., and Potter, V. R., J. Biol. Chem., 147,699 (1943).
9. Sutherland, E. W., in McElroy, W. D., and Glass, B., Phosphorus metabolism,
Baltimore, 1, 53 (1951).
10. Fischer, E. H., and Krebs, E. G., J. BioZ. Chem., 216, 121 (1955).
11. Sutherland, E. W., Ann. New York Acad. SC., 64, 693 (1951).
12. Cori, C. F., Cori, G. T., and Green, A. A., J. BioZ. Chem., 161, 39 (1943).
13. Krebs, E. G., Biochim. et biophys. a&, 16, 508 (1954).
14. Lohman, K., and Schuster, P., Biochem. Z., 272,24 (1934).