Enzymes of Glycogen Metabolism in White Blood Cells
I. Glycogen Phosphorylase in Normal and Leukemic
Human Leukocytes*
ADEL A. YUNIS| AND GRACE K. ARIMURA
(Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri)
SUMMARY
Glycogen phosphorylase has been assayed in normal leukocytes and in those ob
tained from patients with chronic granulocytic and chronic lymphocytic leukemia.
Assay was performed in the direction of both glycogen degradation and glycogen syn
thesis with and without adenylic acid. The results indicate that (a) a major portion
of phosphorylase activity exists in the active or a form as evidenced by 30-70 per cent
activity in the absence of adenosine-S'-phosphate, (6) wide variations in activity were
noted in leukocytes from different donors, (c) leukocyte phosphorylase activity in leukemic cells did not differ significantly from the normal. Some of the properties of
leukocyt9 glycogen phosphorylase and its relation to phosphorylase from liver and
muscle are discussed.
Leukocytes contain significant amounts of glycogen
(19, 20). Little has been done, however, to elucidate the
various factors concerned in controlling the glycogen level
in these cells. As in the case of muscle cells where
mechanical work is made possible through energy-yielding
metabolic reactions, one of the primary functions of
leukocytes, phagocytosis, has been shown to be an energydependent process (14). Whether glycogen plays an
essential role as an energy store in leukocytes as it does in
muscle cells is not clear. Leukocyte glycogen is dimin
ished in certain hématologiediseases such as chronic
granulocytic leukemia and elevated in polycythemia vera
and in leukocytes of infection (17). Similarly, leukocyte
glycogen is elevated in certain types of glycogen storage
disease (21). A change in leukocyte glycogen level may
reflect a change in glycogen synthesis or in glycogen
degradation. It seems important then to investigate the
control mechanisms involved in glycogenesis and glycogenolysis in leukocytes. We have accordingly initiated
studies concerned with enzymes of glycogen metabolism
in these cells. The recent studies demonstrating a
UDPG-linked pathway for glycogen synthesis have
provided strong evidence for the existence in tissues of a
metabolic cycle in which the UDPG-pathway is linked with
the synthesis and phosphorylase catalyzes the degradation
* Supported by grants from U. S. Public Health Service # CA
06213-02,HE 00022-17;by American Cancer Society Institutional
Research Grant to Washington University #IN36D ; and by gifts
in memory of Philipp Hunkel, Mark Edison, Bill Burns, and Joe
Blanchard.
t Leukemia Society, Inc., Scholar.
Received for publication October 7, 1963.
of glycogen (8, 9, 13, 15, 18). This paper describes the
glycogen phosphorylase levels in normal leukocytes and
those of chronic granulocytic and chronic lymphocytic
leukemia and some preliminary observations on the
properties of this enzyme. It will be seen that (a) a major
portion of leukocyte glycogen phosphorylase exists in the
active or a form (active without adenylic acid); (6)
phosphorylase levels in leukemic leukocytes did not differ
significantly from the normal.
MATERIALS AND METHODS
Preparation of leukocyte extracts.—Bloodwas collected in
siliconized 50-ml. graduated test tubes, and neutral NaEDTA1 was added to a final concentration of 5 X IO"3M.
Leukocytes were isolated by Dextran sedimentation, and
contaminating red cells were lysed by transient exposure to
a hypotonie medium as described by Walford (22). The
leukocyte button was homogenized in 40 volumes of
distilled water at 0°C. for 1% minutes in a Virtis 45
homogenizer set at 45,000 r.p.m. The homogenate was
centrifuged at 15,000 X g for 15 minutes, and the clear
colorless supernatant was used for phosphorylase assay.
Phosphorylase assay.—Phosphorylase assay was per
formed by the spectrophotometric method of Wu and
Racker as described by Hülsmannet al. (4). In this
method the rate of reduction of triphosphopyridine
nucleotide (TPN+) is measured in the presence of excess
inorganic phosphate,
glycogen, phosphoglucomutase2
1The following abbreviations are used in this paper: EDTA =
ethylenediaminetetraacetic acid; AMP = adenosine-5'-phosphate;
TPN+ = triphosphopyridine nucleotide.
2The authors are indebted to Dr. V. Najjar for his generous
supply of phosphoglucomutase in the early phase of this study.
489
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490
Vol. 24, April 1964
Cancer Research
TABLE 1
PHOSPHORYLASE
ACTIVITIESOF EXTRACTSFROMNORMALAND LEUKEMICLEUKOCYTES
NOIXAIDonor123456789/imole
Glucose-1-P/hr/mg
proteinWith
LEUKEMIADonor12345678910111213Minole
GRANULOCYTIC
LEUKEMIADonor12345678910ornóle
LYMPHOCYTIC
Glucose-1-P/hr/mg
proteinWith
Glucose-1-P/hr/mg
proteinWith
VERADonor12Ã-imole
Glucose-1-P/hr/mg
proteinWith
AMP0.0820.1110.0500.0750.0620.1130.0460.1510.097CHRONIC
AMP0.1640.2010.2940.1880.1130.1350.2240.1000.2520.1790.1590.1370.214Without
AMP0.0980.0730.1150.0300.0690.0690.1130.0580.0950.1250.0780.1000.167CHRONIC
AMP0.1840.2170.1210.2300.1700.3540.1080.2020.170Without
AMP0.1800.2550.2490.3030.3350.3000.2270.2660.3130.211Without
AMP0.1250.1580.1310.2070.1030.0850.0700.2170.134POLYCYTHEM
AMP0.2740.090Without
AMP0.1830.060
(prepared by the method of Najjar [11]), glucose-6-phosTABLE 2
phate dehydrogenase (Zwischenferment), and TPN+ at
LEUKOCYTE
PHOSPHORYLASE
ACTIVITIESAS ASSAYEDIN THE
DIRECTIONOF POLYSACCHARIDE
SYNTHESIS
pH 7.5, phosphorylase (leukocyte extract) being the
limiting enzyme. Excess phosphoglucomutase and gluLYMPHOCYTIC
cose-6-phosphate dehydrogenase were always checked by NORMALDonor12 GSANULOCYTICLEUKEMIADonori2
LEUKEMIADonor1
replacing glycogen and the leukocyte extract with glucose1-phosphate. The assay was performed with AMP (10~3
P/30min/107
cellsWith
P/30 min/10'
cellsWith
P/30 min/10'
cellsWith
M) and without. The reaction was started by the addition
of TPN+ and is some instances by the addition of leu
AMP20.2
out
out
AMP7.2
out
AMP26.2
kocyte extract. Protein concentration in the extract was
AMP11.2
AMP8.9
AMP5.0
measured by the method of Lowry (10). Phosphorylase
activity was expressed as mamóles glucose-1-phosphate/
(62.08)*
(54.2)*
hour/mg protein, with a molecular extinction coefficient for
25.3 12.6
28.6
20.02 2,ig 8.1 (61.6)*With 4.9
TPNH of 6.22 X 10" sq. cm. X mole~l at 340 im< (3).
345678eg
25 14.8 3ng 24.4 (70.3)*With
13.2CHRONIC
265237.2527.321.1With
14.221.81020.410.5CHRONIC
In some instances phosphorylase was assayed in total
leukocyte homogenates and extracts in the direction of
glycogen synthesis by a modification of the method of
Sutherland as described by Williams and Field (23). The
activity was expressed as ¿uginorganic phosphate (Pi)
liberated/30 min/107 cells as determined on total homog
* Values in parentheses represent /ig P/30 min/mg protein in
enates or ng Pi/30 min/mg protein as determined on cell the extract.
extracts.
slightly higher than that of leukocytes from chronic
RESULTS
myelocytic leukemia and normal donors, there was a
Phosphorylase activity was assayed in leukocytes significant overlap in the individual cases. Leukocytes
obtained from nine normal donors, ten patients with from two patients with polycythemia vera showed normal
chronic lymphocytic leukemia, thirteen patients with phosphorylase activity.
chronic granulocytic leukemia, and two patients with polyIn Table 2 are listed the phosphorylase activities as
cythemia vera. The results of the assays are shown in measured in the direction of polysaccharide synthesis.
Table 1. A wide variation is noted in the levels of phos
The results in eight normal donors and in three patients
phorylase activity in leukocytes obtained from different with chronic granulocytic leukemia were similar to those
donors. The ratio of phosphorylase activity with AMP to obtained in Table 1: (a) wide variations were noted in the
that without AMP ranged between 0.26 and 0.75, indicat
activity levels of leukocytes from different donors, (6)
ing that a significant portion of enzyme exists in the active significant proportions of phosphorylase a activity were
or a form (active without AMP). The level of enzyme found, and (c) there was no significant difference as to
activity in leukemia cells did not differ significantly from enzyme activity between normal and leukemic leukocytes.
the normal. Although the mean activity level in lympho
It will be noted that, when activity is expressed as /ig Pi/
cytes from donors with chronic lymphocytic leukemia was 30 min/107 cells as measured on total homogenates, the
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TUNIS ANDARIMURA—Glycogen
Phosphorylase
lymphocyte appeared to possess an activity about onefourth to one-fifth that of the granulocyte. However,
when expressed as jugPi/30 min/mg protein in the extract
both cell types appeared to have comparable activities.
Leukocyte phosphorylase activities as measured by both
methods was not significantly affected by the addition of
cysteine.
491
leukocyte phosphorylase shows considerable activity hi the
absence of AMP and is activated by glucagon (23), and
cysteine does not stimulate its activity. In addition, low
leukocyte phosphorylase has been demonstrated in cases of
glycogen storage disease caused by deficiency of liver
phosphorylase (4, 23). This latter finding indicates that
both enzymes may share a common genetic control.
Further characterization of this relation should await puri
fication of leukocyte phosphorylase.
The results of phosphorylase assays in normal leukocytes
described here are in close agreement with those of Hüls
mann (4) and Williams and Field, who reported on leuko
cyte phosphorylase activity hi glycogen storage disease
and normal controls (23). Wide variations in activity
were also noted by these authors. Hülsmannattributes
these variations to possible differences in the secretion of
epinephrine and glucagon. Williams and Field were able
to demonstrate considerable activation of leukocyte phos
phorylase by glucagon in vitro (23).
The normal phosphorylase activity in leukemia cells is
of interest in view of the recent report by Nirenberg ( 12)
describing the absence of glycogen phosphorylase activity
in some ascites tumors. More interesting is the fact that
cells from these tumors were devoid of glycogen but con
tained normal amounts of phosphorylase kinase. Although
the present study reveals no difference in phosphorylase
activity between normal and leukemic leukocytes, it is
possible that future studies may reveal differences in the
mechanisms controlling glycogenolysis in these cells. Fur
thermore, other types of human leukemias may be of in
terest to study from this aspect.
DISCUSSION
Cori, Cori, and Green (1) found that glycogen phos
phorylase from rabbit skeletal muscle exists in two forms:
One designated as phosphorylase a exhibits 65 per cent of
its maximal activity in the absence of AMP; the other,
phosphorylase 6, is inactive in the absence of the nucleotide. Extracts of resting muscle were found to contain the
enzyme virtually completely in the b form (5). Similar
results were obtained with rabbit heart muscle and human
autopsy skeletal muscle (24, 25). Phosphorylase 6 can be
readily converted to phosphorylase a; the mechanism of
conversion has recently been elucidated through the work
of Krebs et al. (7). It involves phosphorylation and
dimerization to form phosphorylase a according to the
Mg++
following reaction: 2 phosphorylase b + 4ATP
>phos
phorylase a + 4 ADP. The enzyme catalyzing this
reaction has been termed phosphorylase 6 kinase. When
assayed in resting muscle, phosphorylase kinase is found to
be inactive at pH 7.0 but shows its maximal activity at pH
8.6. This inactive kinase can be activated in several ways :
by Ca4-*-ions, Mg-ATP, cyclic AMP, or Mg-ATP-cyclic
AMP (6) ; the enzyme now exhibits activity at pH 7.0 and
is called active kinase. The rate of glycogenolysis in a
REFERENCES
given tissue presumably depends upon the ratio of phos
1. CORI, C. F.; CORI, G. T.; ANDGREEN, A. A. Crystalline
phorylase a to 6 which in turn depends upon the state of
Muscle Phosphorylase III. Kinetics. J. Biol. Chem., 161:39-55
kinase activity. The physiologic mechanism which trig
1943.
gers phosphorylase kinase activation leading to glycogen
2. HBNION, W. F., AND SUTHERLAND,E. W. Immunological
olysis is the subject of extensive study and is not completely
Differences of Phosphorylases. J. Biol. Chem., 224:477-88
understood at present. The existence of phosphorylase
1957.
3. HORECKER,B. L., ANDKORNBERG,A. The Extinction Coeffi
in the 6 form in resting muscle is compatible with the pres
cients of the Reduced Band of Pyridine Nucleotides. J. Biol.
ence of inactive kinase.
Chem., 175:385-90, 1948.
If one assumes that leukocyte phosphorylase a also ex
4. HÃœLSMANN,
W. C.; OEI, J. L.; ANDCREVELD,S. V. Phosphoryl
hibits 65 per cent of its maximal activity without AMP,
ase Activity in Leukocytes from Patients with Glycogen
Storage Disease. Lancet, Sept. 9, pp. 581-83, 1961.
then the present study shows that leukocyte phosphorylase
5.
KREBS,E.
G., ANDFISCHER,E. H. Phosphorylase Activity of
exists almost entirely in the a form. It is of interest that
Skeletal Muscle Extracts. J. Biol. Chem., 216:113-20, 1955.
preliminary observations in this laboratory indicate that
6. KREBS, E. G.; GRAVES,D. J.; ANDFISCHER,E. H. Factors
leukocytes also contain phosphorylase kinase which readily
Affecting the Activity of Muscle Phosphorylase 6 Kinase. J.
Biol. Chem., 234:2867-73,1959.
converts crystalline rabbit muscle phosphorylase b to the
7. KREBS,E. G.; KENT, A. B.; ANDFISCHER,E. H.: The Muscle
a form at pH 7.O. The implications of these observations
Phosphorylase 6 Kinase Reaction. J. Biol. Chem., 231:73-83,
have to await further studies. Extensive studies on liver
1958.
phosphorylase by Sutherland and co-workers have demon
8. LARNER,J., ANDVILLARPALASI,C. Enzymes in a Glycogen
strated wide differences in the properties of this enzyme
Storage Myopathy. Proc. Nati. Acad. Sci., 46:1234-35, 1959.
and that of muscle (16). Thus, liver phosphorylase ex
9. LELOIR,L. F., ANDCARDINI,C. E. Biosynthesis of Glycogen
from Uridine Diphosphate Glucose. J. Am. Chem. Soc., 79:
hibits over 50 per cent of its activity without AMP, is
6340-41, 1957.
activated by glucagon, its activity is not stimulated by
10. LOWKY,O. H.; ROSEBRAUGH,
N. J.; FARE, A. L.; ANDRAN
cysteine or glutathione, and it can be phosphorylated by a
DALL,R. J. Protein Measurement with the Folin Phenol
Reagent. J. Biol. Chem., 193:265-75, 1951.
kinase enzyme but without dimerization. In addition,
liver and muscle phosphorylase have been shown ro be im- 11. NAJJAR, V. A. Phosphoglucomutase from Muscle. Methods
Enzymol., 1:294-99, 1955.
munologically different (2).
12.
NIRENBERG,
M. W. A Biochemical Characteristic of Ascites
Evidence from other studies and the present one in
Tumor Cells. J. Biol. Chem., 234:3088-93, 1959.
dicates that leukocyte phosphorylase is closely related to 13. ROBBINS,P. W.; TRAUT,R. R.; ANDLIPMANN,F. Glycogen
Synthesis from Glucose, Glucose 6-Phosphate and Uridine
liver phosphorylase rather than to that of muscle. Thus,
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1964 American Association for Cancer Research.
492
14.
15.
16.
17.
18.
19.
Cancer Research
Diphosphate
Glucose in Muscle Preparations.
Proc. Nati.
Acad. Sci., 45:6-12,1959.
SBARBA,A. J., ANDKARNOVSKT,M. L. The Biochemical Basis
of Phagocytosis I. Metabolic Changes during the Ingestion of
Particles by Polymorphonuclear
Leukocytes. J. Biol. Chem.,
234:1355-62,1959.
SCHMID,R.; ROBBINS, P. W.; ANDTRAUT, R. R. Glycogen Syn
thesis in Muscle Lacking Phosphorylase.
Proc. Nati. Acad.
Sci., 46:1236-40, 1959.
SUTHERLAND,E. W.; ANDWOSILAIT, W. D. Liver Phosphoryl
ase; Preparation and Properties. J. Biol. Chem., 218:459-68,
1956.
VALENTINE,W. N.; FOLLETTE,J. H.; ANDLAWRENCE,J. S. The
Glycogen Content of Human Leukocytes in Health and in
Various Disease States. J. Clin. Invest., 32:251-57,1953.
VILLAR, PALASI C., AND LARNER, J. A Uridine Coenzymelinked Pathway of Glycogen Synthesis in Muscle. Biochim.
Biophys. Acta, 30:449, 1958.
WACHSTEIN,M. The Distribution of Histochemically Demon
20.
21.
22.
23.
24.
25.
Vol. 24, April 1964
strable Glycogen in Human Blood and Bone Marrow Cells.
Blood, 4:54-59, 1949.
WAGNER, R. Studies on the Physiology of the White Blood
Cells: The Glycogen Content of Leukocytes in Leukemia and
Polycythemia. Blood, 2:235-43, 1947.
. Glycogen Content of Isolated White Blood Cells in
Glycogen Storage Disease. Am. J. Dis. Child., 73:559-64, 1947.
WALFORD,R. L.; PETERSON, E. T.; ANDDOYLE, P. Leukocyte
Antibodies in Human Sera and in Immune Rabbit Sera. Blood,
12:953-71, 1957.
WILLIAMS,H. E., ANDFIELD, J. B. Low Leukocyte Phosphor
ylase in Hepatic Phosphorylase-Deficient
Glycogen Storage
Disease. J. Clin. Invest., 40:1841-45, 1961.
YUNIS, A. A.; FISCHER, E. H.; ANDKREBS, E. G. Crystalliza
tion and Properties of Human Muscle Phosphorylases a and 6.
J. Biol. Chem., 236:3163-68,1960.
. Comparative Studies on Glycogen Phosphorylase. IV.
Purification and Properties of Rabbit Heart Phosphorylase.
Ibid., 237:2809-15, 1962.
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Enzymes of Glycogen Metabolism in White Blood Cells: I.
Glycogen Phosphorylase in Normal and Leukemic Human
Leukocytes
Adel A. Yunis and Grace K. Arimura
Cancer Res 1964;24:489-492.
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