Download Hexokinase Isozyme Patterns of Experimental

[CANCER RESEARCH 29, 1437-1446, July 1969]
Hexokinase
Isozyme Patterns
of Experimental
Hepatomas
of Rats1
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'.
Shigeaki Sato, Taijiro Matsushima, and Takashi Sugimura
Biochemistry Division, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, Japan
SUMMARY
Hexokinase isozymes in rat tissues were electrophoretically
separated on cellulose acetate membrane. The method was
very quick and gave reproducible results. By using this
method, hexokinase isozyme patterns were studied on normal
rat liver and experimental hepatomas with differing growth
rates and degrees of differentiation.
In normal rat liver, the hexokinase pattern obtained on cellu
lose acetate membrane was identical with that obtained on
starch gel by previous workers. There were four types of hexokinases, which corresponded to Types I, II, III, and IV hexokinases according to Katzen and Schimke, in order of increas
ing mobility from the origin to the anode. Type IV hexokin
ase, which was stained most intensely, was glucokinase, and
duplication of Type IV hexokinase into two bands (IVS, slow
form, and IVf, fast form) was occasionally observed.
Morris hepatomas, as a slowly growing and less deviated hepatoma group, showed relatively predominant Type II hexo
kinase in addition to Types I and III hexokinases, and they
also had a faint Type IV hexokinase band. A rapidly growing
and highly deviated hepatoma group, Yoshida ascites hepa
tomas were characterized by the presence of only Type I hexo
kinase and marked Type II hexokinase and by the loss of Type
IV and Type III hexokinases. Yoshida sarcoma showed a simi
lar hexokinase pattern to that of Yoshida ascites hepatomas. A
slowly growing substrain of Yoshida sarcoma, LY 5, had also
Type III and Type IV hexokinases, resembling Morris hepa
tomas. These observations suggested that the genes for Types
III and IV hexokinase isozymes were not deleted, but their
expressions were blocked in the original Yoshida sarcoma.
The regenerating liver was analogous to Morris hepatomas in
hexokinase pattern. In fetal liver, Type II hexokinase was
dominant, in addition to Types I, III, and weak IV hexo
kinases.
tive factor for the rate of glycolysis in various tumor strains
(29). Meanwhile Gonzalez et al. (6) in 1964 separated rat liver
hexokinase into four types with diethylaminoethyl cellulose
column chromatography. Katzen and Schimke (12) in 1965
succeeded in separating four types of hexokinase in rat tissues
with starch gel electrophoresis and mentioned the presence of
the specific isozyme pattern in the specific tissue. Following
these pioneering works, the hexokinase isozyme patterns of
many tissues, including human materials, have been elucidated,
and the enzymatic properties of each type of isozyme have
been studied (3, 7, 10, 23). While many papers on isozyme
patterns of hexokinase in the normal tissues have been pub
lished, there have been only recent reports by Gumaa and
Greenslade (8) and Shatton et al. (25) on the hexokinase iso
zyme patterns of experimental hepatomas. From the studies
using various concentrations of glucose, Sharma et al. (24) also
reported relatively increased "low-iCm hexokinase" activities
and decreased "highJCm -specific glucokinase" activity during
experimental hepatocarcinogenesis in rats. We suggested the
presence of two types of hexokinases in Yoshida ascites hepa
toma cells by using different concentrations of glucose (27).
In this paper we describe a new method for electrophoretic
separation of hexokinase isozymes on cellulose acetate mem
brane. By using this method, the hexokinase isozyme patterns
were investigated on two groups of experimental hepatomas of
rats, Morris hepatomas as a less deviated and slowly growing
group, and Yoshida ascites hepatomas as a highly deviated and
rapidly growing one. Yoshida sarcoma and its subs trains, in
cluding a strain with slower growth rate than the original
strain, were also investigated. The hexokinase isozyme patterns
thus obtained on hepatomas were compared with those in the
normal liver, the regenerating liver, and fetal liver. The prob
lem of cellular disdifferentiation on carcinogenesis based on
the experimental results will be discussed.
MATERIALS
AND METHODS
INTRODUCTION
An increased rate of anaerobic glycolysis had been regarded
for long to be one of the characteristics of tumor tissues. In
1966, however, Weinhouse (29) pointed out that increased
glycolysis was not always observed in Morris hepatomas. He
described also that the level of hexokinase [ATP:D-hexose
6-phosphotransferase (EC.2.7.1.1)] activity was a determina-
Supported in part by a grant from the Ministry of Education and
Ministry of Health, Japan.
Received October 7, 1968; accepted March 26, 1969.
JULY 1969
Tumors. Morris hepatomas, Nos. 7316A, 7793, 7794A, and
7795, were transplanted intramuscularly in both hind legs of
about 9- to 10-week-old Buffalo strain rats. They were main
tained on diet CE-2 (diet from the Central Laboratory of Ex
perimental Animal, Japan). Three to four weeks after trans
plantation, rats were killed by decapitation, and tumors were
removed; necrotic, hemorrhagic, and nontumorous materials
were carefully trimmed off.
About one million cells of Yoshida ascites hepatomas or
sarcomas were intraperitoneally
transplanted into Donryu
strain male rats 9 to 10 weeks old. Five to ten days later, the
rats were killed by decapitation. The ascitic fluid was aspirated
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S. Sato, T. Matsushima, and T. Sugimura
and was rapidly diluted about five times with cold saline, and
tumor cells were harvested by centrifuging at 100 X g for 3
minutes. After three to four repetitions of washing in this way,
the tumor cells became almost free from contaminating red
blood cells, leukocytes, or peritoneal cells, as determined by
examination under a light microscope. Tumor cells were final
ly well packed by centrifuging at 1,400 X g for 5 minutes.
Normal Adult Regenerating and Fetal Livers. For the nor
mal control, Donryu strain rats 9 to 10 weeks old were mainly
used. Buffalo and Wistar strain rats were also checked, but not
much difference was detected among these three strains. The
regenerating liver was obtained 24 hours after the removal of
about two thirds of the liver of a male Donryu strain rat 9 to
10 weeks old. The fetal liver was collected from fetuses from a
pregnant rat of the same strain 2 to 5 days prior to the ex
pected parturition. The normal and the regenerating livers
were thoroughly perfused with cold saline, and the fetal liver
was carefully rinsed with cold saline.
Preparation of Enzyme Extracts. The livers or the tumor
tissues of Morris hepatomas were homogenized in an equal
volume of cold 0.1 M Tris-HCI buffer (pH 7.4) containing 5
ntiM EDTA2, 5 mM 2-mercaptoethanol,
and 10 mM glucose
with a Potter-Elvehjem type homogenizer with a Teflon pestle.
The cells of Yoshida ascites hepatomas and sarcomas were
homogenized in three volumes of the same buffer with a VirTis 45 homogenizer operating at 25,000 rpm for 4 minutes.
These homogenates were centrifuged at 105,000 X g for 60
minutes or at 164,000 X g for 40 minutes. The supernatant
thus obtained was used as an enzyme extract for the assay of
hexokinase activity and for the electrophoresis of hexokinase
isozymes.
Enzyme Assay. The sum of hexokinase and glucokinase ac
tivities was assayed spectrophotometrically
at 30°Cby measur
ing the rate of NADPH2 formation at 340 m/i. One ml of the
reaction mixture contained the following at the final concen
trations indicated: 0.1 M glucose, 5 mM ATP sodium salt, 5
mM MgCl2, 0.4 international unit/ml G6PD, 5 mM NADP
tetrasodium salt, 5 mM 2-mercaptoethanol, and 0.3 M TrisHCl buffer (pH 7.4). The reaction mixture included also 0.1
ml of the appropriately diluted enzyme extract. Continuous
recording of the optical density was performed with a Gilford
multiple sample absorbance recorder, model 2000. One unit of
hexokinase activity was expressed as one micromole of glucose-6-phosphate formed per minute. This assay method is a
slight modification of that described by Salas et al. (21). Pro
tein was measured by the method of Lowry et al. with crystal
line bovine serum albumin as a standard (14).
Electrophoresis. Three or six jul of the enzyme extract was
placed at the center of a cellulose acetate membrane (Gelman,
1 x 6-f inches). Electrophoresis was carried out at 0°C,using
veronal buffer (pH 8.6, I = 0.05) containing 5 mM EDTA, 1
2The
abbreviations
used
are:
NADP,
nicotinamide
adenine
dinucleotide phosphate; NADPH2, reduced nicotinamide adenine
dinucleotide phosphate; G6PD, gJucose-6-phosphate dehydrogenase;
EDTA, ethylenediaminetetraacetic acid.
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mM 2-mercaptoethanol, and 10 mM glucose for two hours at
25 volts/cm. After electrophoresis, bands of hexokinase iso
zymes were stained by placing the membrane en a 1% agar gel
plate which had been laid on a glass and contained 0.1 M or
0.5 mM glucose, 5 mM ATP, 5 mM MgCl2, 0.4 lU/ml G6PD, 1
mM NADP, 2 mM KCN, 25 Mg/ml phenazine methosulfate, 0.4
mg/ml nitroblue tetrazolium, and 0.1 M Tris-HCl buffer (pH
7.4). Staining was carried out for 40 minutes in the dark at
37°C.Densitometry of hexokinase isozyme bands on a mem
brane was performed with a Densicord, Model 542 of Photovolt with a 525 m/Z filter. As a blank, glucose and ATP were
omitted from the agar gel plate for staining.
RESULTS
Hexokinase Activity. The sum of activities of hexokinase
and glucokinase assayed with 0.1 M glucose on the enzyme
extracts of the normal rat liver, Morris hepatomas, Yoshida
ascites hepatomas, Yoshida sarcomas, the regenerating liver,
and the fetal liver are shown in Table 1. The value was ex
pressed as the specific activity. Table 1 also includes the data
on the survival time of the rat bearing each tumor. The specific
activities of Morris hepatomas were much lower than that of
the normal liver, in accordance with the results obtained by
Weinhouse (29), while the activities of all strains of Yoshida
hepatomas and Yoshida sarcomas were markedly higher than
that of the normal liver. The activity of the regenerating liver
was almost the same as that of the normal liver, and the fetal
liver had less hexokinase activity than the normal liver.
Patterns of Hexokinase Isozymes on Electrophoresis. In
Chart 1, schematic patterns of hexokinase isozymes on the
normal liver, all strains of Morris hepatomas, Yoshida ascites
hepatomas, and Yoshida sarcomas, the regenerating liver, and
the fetal liver stained with 0.1 M glucose are given.
Photographs of the typical hexokinase isozyme patterns of
the normal liver, Morris hepatomas, Yoshida ascites hepa
tomas, Yoshida sarcomas, the regenerating liver, and the fetal
liver are given in Figs. 1—3,and their densitometoric tracings
on membrane are illustrated in Charts 2—4.The normal rat
liver showed four bands migrating to the anode and one faint
band on the cathodic side. Since the cathodic band was stained
even in the absence of glucose and ATP in the staining gel, this
band was not regarded as an isozyme of hexokinase. This band
may correspond to that described as alcohol dehydrogenase by
Kaplan and Beutler (2, 10). The four anodic bands were Types
I, II, HI, and IV hexokinases in order of increasing mobility
according to Katzen and Schimke (12).
Types I, II, and III hexokinases were stained throughout the
whole experiment at glucose concentration of either 0.1 M or
0.5 mM. The color intensities of Types I and II hexokinases
did not change with both concentrations of glucose. Type III
hexokinase was stained slightly more intensely at 0.5 mM glu
cose than at 0.1 M glucose. The band of Type IV hexokinase
was very weak at 0.5 mM glucose, but markedly stained at 0.1
M glucose concentration. From these observations it is evident
that Types I, II, and III bands are lowiCm hexokinases, while
Type IV band is a hexokinase with a high Km value for glu
cose, i.e., glucokinase as described by Katzen and Schimke
(12).
CANCER RESEARCH VOL. 29
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Rat Hepatoma Isozyme Patterns
Table 1
Activities
(units/gm protein)
Normal liver
16.2" (11.3,12.7,16.4,
Morris hepatoma 7316A
Morris hepatoma 7793
Morris hepatoma 7794A
Morris
7795Yoshida
hepatoma
(2.8, 3.9, 4.9, 5.9, 6.1)
6.8 (2.3, 4.6, 8.1, 12.2)
4.8 (1.7,7.8)
(2.6,3.0)20.131.338.2
2.8
Survival times
(days)
18.1, 22.4*)
28-35
90-180
287821
ascites hepatoma
13AH
AH
44AH62F
AH65CAH
66AH66FAH84AAH
127
AH 130
AH 150A
AH225A
272AH
AH
7974Yoshida
(22.9, 25.0, 32.2, 54.4, 56.3)
13.410.638.038.424.3
(10.6,22.2,40.1)
38.6 (13.6,63.5)
63.2 (65.4,61.0)
18.4 (15.6,21.2)
30.327.3
(11.0,12.3,29.0,55.8)39.143.7
101171018
11
13
125-7127-830
sarcomaLY5LY52LY54LY80Regenerating
(33.7,40.2,57.3)
52.744.122.320.7
liver
Fetal liver4.7
7-107-107-10
(18.3,20.9,22.8)
4.7 (4.2,5.2)21
Total hexokinase activities of normal liver, tumors, regenerating and fetal livers, and survival times
of tumor-bearing rats.
"Average value.
* Value of individual determination.
Hexokinase Patterns of Hepatomas and Sarcomas. All strains
of Morris hepatomas examined (Nos. 7316A, 7793, 7794A,
and 7795) had Types I, II, III, and IV hexokinases, among
which Type II hexokinase was predominant. Type IV band in
Morris hepatomas was faint and slightly shifted to the anode
from the position of Type IV in the normal liver. This band
was weaker with 0.5 mM glucose than with 0.1 M glucose.
Four strains of Morris hepatomas had almost the same patterns
of hexokinase isozymes. A faint cathodic band which was re
garded nonspecific was also detected in all cases.
In all strains of Yoshida ascites hepatomas, Types I and II
hexokinases were recognized, and some strains showed a weak
band of Type III hexokinase. No strain had Type IV hexo
kinase. Type II hexokinase was remarkable in all strains. It
may be noticed that rats bearing Yoshida ascites hepatomas
with Type III hexokinase exhibited longer survival times than
those without it.
Yoshida sarcoma and the derived strains were subjected to
analyses of hexokinase isozyme patterns. Yoshida sarcomas,
the original strain and the derived strains, LY 52, LY 54, and
LY 80, showed almost the same hexokinase isozyme patterns
as Yoshida hepatomas. The rats bearing these tumors survived
about a week after transplantation. Exceptionally, a strain, LY
5, which allows the rats bearing this tumor to survive for about
a month, showed relatively intense Type III hexokinase as well
as weak Type IV hexokinase.
JULY 1969
Hexokinase Patterns of Regenerating Liver and Fetal Liver.
Fig. 3 represents the hexokinase isozyme patterns of the regen
erating and the fetal livers. In both cases, four types of hexo
kinases were present, but Type IV was weak and Type II was
relatively intense.
Duplication of Type IV Hexokinase. The duplication of
Type IV band was often observed on the cellulose acetate
membrane, and slow and fast forms were designated IVS and
IVf respectively. Hansen et al. (9, 18) reported that the band
of Type IV hexokinase duplicated on starch gel electrophoresis
when it was carried out in the absence of EDTA in veronal
buffer. In our experiments, the duplication of Type IV hexo
kinase was found either with or without EDTA in veronal
buffer in some normal livers, in pregnant rat livers, and in
some livers of hepatoma-bearing rats, but in some normal livers
the duplication was not observed under any condition, even in
the absence of EDTA in the extracting buffer.
DISCUSSION
A method for the rapid, distinct, and reproducible demon
stration of hexokinase isozymes in animal tissues by electro
phoresis on cellulose acetate membrane was established. In
contrast to starch gel electrophoresis for separation of hexo
kinase isozymes, cellulose acetate membranes are readily avail
able, and much time required for electrophoresis and staining
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S. Sato, T. Matsushima, and T. Sugimura
- Origin
I1' *
M IV
il
Normal liver
\A
Morrishepatoma 7316A
Morris hepatoma 7793
Morris hepatoma 7794A
Morris hepatoma 7795
I D
i a
eo
o i BD
1
IKD1 1
Yoshidaascites hepatoma
AH 13
AH 44
AH 62F
AH 65C
AH 66
AH 66F
A H 84 A
AH 127
AH 130
AH I50A
A H 225 A
A H 272
A H 7974
Liver
Yoshidasarcoma
LY5
LY52
LY54
LY80
Regenerating liver
Fetal liver
experiments, some livers showed the duplication of Type IV
hexokinase even in the presence of EDTA in veronal buffer.
This was also observed by Shatton et al. (25). The band of
Type IV hexokinase was detected also in Morris hepatomas, a
strain of Yoshida sarcoma and the fetal liver. In these cases the
band shifted a little further to the anode as represented in
Chart 1, but it remains to be investigated that these bands
D
Morris Hepatoma
73I6A
I
Chart 1. Schematic representation of hexokinase isozyme patterns
of normal liver, Morris hepatomas, Yoshida ascites hepatomas, Yoshida
sarcomas, and regenerating and fetal livers. Biack, hatched, and open
bars indicate intensely, moderately, and weakly stained bands at 0.1 M
glucose respectively.
Yoshida Hepatoma
is saved. A number of samples can be applied at the same time
in a single chamber, and much smaller amounts of the reagents
were required for staining the isozyme bands. Moreover, the
pattern of hexokinase isozymes of the normal liver obtained
with this method is reproducible and coincides well to that on
starch gel electrophoresis reported by Katzen and Schimke
The duplication of Type IV hexokinase was often detected
in the livers of normal, pregnant, and some hepatoma-bearing
rats. Pilkis and Hansen (18) described an immunologie differ
ence between Types IVf and IVS hexokinases, and they noted
that Type IVf disappeared in fasting and diabetes. Type IVS
hexokinase was also found in the kidney and testis. In our
1440
AH 127
Chart 2. Densitometric tracings of hexokinase isozyme bands on cel
lulose acetate membranes of normal liver, Morris hepatoma 7316A, and
Yoshida ascites hepatoma AH 127. Hexokinase isozyme bands were
stained at 0.1 M glucose.
CANCER RESEARCH VOL. 29
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Rat Hepatoma Isozyme Patterns
The hexokinase isozyme patterns of Morris hepatomas used
in the present experiment are not so deviated from that of the
normal liver except for the relative predominance of Type II
hexokinase and the decrease of Type IV hexokinase.
Yoshida ascites hepatomas are derived from hepatomas in
duced by azo dyes, dimethylaminoazobenzene,
or 3'-methyl-di-
Liver
methylaminoazobenzene
(30). Their morphologic and bio
chemical properties are fairly deviated from those of the nor-
in
Liver
Yoshida Sarcoma
Regenerating Liver
Yoshida Sarcoma
LY5
Chart 3. Densitometric tracings of hexokinase isozyme bands of nor
mal liver, Yoshida sarcoma, original strain, and Yoshida sarcoma LY 5.
Hexokinase isozyme bands were stained at 0.1 M glucose.
correspond to Type IVf hexokinase. In any case, these findings
indicate the existence of glucokinase also in some tumor
tissues.
Shatton et al. (25) also detected Type IV hexokinase in
Morris hepatomas in addition to Types I, II, and III. The dupli
cations of Type I and II hexokinase bands have been also
observed with starch gel electrophoresis (10—12), but these
were not noticed in our experiment.
Fetal Liver
Chart 4. Densitometric tracings of hexokinase isozyme bands of nor
mal, regenerating, and fetal livers. Hexokinase isozyme bands were
stained at 0.1 M glucose.
JULY 1969
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1441
S. Sato, T. Matsushima, and T. Sugimura
mal liver. They grow very fast, and the survival times of rats
bearing them are four days to two weeks. Among seventy
strains of Yoshida ascites hepatomas now available, we ex
amined the hexokinase isozyme patterns in 13 strains, each of
which showed different characteristics in survival times of rats
bearing them, in the form of cell aggregation (free or island
forming), in chromosome number, or in sensitivity to chemotherapeutic agents. All strains investigated had high hexokinase
activities, and they showed Type I and predominant Type II
hexokinase isozymes. The appearance of Type III hexokinase
was noticed in some strains of Yoshida ascites hepatomas, and
the rats bearing these tumors survived a fairly longer period.
No correlation was observed between the form of cell aggrega
tion or the chromosome number and the presence of Type III
hexokinase.
Yoshida sarcoma was originally regarded as a reticulum cell
sarcoma, but recently it is assumed to be a hepatoma. Hexo
kinase isozyme patterns of Yoshida sarcomas were quite simi
lar to those of Yoshida ascites hepatomas. However, in the
cells of LY 5, which grow slowly and kill animals about a
month after the transplantation, Type III hexokinase was de
tected in addition to Types I and II. Furthermore, it was of
interest to note the presence of Type IV hexokinase. This
pattern was really close to those of Morris hepatomas. From
these observations it may be suggested that the absence of
hexokinases III and IV in the original Yoshida sarcoma cells
does not necessarily mean the deletion of genes for these hexo
kinase isozymes, but rather indicates blockage of their expres
sion. Results obtained in our experiments coincide well with
recent observations of Gumaa and Greenslade (8), who also
reported predominant Type II hexokinase in experimental
solid hepatomas and ascites tumors, and also with Shatton et
al. (25), who described hexokinase isozyme patterns in hepa
tomas and other tumors.
Nakamura and Hosoda (16) detected a very low or almost
nondetectable concentration of glucose in ascitic fluid of
Ehrlich ascites tumor. This may be also true for Yoshida
ascites hepatomas and Yoshida sarcomas. In the intramuscular
ly transplanted Morris hepatoma tissues, the vascularization is
supposed to be insufficient compared with the normal livers,
and the exogeneous supply of glucose is also thought to be
small. Under such circumstances, it seems reasonable that low
Km hexokinases would become dominant in such deviated tu
mors as Yoshida ascites and Morris hepatomas.
In hepatocarcinogenesis
the gene for high Km hexokinase
might be switched off, and, in turn, the expression of the
genes for low Km hexokinases, especially for Type II hexo
kinase, might be exaggerated. Similar observations have been
reported on aldolase (1, 15, 27), pyruvate kinase (26, 28), and
fructose 1,6-diphosphatase (22). Such a phenomenon has been
referred to as "switch-off and -on mechanism of gene on carcinogenesis" or "disdifferentiation"
(27). Potter recently ex
pressed this as "oncogeny as blocked ontogeny" (19). An in
crease of hexokinase activity has been described also
cultured mammalian cells transformed to a malignant type
polyoma virus (17).
The presence of the particle-bound hexokinase in ascites
mor cells and experimental hepatomas has been reported
many workers (4, 5, 13, 20). We also observed the presence
1442
in
by
tu
by
of
the paniculate hexokinase in Yoshida ascites hepatomas. The
molecular species of hexokinase, released by Triton X-100,
were the same as those of the soluble fraction.
The regenerating and the fetal livers also possessed hexoki
nase isozyme patterns similar to those of hepatomas. Since
they are also rapidly proliferating, the possibility still remains
that the predominance of Type II hexokinase might be merely
a reflection of the rapid growth of cells.
ACKNOWLEDGMENTS
The authors are indebted to Dr. Hiroshi Sato, Sasaki Institute, Tokyo,
and to Dr. Harold P. Morris, NIH, Bethesda, Md., for giving us the
strains of transplantable hepatomas of rats. The authors acknowledge
the valuable discussion held with Dr. Waro Nakahara, Director of this
Institute, and with Dr. Takashi Kawachi of this department. They wish
to express sincere thanks to Dr. Sidney Weinhouse, Fels Research Insti
tute, Philadelphia, Pa., for reading this manuscript.
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1443
5. Sato, T. Matsushima, and T. Sugimura
3 2 l origin
+"i 2 3 4 cm
Liver
Morris Hepatoma
73I6A
Morris Hepatoma
7793
Yoshida Hepatoma
AH7974
AH 62 F
AH 127
i
Fig. 1. Hexokinase isozyme patterns of normal liver, Morris hepatomas, and Yoshida ascites hepatomas. Hexokinase isozyme bands were stained
at 0.1 M glucose.
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CANCER RESEARCH VOL. 29
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Rat Hepatoma Isozyme Patterns
32
origin
+
l * i 2 3 4 cm
Liver
Yoshida Sarcoma
Yoshida Sarcoma
LY 52
LY54
LY5
2
Fig. 2. Hexokinase isozyme patterns of normal liver and Yoshida sarcomas. Hexokinase isozyme bands were stained at 0.1 M glucose.
JULY 1969
1445
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S. Sato, T. Matsushima, and T. Sugimura
origin
+
3•2i li 4i i 2 3 4 cm
Liver
Regenerating
Liver
3
Fetal Liver
Fig. 3. Hexokinase isozyme patterns of normal, regenerating and fetal livers. Hexokinase isozyme bands were stained at 0.1 M glucose.
1446
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Hexokinase Isozyme Patterns of Experimental Hepatomas of
Rats
Shigeaki Sato, Taijiro Matsushima and Takashi Sugimura
Cancer Res 1969;29:1437-1446.
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