The Hexose Monophosphate Shunt in Glucose
Catabolism in Ascites Tumor Cells
CHARLESE. WENNER, JOHN H. HACKNEY,ANDFRANCISMOLITERNO
(Department of Experimental Biology, Roswett Park Memorial Institute, Buffalo 3, N.Y.)
In the neoplastia cell there are at least two
major pathways for the utilization of carbohy
drates—the Embden-Meyerhof glycolytic path
way, which is quantitatively the more important
(27), and the hexose monophosphate shunt (1,
17, 27). The present study was carried out to
determine the physiological role of the hexose
monophosphate shunt in ascites tumor cells and
the factors which control its operation. Recent
evidence has suggested that the hexose monophosphate shunt provides both intermediates and
reducing capacity in the form of TPNH1 for
reductive synthesis (cf. review in 13, 15, 18).
This paper lends strong supporting evidence for
the latter concept of the function of the hexose
monophosphate shunt. The capacity of intact tu
mor cells to produce TPNH can be demonstrated
by the addition of artificial electron acceptors
which markedly stimulate a TPN-dependent oxi
dation of carbon-1 of glucose. The present study
affirms the idea that the TPNH is utilized for
reductive synthesis by a demonstration of an
anaerobic oxidation of carbon-1 of glucose to car
bon dioxide, which can be stimulated to its aerobic
level by the addition of a physiological electron
acceptor such as pyruvate.
The effect of glucose concentration on COz
formation via the hexosemonophosphate shunt
was studied, since Racker (22) had found that
high glucose concentrations stimulated carbon-1
oxidation by Ehrlich ascites cells. However, the
most important rate-limiting step in the operation
of this pathway that was found was the oxidation
of TPNH.
MATERIALS
AND METHODS
Tumors.—The mouse tumors used in this study are those
described in Table 1. Source references and details of most
of these tumors have been listed by Hauschka et al. (11, 12).
'Abbreviations used: DPN and TPN = oxidized diphospho- and triphosphopyridine nucleotide, respectively; DPNH
and TPNH = reduced diphospho- and triphosphopyridine
nucleotide, respectively; ATP = adenosine triphosphate; DNP
= dinitrophenol; Q = ¿»liters/rag
dry wt/hour.
Received for publication June 5, 1958.
Incubation of tissue.—The tumor cells were removed from
the peritoneal cavity 7-10 days after implantation, at which
time significant growth had occurred. The cell suspensions
were centrifuged at 1000 X g for 10 minutes at 5°C., resuspended in calcium-free Ringer phosphate (0.1 M, pH 7.4),
and centrifuged again. The initial sediments of lymphoma and
K2D ascites cells were resuspended free of the lower red
blood cell pellet. Two or three repetitions removed the erythrocyte contamination. Cell suspensions were adjusted to contain
200 mg. of packed cells/ml (approximately 25 mg. dry weight)
and dispersed with a very loose-fitting Potter-Elvehjem homogenizer which did not break the cells. For most experiments,
1.0 ml. was added to 2.0 ml. of calcium-free Ringer phosphate
solution in a Warburg flask containing substrates and factors
as required. Tissue slices were made with a Stadie-Riggs
slicer. Approximately 200 mg. of cells (fresh weight) were
then added to a final volume of 3.0 ml. of Ringer phosphate
containing substrates and factors in Warburg vessels.
For broken-cell preparations, the following methods were
used: Solid tumors derived from hyperdiploid Ehrlich (EL)
ascites cells were homogenized in a Potter-Elvehjem glass
homogenizer. Ascites cells, after being washed with calciumfree Krebs-Ringer buffer, were homogenized in either of two
ways. For the experiments reported in this paper, the cells
were homogenized in the Servali Omni-Mixer at full speed
at 3°C. for 3 minutes, at which time few intact cells remained.
Since homogenization by this method breaks up the nuclei
to a considerable degree, it was of interest to compare the
properties of the soluble fraction obtained by a more gentle
procedure. Similar results to those reported here were obtained
with the soluble fraction obtained by a method in which
a 75 per cent recovery of nuclei was obtained. This technic
is a modification of the procedure of Lamanna and Malette
as described in Methods and Enzymology for the rupture
of yeast cell suspensions (3). The ascites cells were disintegrated
through mechanical agitation in the presence of grade 10
glow beads (60-80 mesh, 200 m^ average diameter, obtained
from Minnesota Mining and Manufacturing Co., Minneapolis).
Eighteen to 20 gm. of glass beads were placed in the Servali
Omni-Mixer with 10 ml. of a 10 per cent cell suspension
in isotonic sucrose. The cells were then disrupted for a period
of 2 minutes at a rotor speed of 5000 r.p.m., at which speed
the rotor knife blades did not break up the cells in the absence
of the beads. When the nuclei obtained by this procedure
were examined under the phase microscope, very little cytoplasmic contamination was present.
Differential centrifugation of the homogenate
was carried out in the following manner. The
ascites cells, homogenized in 5 volumes of ice-cold
calcium-free Krebs-Ringer solution, were centri
fuged at 20,000 X g for 20 minutes at l°-3°
C.
in the Servali refrigerated centrifuge, and the
1105
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
1106
Vol. 18, October, 1958
Cancer Research
supernatant
was decanted (supernatant
I). Fur
ther centrifugation
of supernatant
I was carried
out at 40,000 X g for 50 minutes in the Spinco
Preparative Model L centrifuge, and the decanted
supernatant
was designated as supernatant II.
The gas phase was air for the cell suspensions
and 100 per cent oxygen for the tissue slices.
After equilibration for 10 minutes at 38°C., sub
strate was tipped in, and the flasks were shaken
for 1 hour unless designated otherwise. Dilute
sulfuric acid was then tipped in. Liberated CO?
was trapped in the center well and isolated at
the end of the reaction as BaCOs following the
addition of 0.6 millimoles of carrier Na2C03. The
BaCOj was counted in "infinitely thick" layers
with a Micromil thin-window counter.
tions were made as described previously
Calcula
(27).
Preliminary
experiments
were carried out in
which the incorporation of C14 of glucose, labeled
in positions 1 or 6, into the respiratory
COZ
was measured. Carbon-6 oxidation was relatively
unaffected
by glucose concentration.
Carbon-1
oxidation was found to be stimulated by increasing
the glucose concentration
from 0.001 to 0.03 M,
provided the incubation period was at least 15 min
utes. The stimulation was insensitive to malonate,
further evidence that the increased C-l oxida
tion was due to a stimulation of the initial enzymes
of the hexose monophosphate
shunt pathway.
However, no stimulation was observed for incu
bation periods of less than 15 minutes.
The lack of stimulation
of C-l oxidation at
the short incubation periods suggested that per
haps at the lower glucose concentrations
the rate
TABLE1
DESCRIPTION
OFMOUSE
ASCITES
TUMORS
STUDIED
Ascites tumors:
Anaplastic carcinomas:
Hyperdiploid Ehrlich (EL)
Hypotetraploid Ehrlich Clone 2 (E2)
Hypotetraploid Krebs-2 Clone D (K2D)
Lymphomas:
A #1 lymphoma
6C3HED lymphosarcoma
DBA/2 lymphoma
P288 lymph node leukemia
Sarcomas:
MC1M (fibrosarcoma)
Solid tumors:
Hyperdiploid Ehrlich (EL) carcinoma
Host strain
Routine serial
passage in:
Ha/ICR Swiss
Ha/ICR Swiss
Ha/ICR Swiss
Females
Males
A/Ha
C3H/St
DBA/2
DBA/2
Females
Females
Females
Females
C3H,/He
Males
Ha/ICR Swiss
Males
Males
Lactic acid was determined enzymatically
by
the method of Horn and Bruns (14); pentose
and sedoheptulose
were assayed by the use of
the orcinol reagent (6, 28); and glucose was de
termined by the anthrone method (24) .2
of COi formation via the hexose monophosphate
shunt was linear for only a short time. Therefore,
the effect of glucose concentration
on carbon-1
oxidation was measured in the presence of 0.017 M
malonate, which completely inhibits oxidation of
carbon-6. Under these conditions, carbon-1 oxida
RESULTS
tion is assumed to represent COt formation via
Effect of glucose concentration on hexose monothis pathway. As seen in Chart la, the initial
phosphate shunt.—In view of the observation of rate of oxidation of carbon-1 of glucose by the
Racker (22) that the ratio of C-l/C-6 oxidation
hyperdiploid Ehrlich ascites cells was independent
of glucose concentration
in the range 5 X IO"3 M
of glucose by Ehrlich ascites cells is dependent
to 5 X 10~4 M. However, a decline in the rate
on the concentration of glucose, we first examined
the controlling effect of glucose concentration
on of oxidation of carbon-1 was observed at the
the rates of COz formation via the alternate oxi- lower glucose concentrations,
which can be ex
dative pathway by ascites tumor cells.
plained by the rapid initial disappearance
of glu
1Uniformly labeled glucose (glucose-U-C14) and glucose- cose, as plotted in Chart Ife. The glucose which
2-C" were obtained from H. S. Isbell of the National
Bureau of Standards, and glucose-6-C" and glucose-1-C14 disappeared could be accounted for as lactic acid,
although some lactic acid was found to disappear
were purchased from the Volk Radiochemical Company. Lactic
acid dehydrogenase was obtained from Worthington Biochemi
after the first 10 minutes of reaction. Thus, it
cal Company. Malonate and dinitrophenol were recrystallized
would appear that the operation of the alternate
from commercial preparations. Phenazine methosulfate, pyripathway is not limited by glucose concentrations
dine nucleotides, and 2X-crystalline yeast alcohol dehydrogen
above 5 X 10~4 M except under conditions where
ase were obtained from the Sigma Chemical Company.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
WENNER et al.—Glucose Catabolism in Ascites Tumor Cells
the substrate is removed by the glycolytic en
zymes.
An experiment with less tissue was carried
out to determine the effect of a wide range of
glucose concentrations on the rate of glycolysis
and CÛ2 production via the shunt. Since this
experiment, described in Table 2, was carried
out for very short incubation periods in which
oxidation of carbon-6 was negligible, it was found
unnecessary to add malonate to determine COs
formation via the shunt. The rate of oxidation
of carbon-1 of glucose, which is assumed to be
derived entirely from the shunt, was independent
of substrate concentrations as low as 2.5 X 10~5M.
Thus, the limitation in hexose monophosphate
1107
experiment; i.e., for glycolysis, 0.17 jumóleglucose
was utilized per 10 minutes of incubation, and,
for carbon-1 oxidation, only 0.005 /amolé
was uti
lized per 10 minutes.
As will be described in another section, pyruvate
stimulates oxidation of carbon-1 of glucose by
acting as an electron acceptor. This introduces
the complication that the glucose concentration
o
O
X 150.005M/
/
u
o
co
oc
/0.0025M
10-
u
o
_J
o
O.OOIM
5-
0.0005M
IO
20
TIME (MINUTES)
¿C
O
IO
20
30
TIME (MINUTES)
CHAKT16.—Ascitescells (200 mg. fresh weight) were incu
bated in calcium-free Krebs-Ringer phosphate buffer for the
designated time at 87.8°C. with air as the gas phase. Malonate
was present in a final concentration of 0.017 M. Hexose monophosphate shunt decarboxylation is considered to be equiva
lent to the amount of carbon-1 oxidation in this system.
shunt decarboxylation cannot be attributed to
a low substrate affinity. The rate of glycolysis
was also maximal at low substrate concentrations,
although at somewhat higher concentrations than
2.5 X 10~6 M. Despite the higher concentrations
might influence the shunt by its effect on the
pyruvate level. To avoid this complication, the
effect of glucose was measured under conditions
in which the concentration of electron acceptor
was not such a crucial factor. That is, in the
presence of méthylène
blue, which is a more ef
fective electron acceptor than pyruvate, the rate
of carbon-1 oxidation was still found to be inde
pendent of substrate at concentrations as low
as 2 X IO-6 M.
necessary for optimal rate, the glycolytic enzymes
have a marked competitive advantage, as seen
by a comparison of the optimal velocities in this
Effect of artificial electron acceptors on oxidation
of glucose-C1* by ascites cells.—Since glucose con
centration did not limit the initial rate of COs
CHABTlo.—Effect of time and substrate concentration on
hexose monophosphate shunt decarboxylation by hyperdiploid
Ehrlich ascites cells. The molarities represent initial concen
trations of glucose-1-C14.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
1108
Vol. 18, October, 1958
Cancer Research
production via the shunt, the possibility that the
electron transport system was a rate-limiting step
was examined by the use of artificial electron
acceptors. Méthylène
blue, phenazine methosulfate, and 2-methyl-l,4-naphthoquinone were found
to stimulate respiration from two- to five-fold,
but the most marked effect of these substances
was the preferential stimulation of oxidation of
carbon-1 of glucose. As seen in Experiment I,
Table 3, there was a minor enhancement of carbon6 oxidation by the addition of méthylène
blue,
but oxidation of carbon-1 was stimulated seven
fold. Furthermore, the stimulation of carbon-1
oxidation was malonate-insensitive,
indicating
that the stimulation might be attributed to the
initial reactions of the hexose monophosphate
shunt. The most effective electron acceptor was
phenazine methosulfate, which gave a 15- to 30fold stimulation of oxidation of carbon-1 by the hyperdiploid and hypotetraploid Ehrlich and Krebs2D ascites cells.
The manifold stimulation of carbon-1 oxidation
by dyes suggests that one of the rate-limiting
steps for the reactions of the shunt may be the
oxidation of TPNH. Therefore, it was of in
terest to examine the effect of 2-methyl-l,4naphthoquinone, which has been shown to oxidize
TPNH more rapidly than DPNH (7). As seen
in Experiment 5, this compound markedly in
creased carbon-1 oxidation but had no effect on
the oxidation of carbon-6, suggesting that it stimu
TABLE 2
lated the hexose monophosphate shunt solely.
These electron acceptors also were found to
EFFECTOFGLUCOSE
CONCENTRATION
ONAEROBIC
GLYCOLYSIS
ANDCO2FORMATION
VIATHEHEXOSE
MONO- stimulate the oxidation of carbon-2, which ap
PHOSPHATE
SHUNTPATHWAY
peared to be related to the extent of enhancement
Flasks containing 10 mg. of EL ascites cells (fresh tissue of carbon-1 oxidation. Since carbon-2 can give
weight) were equilibrated for 10 minutes at 38°C. in calciumrise to carbon-1 if recycling of the pentose cycle
free Krebs-Ringer phosphate, after which procedure glucoseoccurs, it is presumed that the electron acceptors
1-C" was added from the side arm. Flasks of each glucose con
centration were then incubated for 5, 10, and 15 minutes. The
stimulate the pentose cycle. Evidence compatible
values recorded in this table represent the rate for the 10with the stimulation of the recycling process was
minute period, at which time the velocity was linear.
obtained by the assay of pentose and sedoheptuGlucose
lose in the experiments with méthylène
blue. There
concentration
Lactic acid
Glucose-C14 to C14Os
(Final molarity)
(/imoles)
(/¿atoms)
was no increase of either sugar (measured by the
1.0X10-«
0.34
0.005
orcinol reaction) in the presence of méthylène
2.5X10-»
0.32
0.005
5.0X10-4
0.30
0.004
blue. The lack of accumulation of these inter
2.5X10-'
0.30
0.004
mediates
suggests that reactions subsequent to the
1.0X10-4
0.26
0.004
initial dehydrogenations were not rate-limiting.
5.0X10-»
0.20
0.005
2.5X10-5
0.16
0.005
To determine whether the stimulatory effects
TABLE 3
EFFECTOFARTIFICIAL
ELECTRON
ACCEPTORS
ONGLUCOSE-C"
OXIDATION
BYINTACTASCITES
CELLS
are basedphenazine
on tissue methosulfate
used (200 mg.(3fresh
weight).2-methyl-l,4-naphthoquinone(l
The final concentrations were as
(0.01 M),
méthylène
blueValues
(7 X IO-1M),
X 10~4M),
X follows:
10~4M), glucose
dinitrophenol
(1 X
10-'M).
*
EL
30
1
EXPERIMENTno.:
EL
Iff
ASCITES:
TIKE IMI-,. :
Méthyl
ène
Blue
ADDITION:
Méthyl
ène
Blue
None
10
Phenaphenol zinefimoles
3.9
Glucose carbon
Total
C-l
C-2
C-6
3.6
6.6
8.8
zineconsumed
of oiygen
1.0
0.5
3.04E210Phena 3.4
Dye
1.7
S
K2D
SEL10Dinitro-
Phenazme
None
2-Me-l,4-Naphthoquinone
0.75
2.9
1.5
/latoms of glucose carbon to COi
0.9
2.4
0.10
0.61
0.19
0.35
0.05
0.10
1.2
3.0
4.3
0.19
1.2
0.35
0.09
0.24
0.22
0.29
4.28
0.06
1.68
0.02
0.79
0.004
0.09
1.23
3.83
0.10
1.58
0.03
0.99
0.03
0.02
0.37
1.40
3.40
0.08
1.06
0.70
0.05
0.78
0.24
0.02
0.18
0.03
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
WENNER et al.—Glucose Catabolism in Ascites Tumor Cells
of the dyes could be attributed
to an uncoupling
of phosphorylation
rather than their serving as
electron acceptors, carcinoma cells were incubated
with 1 X 10~6 M dinitrophenol
in the presence
of 0.01 M glucose-C14 labeled in positions 1 or
<5.As seen in Experiment 2, Table 3, no preferen
tial stimulation
of carbon-1 was observed, but
oxidation of both carbons-1 and -6 was stimulated
to the same extent, suggesting that a symmetrical
cleavage of glucose and oxidation via the citric
acid cycle occurred. Thus it would appear that
the stimulatory
effects observed with electron
acceptors could not be attributed to an increased
availability of phosphate or phosphate acceptors.
Localization of a TPN-dependent
hexose monophosphate shunt decarboxylative activity in the soluble
1109
6-phosphogluconic
dehydrogenase in the superna
tant fraction of tissues from solid neoplasms (9).
As seen in Table 5, no oxidation of carbon-1
by the hyperdiploid
Ehrlich ascites particulatefree supernatant
could be demonstrated
unless
pyridine nucleotides were present. When glucose6-phosphate served as substrate, there was appre
ciable oxygen consumption when TPN was added.
However, no oxygen consumption
was observed
if DPN was added unless ATP was also present.
Since TPN has been shown to be synthesized from
DPN and ATP in mammalian
systems (20), it
is conceivable that formation of TPN by this
system permits appreciable oxygen consumption.
Preliminary experiments indicate that a DPN kinase is present in the soluble fraction.
TABLE 4
INTRACELLULAR
DISTRIBUTIONOF HEXOSE MONOPHOSPHATESHUNT DECARBOXYLA
TIVE ACTIVITYOF HYPERDIPLOIDEHRLICH ASCITESCELLS
The following substances were in the designated final concentrations: MgSOj, 3 X 10~3M; cytochrome c, 4 X 10-* M; potassium chloride, 0.14 M; phosphate buffer, pH 7.4, 6 X IO"3M; TPN,
3 X 10-«
M; yeast hexokinase, 330 K.M. units at 25°C.; ATP, 2 X lp~3 M; glucose, 0.01 M;phenazine methosulfate, 2 X 10""*M; and 0.6 ml. of the tissue suspension in calcium-free Krebs-Ringer
buffer representing 120 mg. of tissue (fresh weight). The volume was brought to 1.6 ml., and the
cells were incubated for 20 minutes with air as the gas phase at 38°C. The values are based on
time of incubation per tissue used. Hexokinase increased C-l oxidation of supernatant by 20%.
MATOMP OF GLUCOSE
CAKBON
TO COl*
Total
C-I
C-i
FRACTION
Oz UPTAKE
Whole cells
3.9
3.35
1.86
0.12
Homogenate
4.85
1.80
0.43
4.4
Supernatant I
2.79
1.81
0.07
4.0
Supernatant II
4.2
3.10
1.96
0.17
Part ¡culate
0.4
0.10
* Headings for separate columns indicate position of radioactive label in substrate used.
fraction.—Disruption
of the cell membrane per
mitted further study as to the localization and
the establishment
of a TPN-dependence
of the
stimulation of carbon-1 oxidation by phena/ine.
Preliminary experiments with 0.25 M isotonic su
crose as the homogenization
medium indicated
that carbon-1 was oxidized primarily by the sol
uble fraction of the ascitic homogenate. Further
distribution studies were carried out with isotonic
salt solution as suspending medium. As seen in
Table 4, appreciable oxidation of glucose by the
hyperdiploid Ehrlich ascites homogenate was ob
served with supplements of phenazine methosul
fate, pyridine nucleotides, and ATP.8 The rate
of oxidation of glucose carbon-1 by the homog
enate preparation
was similar to that observed
in the intact cells. Furthermore,
all the activity
of the homogenate
could be accounted for by
the soluble fraction. It is presumed that the lo
cus of hexose monophosphate
shunt decarboxylation is the nonparticulate
cytoplasmic fraction,
which is in agreement with the compartmentation of glucose-6-phosphate
dehydrogenase
and
C-6
0.05
0.09
0.04
0.05
Further data which are compatible with the
TPN specificity for C-l oxidation are also shown
when glucose-1-C14 serves as substrate. Since ATP
is required for the hexokinase reaction, addition
of pyridine nucleotides alone does not permit
optimal oxidation of carbon-1. Therefore, a study
of the concentrations
of DPN or TPN required
to give optimal oxidative activity was made in
the presence of ATP. TPN was more effective
in stimulating hexose monophosphate
shunt decarboxylation
than DPN, since a greater conver3It is somewhat surprising that carbon-6 oxidation was
as rapid in the soluble portion as in the whole cells. However,
this finding is not necessarily contradictory to the established
fact that mitochondria are involved in C-6 oxidation. There
is a significant time lag for incorporation of C14from carbon-6
of glucose into the respiratory CO2 by the intact ascites cell;
this can probably be attributed to the dilution of the radio
active intermediates by the numerous intermediary metabolites
involved in citric acid cycle oxidation. In view of the short
incubation period in this experiment, only a relatively small
amount of C-6 oxidation is observed. This oxidation can prob
ably be attributed to hexose monophosphate shunt decarboxylation by a randomization of the isotope, e.g., resynthesis
of hexose from symmetrical 3-carbon units.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
TABLE 5
TPN-DEPENDENCE
FOR GLUCOSE OXIDATION BY EL ASCITES SUPERNATANT II
The reaction mixture was essentially the same as described in Table 4, except that hexokinase was not present and nucleotides were added as specified below. The values are based
on the time of incubation (1 hour). Substrate concentrations: glucose-6-PO«
and glucose-1-C",
0.01 M each.
SUBSTBATÕ: GLUCOSE-e-PO,Oxygencon
ADDITIONS
DPN
(«)
ATP
(u)
1to
sumed(/¿moles)000.88.28.6GLUCOSB-I-C"Oiygencon
sumed(pinoles)00411.93.33.3GlucosecarbonCOj(/¿atoms)00.041.380.871.511.53
TPN
(M)
0.001
0.0014
0.001
0.001
0.0014
0.00014
0.0014
0.0014
0.00014
0.001
0.001
TABLE 6
ANAEROBICFORMATIONOF CO2 IN ASCITESCELLS*
GAS
PHAS*
ASCITES
Total
GLUCOSE
CARBON
TOCOat
(iiatoms/hour/flask)
C-I
OÃ-
C-«
6C3HED lymphoma
Nt
0.20
0.19
0.01
0.01
Air
0.58
0.30
0.07
0.07
K2D carcinoma
NI
0.22
0.17
0.003
0.02
Air
1.53
0.45
0.17
0.15
Hyperdiploid (EL) carcinoma
Nj
0.19
0.10
0.003
0.004
Air
1.55
0.25
0.11
0.10
MC1M fibrosarcoma
Nj
0.37
0.20
0.05
0.06
Air
4.04
0.79
0.57
0.44
DBA/2 lymphoma
Nt
0.25
0.15
0.007
0.02
Air
0.62
0.29
0.06
0.13
P288 lymph node leukemia
Nt
0.38
0.24
0.02
0.02
Air
1.42
0.48
0.22
0.19
* Each flask contained 100 mg. (wet wt.) ascites cells in 3 ml. calcium-free Krebs-Ringer phos
phate buffer. After 10 minutes' equilibration, glucose was added from the side arm to a final con
centration of 0.01 M.
For anaerobic experiments, Linde High Purity Nitrogen (specified 99.99 per cent), passed
through three successive solutions of alkaline anthroquinone-hydrosulfite (8), was bubbled through
the medium before the experiment and flushed through the flasks for 10 minutes after they were
on the manometers. The subsequent 10-minute equilibration period provided added assurance that
traces of oxygen would be consumed by respiration before the addition of labeled glucose. The re
action was stopped after 60 minutes of incubation at 38°,during which interval no measurable
respiration occurred. CO2trapped in the center well was counted as BaCO. at infinite thickness.
t Headings for separate columns indicate position of radioactive label in substrate used.
TABLE 7
EFFECT OFPYRUVATEON GLUCOSEOXIDATION BYASCITESCELLSAND BY MOUSELIVER
SLICESUNDER AEROBICAND ANAEROBICCONDITIONS
Experimental conditions are the same as described in Table 6 except that in the experiment with mouse liver 200 mg. of
tissue slices (fresh weight) were used.
TISSUE
K -1 ' carcinoma
GAB
PHASE
Ns
Air
PYBOVATB
(0.01 M)
-
GLUCOSE
CABBON
TOCOj*
OXYGEN
C-ÃŽ
C-6
Total
UPTAKE
(fiatoms per hour per flask)
(timóles)
0.120.390.320.410.150.370.290.370.010.020.170.190.0020.040.120.100.010.040.060.050.010.020.100
C-l
0.32
0.12
0.10
1.07
0.29
0.06
0.13
0.62
3.4
3.7
DBA/2 lymphoma
Air
3.0
3.0
Liver
N,
Air
0.17
0.05
0.25
+
0.19
0.03
0.48
* Headings for separate columns indicate position of radioactive label in substrate used.
8.7
9.8
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
WENNERe¿
al.—GlucloseCatabolism in Ascites Tumor Cells
sion of glucose carbon-1 to COj was observed
by the addition of TPN at a concentration of
0.0001 M. Thus, it would appear that oxidation
of carbon-1 is TPN-dependent in this system.
In a similar manner, the distribution and TPN
dependence of the carbon-1 oxidative system was
studied with a solid Ehrlich tumor derived from
the hyperdiploid Ehrlich ascites. Carbon-1 oxida
tion by a Potter-Elvehjem homogenate of this tu
mor could also be attributed to a TPN-dependent
oxidative system localized in the nonparticulate
cytoplasmic fraction.
Further evidence in support of the TPNdependent oxidation of carbon-1 of glucose by the
supernatant fraction was obtained spectrophotometrically. No measurable glucose-6-phosphate
dehydrogenase activity was observed if DPN
served as coenzyme under the conditions of assay
described by De Moss (5). However, when TPN
served as coenzyme, the rate of TPNH production
by the supernatant fraction was QTrNH= 24
at 30°C. Thus, C02 formation by way of the
hexose monophosphate shunt is apparently not
limited by this dehydrogenase. Since hexokinase
increases the phenazine-stimulated C-l oxidation,
it may limit the enhanced decarboxylation.
Effect of oxygen on glucose-C1*oxidation.—Since
the in vitro limitation imposed on the oxidation
of TPNH may be overcome in vivo by a number
of reductive syntheses which require TPNH as
the electron donor, it is important to know the
extent to which oxygen is used as an electron
acceptor for the hexose monophosphate shunt.
In Table 6 are described the results of studies
carried out with a number of ascites tumors on
the rates of oxidation of the different carbon
atoms of radioactive glucose to carbon dioxide
under anaerobic and aerobic conditions. These
studies have revealed that, in all the ascites cells
examined, there is an appreciable anaerobic for
mation of carbon dioxide from glucose.4 There
was a significant oxidation of carbon-1 of glucose,
which accounted for the major portion of the
glucose carbon to CO2. The oxidation of carbon-1
proceeded anaerobically at about 10—40per cent
of the aerobic rate and accounted for approxi
mately 55-90 per cent of the total glucose carbon
oxidized to COS. The anaerobic formation of car
bon dioxide can probably be attributed to the
presence of endogenous substances which can act
as electron acceptors for TPNH oxidation.
As seen in Table 7, the addition of 10~2Msodium
4Although the rate of anaerobic formation of carbon dioxide
is small in relation to the oxygen consumption (S-10 per cent),
it may in part account for respiratory quotients greater than
1 observed by previous investigators (2, 19).
lili
pyruvate in the medium resulted in a threefold
stimulation of the anaerobic oxidation of glucose
carbon-1 by the Krebs-2 carcinoma and DBA/2
lymphoma.6 It is presumed that pyruvate might
serve as an electron acceptor for the TPNH gen
erated by the initial enzymes of the hexose monophosphate shunt. It is striking that pyruvate
stimulates carbon-1 oxidation more than does oxy
gen, and in the presence of pyruvate oxygen
does not increase carbon-1 oxidation. The failure
of pyruvate and oxygen to stimulate additively
suggests that intermediary metabolites such as
pyruvate may serve as the principal electron ac
ceptors for the hexose monophosphate shunt in
ascites cells and that the role of oxygen in this
pathway may be to favor the accumulation of
such intermediates.
The stimulation of carbon-1 oxidation by the
addition of pyruvate under anaerobic or aerobic
conditions was also observed with three other
mouse ascites tumors, namely, the MClM fibro
sarcoma, 6C3HED lymphoma, and the hyper
diploid Ehrlich (EL) carcinoma. Thus, it appears
that the stimulatory effect of pyruvate as well
as the anaerobic formation of COa is a general
property of ascites tumors.
It was also of interest to study anaerobic C-l
oxidation of glucose by mouse liver slices, a nonneoplastic tissue which has hexose monophosphate
shunt activity. As seen in Table 7, no significant
anaerobic CÜ2production from carbon-1 of glu
cose was observed in the presence or absence
of pyruvate. Thus, the anaerobic oxidation which
is observed with neoplastic tissues is not associated
with all tissues having hexose monophosphate
shunt activity.
Coupling of hexose monophosphate shunt dehydrogenases with lactic acid dehydrogenase.—The
mechanisms by which pyruvate might serve as
an electron acceptor are manifold. Of the possible
considerations, TPNH might be reoxidized via lac
tic acid dehydrogenase; or by the TPN-dependent
malic enzyme, which could account for the anaero
bic COj fixation into pyruvate observed by Crane
and Ball (4) for ox retina; or by enzymes producing
propanediol phosphate from pyruvate (10); or
by transhydrogenase as an alternate potential
mediator of DPNH oxidation. Malic enzyme did
not seem to be involved, since the addition of
'An unexpected stimulation of C-6 oxidation was also
observed by the addition of pyruvate, which should be a
competitive substrate for that coming from glucose-6-C14.
It is conceivable that this enhancement of the relatively
minor C-6 oxidation might also be attributed to hexose monophosphate shunt decarboxylation if randomization of isotope
occurred, e.g., the resynthesis of hexose from glycolytic inter
mediates.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
1112
bicarbonate was not required for maximal rate
of carbon-1 oxidation in the presence of pyruvate.
The most likely explanation of the stimulatory
effect of pyruvate on carbon-1 oxidation is that
the TPN-dependent dehydrogenases of the shunt
are linked with the conversion of pyruvate to
lactate. Evidence for a TPN-linked lactic acid
dehydrogenase can be demonstrated under the
experimental conditions in which a stimulation
of glucose carbon-1 oxidation by pyruvate was
observed. As shown in Table 8, the soluble fraction
of ascites cells catalyzes a rapid oxidation of
TABLE 8
OXIDATION
OFTPNH BYPYRUVATE
CATALYZED
BYEL ASCITES
SUPERNATANT*
MHOLSS AK I Ml 3 Horns'
INCUBATION
PrBIDINE
NUCLKOTIDE
Vol. 18, October, 1958
Cancer Research
Net Amóles
pyridine
nucleotide
oxidized
0.90
1.3
INITIAL HATFop
OXIDATION
Net Amóles
lactate
produced
0.80
1.3
OK
PYRIDINE
NUCLEOTIDE
Qoiid.
PNt
TPNH
36
DPNH
940
TPNH+DPN
2
*The reaction vessels contained ascitic supernatant II
(equivalent to 30 mg. of fresh weight of ascites cells) in 3.4 ml.
of calcium-free Ringer phosphate buffer containing 0.01 M py
ruvate. The oxidation of the reduced pyridine nucleotide was
measured by determining the change in optical density at 340
m/¿
at 30°C. in the Gary spectrophotometer. The reaction was
started by the addition of 0.9 /¿moles
of TPNH or 1.8 /¿moles
of DPNH to the sample compartment, and stopped by the
addition of trichloroacetic acid for assay of lactic acid. For ini
tial rate studies, less tissue was used to observe rates which
were linear with respect to enzyme concentration. The rate of
TPNH and DPNH oxidation in the absence of pyruvate was
negligible.
tQ oxid. PN refers to /¿Iof pyridine nucleotide oxidized/mg dry wt/hour.
TPNH by pyruvate. The oxidation of TPNH
is accompanied by an almost stoichiometric pro
duction of lactic acid, which is indicative that the
reaction is due to lactic acid dehydrogenase.6
The enzyme appears to be similar to the TPNlinked lactic acid dehydrogenase of the soluble
fraction of rat liver as described by Navazio
et al. (21). The initial rate of TPNH oxidation
by the tumor enzyme ranged from l/25th to
l/40th the rate of DPNH oxidation, a somewhat
higher relative activity than for the liver enzyme
studied at the same pH. As has also been observed
with the liver preparation, TPNH oxidation was
markedly inhibited by the addition of DPN.
The possibility was considered that the oxida
tion of TPNH was catalyzed by a DPN-specific
lactic dehydrogenase mediated by transhydrogen6This finding might also explain the observation of Kit
(17) that, in the presence of fluoride and pyruvate, the oxida
tion of carbon-1 of glucose by the Gardner and Ehrlich
tumors under aerobic conditions was stimulated.
äse.In order to test for the presence of trans
hydrogenase in the soluble fraction, an experiment,
described in Table 9, was carried out in which
a DPN-specific enzyme, alcohol dehydrogenase,
and its substrate, acetaldehyde, were added to
the supernatant in the presence of TPNH. One
would expect a catalysis of the oxidation of TPNH
if transhydrogenase were present. However, no
oxidation of TPNH was observed under conditions
in which pyruvate was reduced by TPNH. Fur
thermore, transhydrogenase could not be detected
in the supernatant by an assay involving the
oxidation of TPNH by DPN in the presence
of acetaldehyde and alcohol dehydrogenase. There
fore, it is presumed that the oxidation of TPNH
by pyruvate is catalyzed by a TPN-linked lactic
acid dehydrogenase.
Although the enzyme catalyzes a much slower
oxidation of TPNH than of DPNH, it is present
in the ascites supernatant with a capacity to
TABLE 9
ABSENCE
OFTRANSHYDROGENASE
INEL
ASCITES
SUPERNATANT*
ADDITIONS
Substrate
Pyruvate
Alcohol
dehydrogenase
-Hacetaldebyde
-
A OPTICALDENSITY
MINUTEXÕOMG.
FRESHWEIGHT
DPNH
TPNH
0.023
0.011
7.5
0.180
+
3.6
0.015
+
0.170
Pyruvate
+
0.011
DPN
* The reaction vessels contained ascitic supernatant H
(equivalent to 20 mg. fresh weight or less) in a total volume
of 1.1 ml. of calcium-free Krebs-Ringer phosphate buffer.
Additions as described above were made to both reference and
sample cells except that pyridine nucleotide was not added to
the reference cells. The reaction was started by the addition of
reduced pyridine nucleotide to the sample compartment, and
the decrease in optical density at 340 m/¿at 30°C. was re
corded. For the extremely rapid oxidations of DPNH, the ini
tial reaction rate was measured with less tissue, and the value
represented above was calculated for the equivalent of 20 mg.
of tissue from the initial reaction velocity. Concentrations
used:
0.03 M;
alcoholanddehydrogenase
acetalde
hyde, pvruvate,
0".003M; DPN,
DPNH,
TPNH, 1.2 Xand
10~*M.
oxidize TPNH which is more than sufficient to
satisfy the stoichiometric requirements of carbon-1
oxidation. Thus, since two molecules of TPNH
must be oxidized for each molecule of CO2 formed
via the shunt, a QOIM.TPN
= 36 would permit
a rate of CO2 formation from carbon-1 of glucose
equivalent to a Q value of 18. However, the highest
rate of CO2 production from carbon-1 of glucose
by the intact ascites cell in the presence of pyru
vate had a Q value of 1, which is far below
its potential. The rate of formation of TPNH
is equivalent to a Q of 24 as measured either
spectrophotometrically with the ascites superna
tant or by carbon-1 oxidation in the phenazine-
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WENNERet al.—GlucoseCatabolism in Ascites Tumor Cells
1113
transhydrogenase in the soluble fraction of the
tumor cells. Furthermore, Reynafarje and Potter
(23) have reported that TPN-cytochrome c reduc
tase as well as transhydrogenase is virtually absent
in the Novikoff hepatoma.
The availability of electron acceptors, however,
cannot be considered as the only rate-limiting
step in the operation of the hexose monophosphate
shunt in ascites cells. If the mechanism by which
pyruvate stimulates is via the TPN-linked lactic
acid dehydrogenase, consideration of the relative
concentrations of TPNH and DPN must be made.
Since the intracellular concentration of TPNH
is low with respect to DPN, the presence of
DPN in the soluble fraction could readily exert
a regulatory effect on TPN-dependent dehydrog
enases.
Although an anaerobic oxidation of carbon-1
of glucose could not be demonstrated with mouse
liver slices in the presence or absence of pyruvate,
these properties are not unique to the neoplastic
tissues. Kinoshita (16) has reported an anaerobic
DISCUSSION
oxidation of carbon-1 of glucose which could be
This study points out that the competitive
stimulated by pyruvate beyond the aerobic level.
advantage of glycolysis over the shunt pathway
Furthermore, Dr. Leonard Cohen has independ
for glucose utilization by neoplastic ascites cells ently observed this phenomenon in the retina
cannot be attributed to a limitation in substrate
of the 5-day-old rabbit.7
supply. Judging from the marked stimulation of
A possible alternate function of the hexose
carbon-1 oxidation by artificial electron carriers monophosphate shunt in ascites cells is the syn
observed in the present experiments, a more likely thesis of ribose-5-phosphate for nucleic acids and
rate-limiting factor in the in vitro operation of coenzymes. The hexose monophosphate shunt
the hexose monophosphate shunt is the availability
pathway would be a very direct pathway for penof a hydrogen acceptor, in which case the capacity tose formation involving oxidative decarboxyl
of the hexose monophosphate shunt to generate ation of glucose-6-phosphate. However, from our
reduced TPN would exceed the rate of oxidation results, which have been described in a prelim
of TPNH. This is in agreement with the sugges
inary report (26), it seems that these tumor cells
tion (15,18) that the hexose monophosphate shunt synthesize ribose-5-phosphate predominantly by
may function to provide TPNH for directing a C-3, C-2 condensation, presumably from transspecific reductive syntheses.
ketolase and transaldolase reactions.
Evidence compatible with this suggestion is
SUMMARY
obtained by the demonstration of the shunt under
Examination of the rate-controlling factors in
anaerobic conditions, when endogenous substrates
could serve as oxidants for TPNH. The marked the hexose monophosphate shunt pathway in as
stimulation of anaerobic glucose decarboxylation
cites tumor cells was made by studying the incor
by pyruvate provides an example for TPNH oxi poration of C14 of glucose—labeled uniformly or
in carbons-1, -2, and -6—into the respiratory
dation by fermentation intermediates.
Since oxygen does not increase glucose carbon-1 CÛ2 under varied conditions. A study of the
oxidation in the presence of pyruvate, it is pre
effect of glucose concentration on the operation
sumed that intermediary metabolites serve as the of the alternate pathway in a hyperdiploid Ehrlich
principal electron acceptors for the hexose mono- ascites tumor revealed that the initial rate of
phosphate shunt and that oxygen favors the CO2 production by the shunt was independent
accumulation of suitable electron acceptors for of substrate concentration in the range of 2.5 X
TPNH. That oxygen is not directly involved IO"6 M tO 1 X IO"2 M.
in the oxidation of TPNH is also suggested by
Artificial electron acceptors such as méthylène
our failure to observe appreciable activity of TPN7L. IL Cohen and W. K. Noell, Glucose Oxidation in the
cytochrome c reductase in homogenates and of Developing Retina (in preparation).
stimulated intact cell. Therefore, the failure of
pyruvate to stimulate carbon-1 oxidation by the
intact cell to the level of the soluble fraction
cannot be attributed to dehydrogenases as ratelimiting steps. The most likely explanation for
the lack of realization of the full capacity of
carbon-1 oxidation by the addition of pyruvate
is that the intracellular level of DPN is sufficient
to cause an inhibition in the oxidation of TPNH
by pyruvate. As has been shown in Table 8,
equimolar concentrations of DPN inhibit marked
ly TPNH oxidation. Furthermore, the addition
of DPN has also been found to cause a marked
inhibition of C-l oxidation by pyruvate catalyzed
by the EL ascites supernatant. Thus, in addition
to the availability of an electron acceptor such
as pyruvate which might limit the operation of
the shunt, the relative concentrations of DPN
and TPNH would also appear to be important
in the regulation of TPN-dependent dehydrogenations.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
1114
Cancer Research
Vol. 18, October,
1958
blue, menadione, and phenazine methosulfate
stimulated carbon-1 oxidation from six- to 30-fold
with only a slight stimulatory effect on the oxida
tion of carbon-6, suggesting that one of the ratelimiting factors in the operation of the alternate
pathway is the availability of the electron trans
port system. This stimulation was also observed
with homogenates of ascites cells when a source
of TPN was supplied. This TPN-dependent oxidative system for carbon-1 was localized in the sol
uble fraction, which also contained a lactic acid
dehydrogenase that catalyzed the oxidation of
TPNH by pyruvate.
A significant oxidation of carbon-1 of glucose
by intact tumor cells was observed under anaerobic
•conditions.
The rate of oxidation of carbon-1 was
stimulated by pyruvate to that observed in the
presence of oxygen. Oxygen did not increase car
bon-1 oxidation by the intact cells in the presence
of moderate pyruvate levels, indicating that inter
mediary metabolites such as pyruvate may serve
as the principal electron acceptors for the hexose
monophosphate shunt. From the data, it is con
cluded that the prime function of the hexose
monophosphate shunt is to provide reduced triphosphopyridine nucleotide for specific reductive
syntheses.
lism in Mammalian Tissues and Tumors. Biochem. J.,
66:171-75,1954.
10. GHOTH,D. P.; LEPAGE, G. A.; HEIDELBERGER,
C.; and
STOESZ,P. A. Metabolism of Pyruvate in Tumor Homog
enates. Cancer Research, 12:529-34, 1952.
11. HAUSCHKA,
T. S. Cell Population Studies on Mouse Ascites
Tumors. Trans. New York Acad. Se. S. II, Vol. 16:64-73,
1953.
12. HAUSCHKA,T. S., and FURTH, J. The Pathophysiology
and Immunogenetics of Transplan table Leukemia. Proc.
Henry Ford International Symposium, The Leukemias:
Etiology and Pathophysiology, pp. 87-120. New York:
Academic Press, 1957.
13. HORECKEH,B. L., and HIATT,H. II. Pathways of Carbo
hydrate Metabolism in Normal and Neoplastic Cells.
New Eng. J. Med., 258:177-84, 1958.
14. HORN, H. and BRUNS,F. H. Quantitative Bestimmung
von l(+)-Milchsäure mit Milchsäuredehydrogenase. Biochim. & Biophys. Acta, 21:378-80, 1956.
15. KAPLAN,N. O.; SWARTZ,M. N.; FRECH, M. E.; and
CIOTTI,M. M. Phosphorylative and Nonphosphorylative
Pathways of Electron Transfer in Rat Liver Mitochondria.
Proc. Nat. Acad. Se., 42:481-87, 1956.
16. KiNosHiTA,J. H. The Stimulation of the Phosphogluconate
Oxidation Pathway by Pyruvate in Bovine Corneal Epi
thelium. J. Biol. Chem., 228:247-53, 1957.
17. KIT, S. The Role of the Hexosemonophosphate Shunt
in Tumors and Lymphatic Cells. Cancer Research, 16:
70-76, 1956.
18. KREBS, H. A. Considerations Concerning the Pathways
of Syntheses in Living Matter. Bull. John Hopkins Hosp.,
96:19-23, 1954.
19. KUN, E., TALALAY,P., and WILLIAMS-ASHMAN,
H. G.
Studies on the Ehrlich Ascites Tumor. I. The Enzymic
and Metabolic Activities of the Ascites Cells and the
ACKNOWLEDGMENTS
Ascitic Plasma. Cancer Research, 11:855-63, 1951.
The authors are indebted to Dr. T. S. Hauschka for
20. MEHLEB,A. H.; KORNBERG,
A.; CRISOLA,S.; and OCHOA,
furnishing the animals bearing the tumors used in this study,
S. The Enzymatic Mechanism of Oxidation-Reductions
and to Dr. L. H. Cohen for helpful suggestions.
between Malate or Isocitrate and Pyruvate. J. Biol. Chem.,
174:961-77, 1948.
REFERENCES
21. NAVAZIO,F.; ERNSTER,B.; and ERNSTER, L. Studies
on TPN-linked Oxidations. II. The Quantitative Sig
1. ABRABAM,S.; HILL, R.; and CHAIKOFF,I. L. Concerning
1'athways of Glucose Utilization in Mouse Liver and
nificance of Liver Lactic Dehydrogenase as a Catalyzer
of TPNH Oxidation. Biochim. & Biophys. Acta, 26:
Hepatoma. Cancer Research, 15:177-80, 1955.
416-21,1957.
2. BLOCH-FRANKENTHAL,
L., and WEINHOUSE,S. Metabolism
22. RACKER,E. Carbohydrate Metabolism in Ascites Tumor
of Neoplastic Tissue. XII. Effects of Glucose Concen
Cells. Ann. N.Y. Acad. Sc., 63:1017-21, 1956.
tration on Respiration and Glycolysis of Ascites Tumor
23. RETNAFARJE,B., and POTTER,V. R. Comparison of TransCells, Studied with Carbon-14 Labeling. Cancer Research,
hydrogenase and Pyridine Nucleotide-Cytochrome c Rc17:1082-90,1957.
ductase Activities in Rat Liver and Novikoff Hepatoma.
S. COLOWICK,
S. P., and KAPLAN,N. O. General Preparative
Cancer Research, 17:1112-19, 1957.
Procedures. Methods in Enzymology I, p. 69, 1955.
24. SEIFTER,S.; DAYTON,S.; Novic, B.; and MUNTWYLEH,
E.
4. CRANE,R. K., and BALL, E. G. Relationship of C14OZ
Estimation of Glycogen with Anthrone Reagent. Arch.
Biochem., 25:191-200, 1950.
Fixation to Carbohydrate Metabolism in Retina. J. Biol.
Chem., 189:269-76, 1951.
25. WEINHOUSE,S. Studies on the Fate of Isotopically Labeled
5. DE Moss, R. D. Glucose-6-phosphate and 6-PhosphoMetabolites in the Oxidative Metabolism of Tumors. Can
cer Research, 11:585-91, 1951.
gluconic Dehydrogenases from Leuconostoc mesmleroides.
Methods in Enzymology, 1:328-32, 1955.
26. WENNER,C. E.; HACKNET,J.; and HERBERT,J. Pathways
of Glucose Metabolism in Ascites Cells. Proc. Am. Assoc.
0. DRURT, H. F. Identification and Estimation of Pentoses
Cancer Research, 2:259, 1957.
in the Presence of Glucose. Arch. Biochem., 19:455-66,
27. WENNER,C. E., and WEINHOUSE,S. Metabolism of Neo
1948.
plastic Tissue. IX. An Isotope Tracer Study of Glucose
7. ENOLAHD,S., and STRECKER,II. J. Oxidation of Reduced
Catabolism Pathways in Normal and Neoplastic Tissues.
Pyridine Nucleotides in Brain. Fed. Proc., 15:248, 1956.
J. Biol. Chem., 222:399-414, 1956.
8. FIESER, L. Experiments in Organic Chemistry, ad ed.,
28. WOOD.W. A., and SCHWERDT,R. F. Carbohydrate Oxi
p. 393. Boston: Heath, 1950.
dation by Pseudomonas fluorescens. II. Mechanism of
Hexose Phosphate Oxidation. J. Biol. Chem., 206:625-35,
9. CLOCK,G. E., and McLEAN, P. Levels of Enzymes of
the Direct Oxidative Pathway of Carbohydrate Metabo
1954.
Downloaded from cancerres.aacrjournals.org on July 31, 2017. © 1958 American Association for Cancer Research.
The Hexose Monophosphate Shunt in Glucose Catabolism in
Ascites Tumor Cells
Charles E. Wenner, John H. Hackney and Francis Moliterno
Cancer Res 1958;18:1105-1114.
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