[CANCER RESEARCH 33, 2562 2567, November 1973]
Energy-linked Functions of Tightly Coupled Mitochondria
Isolated from Ehrlich Ascites Tumor Cells1
Rupert F. W. Thorne and Fyfe L. Bygrave
Department of Biochemistry, Faculty of Science, The Australian National University, Canberra, A. C. T., Australia
SUMMARY
Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells contain a latent adenosine triphosphatase
(ATPase) that is stimulated by a range of dinitrophenol
concentrations
up to 0.5 mM. Maximal uncoupler-stimulated ATPase activity is approximately 60% of that found
with rat liver mitochondria and is inhibited by oligomycin
and by atractyloside.
Rapid rates of ATPase activity accompany the accumu
lation of Ca2+ by ascites tumor mitochondria in an adeno
sine triphosphate-supported
system. In contrast to Ca2+,
Mg2+ is unable to induce ATPase activity in intact mito
to regulate
the movement
of Ca2+ within the tumor
cell
might also contribute to the imbalance between glycolysis
and respiration.
It is noteworthy that despite extensive
work on Ca2+ accumulation and release by mitochondria
isolated from a wide range of mammalian tissues (18), only
quite recently has it been seriously considered and shown
that this system could in principle serve to modify cytoplasmic metabolic activities sensitive to Ca2* (19). Else
where we have collated evidence in support of such a role
for mitochondria
in modifying the cellular ionic environ
ment (25, 26).
Thus from several points of view it seemed important to
study the interaction of Ca2+ with energy-linked reactions
chondria but can in sonically disrupted preparations.
Pro
longed incubation of the ascites mitochondria with Ca2+
does not lead to irreversible ATPase, swelling, or spon
taneous release of the accumulated Ca2+, events typical
of rat liver mitochondria.
in mitochondria from ascites tumor cells. Apart from any
other consideration,
no reports of this nature have been
made as yet with these cells. Our general approach has
been to examine and characterize the influence of Ca2'1" on
a variety of energy-linked
reactions involved either di
rectly or indirectly with the accumulation
or release of
Ca2+ by mitochondria,
particularly
in the light of ex
INTRODUCTION
periments carried out with mitochondria from rat liver.
In relation to the comparison that is made in this study
between mitochondria
isolated from Ehrlich ascites cells
and rat liver, the following should be borne in mind. Rat
liver mitochondria
are similar in most respects to other
mammalian mitochondria
that have been examined (13).
They have the added advantage that they have been most
thoroughly characterized especially in regard to their inter
action with Ca2+. Thus the comparison that we are making
For many years the energy metabolism of the cancer cell
has been the subject of intense biochemical investigation.
The Ehrlich ascites tumor in particular is known to exhibit
an unusually high rate of aerobic glycolysis in comparison
to normal cells. On the basis of this finding, Warburg (29)
proposed a theory of carcinogenesis in which the interac
tion of the carcinogen with the cell leads to an impairment
of respiration. The cell subsequently adopts a fermentative
mechanism to derive energy or it dies. According to this
theory, mitochondria
are the primary site of metabolic
damage to the cell. Of interest in this regard are reports
from several groups (14, 16, 20) that mitochondria isolated
from some strains of tumor cells fail to exhibit significant
uncoupler-stimulated
ATPase activity, a property very
characteristic
of mitochondria
obtained from different
types of "normal" tissues.
Several years ago one of us introduced the proposition
that Ca2+ might be a factor involved in the control of
metabolic activities such as glycolysis that are inhibited by
this ion (47). The suggestion was made that a relatively
low concentration of Ca2+ in the Ehrlich ascites tumor cell
here is between mitochondria characteristic of a mammalian
cell that has controlled respiratory and glycolytic activities
and mitochondria
isolated from a tumor cell whose ap
parent form of control over these same processes is differ
ent from that existing in normal cells.
In this communication we report our findings on the Ca2+
and uncoupler-induced
ATPase and some swelling char
acteristics of these tumor mitochondria.
As we have in
dicated elsewhere in a brief report (28), these reactions ex
hibit features which are not seen in the well-documented rat
liver mitochondria system. It is our view that these find
ings justify the need for further studies of this nature in
these tumor cells.
might contribute to its high rate of glycolysis. It has oc
curred to us that an impaired ability of tumor mitochondria
MATERIALS
'This study was supported by a grant from the Australian Capital
Territory Cancer Society.
Received March 29, 1973; accepted July 5, 1973.
2562
AND METHODS
The Ehrlich ascites tumor used was a hyperdiploid strain
supplied by Dr. E. L. French, Animal Health Research
CANCER
RESEARCH
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A TPase in Intact Ascites Mitochondria
Laboratories, Parkville, Victoria, Australia. Details of the
method of propagation are described elsewhere (5).
Cells (8- to 11-day tumor age) were harvested from the
peritoneal cavity of mice killed by cervical dislocation and
maintained in a beaker kept on ice. A reasonable yield of
mitochondria could be obtained from cells harvested from
6 to 8 mice (50 to 60 ml fluid).
The method of Wu and Sauer (31) with the Dounce
homogenizer was used, with minor modifications to pre
pare the mitochondria. All manipulations were carried out
at 0-4°.The cells were initially packed by low-speed centrifugation (500 x g) and the cell-free ascitic fluid was
drawn off with a vacuum aspirator and discarded. They
were then washed twice in 3 volumes of 0.25 M sucrose
containing the 1 mM EDTA (pH 5.7) by resuspension and
centrifugation at 500 x g. Care was taken when resuspending the cells not to disturb any packed erythrocytes. The
blood-free cells were then resuspended in 40 ml of the isola
tion medium and transferred to a Dounce tissue homoge
nizer (40-ml size) packed in ice. Rupture of the cells was ac
complished by 12 to 18 vigorous direct passes of the pestle
(Dounce "loose-fitting" pestle). At no stage of the prepara
tion was citrate present (cf. Réf.
31).
The resulting homogenate was then divided between four
15-ml Corex centrifuge tubes and centrifuged for 10 min
at 900 x g in a Sorvall RC-2B refrigerated centrifuge
using the SS-34 rotor. The supernatants were combined
and retained in a 40-ml centrifuge tube packed in ice. The
pellets were combined and resuspended into 60 ml with
isolation medium. The process of cell rupture and cen
trifugation were repeated, the homogenization being much
more gentle and requiring only 6 to 10 passes of the pestle.
The supernatants were again combined in a 40-ml centri
fuge tube and, together with the supernatant from the
1st homogenization, centrifuged for 10 min at 8000 x g.
The pellets were washed with a few ml of 0.25 M sucrose,
combined, and resuspended to about 3 ml using a small
glass Teflon homogenizer. This gave a yield of between
40 and 60 mg protein.
Rat liver mitochondria were prepared as described
elsewhere (25).
Protein Determination. Protein was assayed by a Biuret/
cyanide method (27).
Acceptor Control Ratios. The acceptor control ratio was
determined using a Clark-type oxygen electrode (22) fitted
to a Rikadenki 10-inch pen recorder.
Calcium Ion Movement. Movement of Ca2+ was fol
lowed using 45Ca2+; 0.1 ml of the incubation mixture was
applied to a Millipore filter (0.45-/Õpore size) and the
residue was rapidly washed with 2 ml 80 mM sucrose con
taining 16 mM Tris-HCl (pH 7.4). The filter was then
placed in a glass scintillation vial to which was added 10 ml
ethoxy ethanol scintillation fluid and counted in a Packard
Tri-Carb scintillation counter.
Mitochondria! Volume Changes. Volume changes oc
curring in mitochondria were followed by measuring the
changes in light absorbance at 520 nm using a Unicam
SP800 recording spectrophotometer.
Measurement of ATPase Activity. ATPase activity was
NOVEMBER
determined by measuring either change in pH or the appear
ance of inorganic phosphate in the incubation medium.
Proton movements were monitored by means of a
combination glass electrode fitted to an expanded scale
pH meter (Townson and Mercer, Sydney, Australia)
having a 100-mV recorder outlet (1.4 pH units), which
was in turn connected to a 10-inch Rikadenki recorder
(10 mV full-scale deflection). Under the conditions used,
changes in pH were a linear function of changes in proton
concentration. The apparatus was calibrated by the addi
tion of standard HC1.
When the appearance of phosphate was measured, 0.2 ml
of the incubation medium was placed in an Eppendorf cen
trifuge tube containing 1.0 ml ice-cold 10% trichloroacetic
acid. The mixture was shaken and allowed to stand on ice
for 10 min. After centrifuging in an Eppendorf bench cen
trifuge, 1 ml of the supernatant from this mixture was
removed and assayed for inorganic phosphate by the
method of Baginski et al. ( 1).
In control experiments the extent of H+ ejection was
compared with the appearance of inorganic phsophate
over a 10-min period with Ca2+ and with dinitrophenolstimulated ATPase activities. In the Ca2+-stimulated
system the ratio H* ejected to phosphate release was about
1.6/1.0 and in the dinitrophenol-stimulated system the
ratio was about 0.85/1.0. These values agree well with those
obtained by other workers (2). In the majority of experi
ments, ATPase activity was determined by measuring the
change in pH of the incubation system. Initial rates of ac
tivity were calculated by taking the tangent to the curve at
the point where Ca2+ or dinitrophenol was added to start
the reaction.
Materials. ATP was a product of C. F. Boehringer and
Soehne GmbH, Mannheim, Germany. All other reagents
were of analytic reagent grade. 45CaCl2 (specific radio
activity, 0.507 mCi/mmole) was a product of The Radiochemical Centre, Amersham, Bucks., United Kingdom.
RESULTS AND DISCUSSION
Ehrlich ascites tumor cells are characteristically robust
and possess a tough cell membrane. As a consequence,
relatively harsh procedures are required to disrupt them
[see, for example, Wu and Sauer (31)]. Because the prop
erties of mitochondria! ATPase are to a large extent de
termined by the integrity of the isolated mitochondria and
because harsh isolation procedures can cause mitochon
dria! damage, it was important to establish at the outset
of this study to what extent the mitochondria isolated
from Ehrlich ascites tumor cells in this laboratory were
"intact." The routine assay procedure adopted to test
for mitochondria! integrity was the polarographic mea
surement of the rates of states III and IV respiration and
the determination of the acceptor control ratio. The
polarographic trace shown in Chart 1 represents one of
many obtained with numerous mitochondrial preparations.
In this particular experiment the acceptor control and
ADP/oxygen ratios are 4 and 1.9, respectively. Our
1973
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2563
Rupert F. W. Thome and Fyfe L. Bygrave
ATPase in ascites tumor mitochondria were approximately
60% of that in rat liver mitochondria. These data indicate
that the influence of dinitrophenol concentration on ATPase
of ascites tumor mitochondria is quite different to that on
ATPase of well-differentiated hepatoma 7800 mitochon
dria, where dinitrophenol stimulates maximally at a con
centration of about 10 ¿IM
and above about 40 /¿Mhas
no effect whatsoever on ATPase activity (20).
Sensitivity of ATPase Activity in Ascites Tumor Mito
chondria to Atractyloside. Atractyloside is a potent and
specific inhibitor of the adenine nucleotide translocase
(3, 21, 30). This enzyme system, which permits the move
100 ngatoms
ment of adenine nucleotides across the inner membrane,
oxygen
has been observed in mitochondria isolated from a variety
of tissues. The influence of atractyloside on the dinitrophenol-stimulated ATPase of ascites and of rat liver mito
chondria is shown in Chart 3. ATPase activity in the tumor
1 run
mitochondria is clearly sensitive to the inhibitor with maxi
mal inhibition (in the presence of 1 mM ATP) occurring at
Chart I. Respiratory activity of mitochondria isolated from Ehrlich about 25 fiM; this compares with a value of about 6 ¿uM
ascites tumor cells. The reaction mixture contained 100 mM sucrose,
obtained for rat liver mitochondria. The high sensitivity of
50 mM KC1, 15 mM Tris-HCl (pH 7.4), 10 mM potassium phosphate,
ATPase to atractyloside thus provides good evidence for
2 mM MgCI2, I mM EDTA, and 10 mM succinate in a total volume of
2.0 ml. Mitochondria (100 /il, containing 3 mg protein) and ADP were the existence in these tumor mitochondria of an adenine
nucleotide translocase.
added as indicated. The temperature was 25°.
Influence of (a
on ATPase Activity in Mitochondria
Isolated
from
Ascites
Tumor Cells. A considerable body of
preparations generally exhibited acceptor control ratios information relating to
the interaction of Ca2* with mito
of between 3 and 5; those with values less than 3 were dis
chondria
can
be
obtained
from a buffered incubation sys
carded. Mitochondria that possess acceptor control ratios
tem containing ATP and Ca2^ and by measuring changes
of this high order of magnitude are regarded as being rea
in pH, movement of 45Ca2+, and changes in light scatter
sonably intact (see Refs. 8 and 9 and, for ascites mitochon
ing at 520 nm (see Refs. 11 and 23). The following events
dria, Ref. 24). Further evidence for mitochondrial integ
are known to occur with mitochondria isolated from rat
rity is presented below.
liver. Immediately after Ca2^ is added to the incubation
Effect of ATP and Dinitrophenol Concentrations on mixture, there is a phase of H+ ejection corresponding in
ATPase Activity in Mitochondria Isolated from Ehrlich
the main to the active accumulation of Ca2+ supported by
Ascites Cells. Since as far as we are aware the properties ATP hydrolysis (2). Once all of the Ca2+ has been accu
of mitochondrial ATPase have not been studied previously
mulated the rate of H+ ejection declines for a period the
in these tumor cells, it was essential to determine the con
length of which depends on the amount of Ca2" presented
centrations of ATP required for maximal activity. Data
from these experiments are shown in Chart 2/4 and are
200
200
compared with those obtained with rat liver mitochondria.
The initial rate of ATPase activity stimulated by 50 fM
dinitrophenol increases as the concentration of added ATP
is increased. Maximal activity is reached at about 0.8 mM
100
100
ATP with a half-maximal velocity at about 150 /uMATP;
these values are close to those found with mitochondria
from rat liver.
The 2nd set of data in Chart 2 shows the influence of
dinitrophenol concentration on ATPase activity. This ex
O
1230
50
XX)
periment was undertaken in the light of reports that unmM ATP
pM Dinitrophenol
coupler-stimulated ATPase activity in mitochondria iso
Chart 2. Influence of ATP and dinitrophenol concentrations on the
lated from a variety of hepatomas is considerably lower initial rates of ATPase activity in mitochondria isolated from rat liver and
than in those from rat liver (14, 16, 20). Our data (see ascites cells. The reaction mixture contained 80 mM sucrose, 16 mMTrisHCl (pH 7.4), and 4 mg mitochondrial protein (prol.) in a total volume of
Chart 2B) show that maximal stimulation of the ascites 3.0
ml at a temperature of 25°.In the experiment shown in A, 50 ¿IM
ATPase occurs at about 50 ¿¿M
and levels off at higher
dinitrophenol was present with varying concentrations of ATP as indicated
concentrations. In other experiments it was seen that In the experiment shown in B, 3 mM ATP was present with varying con
ATPase activity in the tumor mitochondria in the pres centrations of dinitrophenol as indicated. The rate of H* ejection was
ence of 500 and 100 ¿/M
dinitrophenol, respectively, was measured as described in "Materials and Methods." The reactions were
approximately equivalent. The data of Chart 2, A and B, started by the addition of dinitrophenol. •¿
mitochondria from rat liver;
show also that maximal rates of uncoupler-stimulated
O, mitochondria from ascites tumor cells.
Mitochondria
2564
CANCER RESEARCH
VOL. 33
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A TPase in intact Ascites Mitochondria
a characteristic shape which depends on whether initial or
final phase ATPase activity is measured (11). The shape
for initial ATPase, i.e., that corresponding to ATP-supported Ca2+ accumulation, is sigmoidal while that for the
irreversible ATPase is hyperbolic. It is of particular rele
vance to the ionic control hypothesis (7, 19) that the ini
tial rate of Ca2* accumulation measured as a function of
Ca2+ concentration is also sigmoidal (11, 12, 26).
The 2 experiments whose data are presented in Chart 6
were carried out to compare the response of the tumor mito
chondria ATPase to Ca2* with that described above for rat
20
30
50
pM Atractyloside
Chart 3. Influence of atractyloside concentration on the uncouplerstimulated ATPase of rat liver and ascites tumor mitochondria. The reac
tion was carried out as described in Chart 2 but with 50 ¿¿M
dinitrophenol,
1 mM ATP, and 6 mg mitochondrial protein present. The concentration
of atractyloside was varied as indicated. •¿,
mitochondria from rat liver;
O, mitochondria from ascites tumor cells.
initially to the mitochondria. During this lag period it
appears that changes in the membrane occur since what
follows is a 2nd phase of rapid H* ejection corresponding
to irreversible ATPase activity (11, 23), release of the ac
cumulated Ca2 +, and swelling of the mitochondria. All of
the events are shown in the experiments indicated in
Charts 4 and 5.
Data in Charts 4B and 5 (continuous line) also show the
events which occur when ascites mitochondria are examined
in a similar system. The pattern of events obtained is dis
tinctly different. The initial phase of rapid H* ejection
(corresponding to accumulation of the Ca2+) is evident.
However, the lag phase of apparent inactivity continues
for up to 15 min without resulting in any release of Ca2+
from the mitochondria. At the same time, the mitochon
dria do not undergo any swelling. Neither the addition of
phosphate nor incubations for up to 30 min in the presence
of 900 ¿IM
Ca2+ resulted in release either of the Ca2+ or
of Ca2+-induced swelling.
The apparent absence in the ascites mitochondria of a
mechanism that brings about a spontaneous release of ac
cumulated Ca2+ led to a consideration of whether uncouplers of oxidative phosphorylation are able to induce
such a release. It is well established that energy is required
by mitochondria to maintain accumulated Ca2+ (15) and
that uncouplers release this Ca2+ by collapsing the
"energy pressure" in the membrane. Data from experi
ments to test this (see Chart 4B) showed that the addition
of carbonyl cyanide w-chlorophenylhydrazone to mito
chondria that have accumulated Ca2+ induce a rapid re
lease of the Ca2+. Thus these mitochondria like other
mammalian mitochondria require energy in order to main
tain the accumulated Ca2+.
Influence of Ca2+ Concentration on ATPase Activity in
Ascites Mitochondria. With rat liver mitochondria, the
curve relating ATPase activity to Ca2+ concentration has
NOVEMBER
si
I ,
o
i
n
B
o
see
Tim«(min)
Chart 4. pH changes associated with ATP-supported Caz* accumu
lation in mitochondria isolated from rat liver and from ascites tumor
cells. The reaction mixture contained 80 mM sucrose, 16 mM Tris-HCl
(pH 7.4), 3 mM ATP, and 4 mg mitochondrial protein. The total volume
was 3.0 ml and the temperature was 25°.The reaction was started by the
addition of "Ca2+, which was added at the concentrations indicated.
At the times shown a sample of the incubation mixture was removed and
the mitochondria were assayed for 45Ca2+ content as described in
"Materials and Methods." A. data obtained with rat liver mitochondria;
B, data obtained with ascites mitochondria. Continuous lines represent
pH traces obtained after the additions of 600 (A) and 900 (B) nmoles
"Ca2*, respectively. A, 45Ca2* in rat liver mitochondria; O, 45Ca2*
in ascites mitochondria. At the point indicated with an arrow in B, 2 ¿tM
carbonyl cyanide m-chlorophenylhydrazone was added to the incubation
mixture.
o(SinCt>M$00.2OÂ0.6Îi iii'i1\\
_
0
10
20
Time (min )
Chart 5. Absence of Ca2*-induced swelling in mitochondria isolated
from ascites tumor cells. The reaction mixture contained 80 mM sucrose,
16 mM Tris buffer (pH 7.4), 3 mM ATP, and 4 mg mitochondrial pro
tein in a final volume of 3.0 ml. The temperature was 25°.At the point
indicated by the arrow 400 nmoles of Ca2* were added.
, trace ob
tained with rat liver mitochondria;
, trace obtained with ascites
tumor mitochondria.
1973
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
2565
Rupert F. W. Thome and Fyfe L. Bygrave
200
300
100
nmotes Co*'
.2» added / mg protein
Chart 6. Effect of Ca2' concentration on initial and final rates of
Ca2+-stimulated ATPase activity in mitochondria from ascites tumor
cells. The reaction mixture was the same as that described in Chart 2 but
with 3 mM ATP and varying concentrations of Ca2+ as indicated in a
final volume of 3.0 ml. The rate of H ' ejection in the initial and final
phases was measured as described in "Materials and Methods." •¿
data
to stimulate the latent ATPase in the untreated mito
chondria but does stimulate in the sonically disrupted
preparations. Dinitrophenol and Ca2^ on the other hand
induce maximal stimulation of ATPase in the untreated
mitochondria. Moreover, NADH that is normally not
oxidized by intact mitochondria was similarly not oxi
dized by our preparations of ascites mitochondria (data
not shown). These observations together further es
tablish that the untreated preparations contained a very
high proportion of intact mitochondria (see also Ref. 17).
Besides providing further evidence for the intactness of
the mitochondria used in this work, these data also sug
gest that the permease in ascites mitochondria respon
sible for the accumulation of Ca2+, like that in rat liver
mitochondria (18), is able to discriminate against Mg2+.
CONCLUSIONS
Several facets of the data obtained in this study are
potentially relevant to the biochemistry of the tumor cell.
First, true uncouplers such as dinitrophenol induce
significant rates of ATPase in intact, tightly coupled
liver mitochondria. The stimulus/response curve for the mitochondria isolated from Ehrlich ascites tumor cells.
initial phase of ATPase activity is clearly sigmoidal with Although the maximal rates were only about 60% of
a rate of saturating Ca2+ concentrations of about 160 those obtainable with rat liver mitochondria, they are
nmoles H+ ejected per min per mg protein. The maxi
very much greater than those found in tightly coupled
mal ATPase activity in the final phase on the other hand hepatoma mitochondria. Second, Ca2+ can be accumu
represents only a small fraction ofthat in the initial phase. lated by ascites tumor mitochondria in a system sup
This situation contrasts sharply with that for rat liver ported by ATP. The rates of the concomitant ATPase ac
mitochondria where the maximal rates for each phase tivity approach but do not reach those obtained in the rat
are about 250 and 160 nmoles H+ ejected per min per liver system. Third, Ca2+ is unable to induce either an
mg protein for the initial and final phases, respectively. irreversible ATPase or swelling of ascites tumor mito
Influence of Disruptive Procedures on Reactivity of chondria. As a consequence, spontaneous release of Ca2+
ATPase from Ascites Mitochondria. Earlier in this work from these mitochondria also does not take place in vitro.
we argued on the basis of acceptor control values that
Although any interpretation of these findings in relation
the preparations of mitochondria used in this work were to the metabolism that typifies the ascites tumor cell is
intact. The following points seen in the data presented in difficult to make at this stage, there is the hint that these
Table 1 confirm this view. First, Mg2+ has little ability mitochondria will accumulate but not so readily release
Ca2+. If one assumes that the events we have seen in
Table 1
vitro also occur in vivo then this would have the effect in
Effect of disruptive treatments on interaction of ascites mitochondria
the simplest sense of increasing the Mg2+/Ca2+ ratio in
with A TPase-inducing agents
the cytoplasm and thus of allowing Ca2+-sensitive meta
The reaction mixture was as described in Chart 2 with 3 mM ATP and
270 /¿M
Ca2* or Mg2*, 50 /IMdinitrophenol, and 5 ¿igoligomycin pres
bolic processes located therein to proceed at faster (un
controlled) rates (19). Studies on Ca2+-induced respiratory
ent as indicated.
activities in these organdÃ-es(Ref. 28; R. F. W. Thome and
ATPase activity (nmoles H*
F. L. Bygrave, manuscript in preparation) support this
ejected/min/mg protein)
view.
calculated from the 1st experiment: O, data calculated from the 2nd ex
periment, pro!., protein.
dis
rupted mitochon
dria
chondriaLiver9
fromAscites10 fromLiver
AdditionsNone
Ca2*
Mg2'
Dinitrophenol
Dinitrophenol plus
oligomycinControl
"Not measured.
2566
We are grateful to Dr. E. L. French for the provision of the initial cells
used in this work.
Ascites18
112
9
120
9mito-
75
56
116
10
60
101
10Sonically
ACKNOWLEDGMENTS
25
53
75
48
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A TPase in Intact Ascites Mitochondria
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NOVEMBER
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
2567
Energy-linked Functions of Tightly Coupled Mitochondria
Isolated from Ehrlich Ascites Tumor Cells
Rupert F. W. Thorne and Fyfe L. Bygrave
Cancer Res 1973;33:2562-2567.
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