[CANCER RESEARCH 30, 2223-2230,
August 1970]
Metabolic-independent Volume Changes and Mg++ Binding in
Mitochondria Isolated from AH-130 Yoshida Ascites
Hepatoma1
Francesco Feo and Antonio
Matli
Institute of General Pathology, University of Turin, Turin, Italy
SUMMARY
Mitochondria
isolated from AH-130 Yoshida ascites
hepatoma appear spontaneously
swollen. In these mito
chondria, the metabolic-dependent
swelling induced by
phosphate and the metabolic-independent
swelling induced
by digitonin occur to a very small extent. In the swollen
mitochondria,
evident
shrinkage
may beATP.
induced
adding
Mg"1"1"
ions in the
absence
of exogenous
This by
shrinkage
has been determined by spectrophotometric
as well as by
water content and packed volume measurements.
The shrinkage, which occurs in metabolizing and in
metabolically inhibited mitochondria, is not reversed either
by diluting the external Mg++ concentration in the mitochondrial suspension or by washing the mitochondria after
incubation in the presence of the bivalent cation.
At the same time as the shrinkage, there occurs a passive
uptake of Mg++ by tumor mitochondria. There is evidence
that a fraction of the Mg++ taken up is firmly bound to the
mitochondria! surface. Both the Mg
binding and the
Mg++-induced shrinkage have some common features, chiefly
their sensitivity to inhibition by KC1 present in the medium.
Our findings suggest that the Mg+ -induced shrinkage in
tumor mitochondria is related to the irreversible binding of
the bivalent cation to the membranes of the mitochondria.
INTRODUCTION
The mitochondria isolated in isotonic sucrose from AH-130
Yoshida ascites hepatoma appear spontaneously swollen (11,
12). This spontaneous swelling may be in part responsible
for the inability of tumor mitochondria to undergo extensive
volume changes in hypotonie conditions or in conditions
that
induce
large-amplitude,
metabolic-dependent,
or
metabolic-independent
swelling in normal liver mitochondria
(11). In the latter mitochondria,
it is not possible to
counteract the swelling extensively by addition of Mg++
ions, in the absence of exogenous ATP; however, in tumor
'The present investigation has been aided by a grant from the
National Research Council of Italy.
Received October 27, 1969; accepted April 23, 1970.
mitochondria,
appropriate amounts of Mg++ induce the
complete reversal of the swelling by an oligomycin-insensitive
mechanism (11, 12).
The Mg++-induced shrinkage is nonosmotic in nature (12)
and is unrelated, in its rapidity and its response to metabolic
inhibitors, to the well-known shrinking effect of ATP plus
Mg++ (18).
The shrinkage caused by Mg++ on mitochondria from
ascites hepatoma is paralleled by the uptake of the bivalent
cation. The hypothesis has been advanced that this uptake is
responsible for the shrinkage (12). In this paper, experi
mental evidence is presented in support of this hypothesis.
MATERIALS AND METHODS
Cells from Long-Evans rats inoculated 5 days previously
with AH-130 Yoshida ascites hepatoma were rapidly collected,
separated from ascitic fluid by centrifugation, and chilled in
ice. The tumor cells were freed of erythrocytes
by
suspending them for 30 sec in cold, doubly distilled water;
the isotonic conditions were restored by the addition of
appropriate amounts of l N NaCl.
The cells were centrifuged at 10,000 X £m¡n,and the
packed sediment was weighed and transferred in a PotterElvehjem homogenizer, adding at the same time 1 to 2 ml of
the isolation medium (0.29 M sucrose-0.02 M Tris-Cl, pH
7.4-0.001 M EDTA-1 /oo bovine serum albumin). They were
then homogenized with 3 strokes at 1,500 to 2,000 rpm; the
homogenate was diluted to 20% (w/v) and centrifuged at
6,000 X gm¡n. The sediment was rehomogenized, and the
homogenate was diluted to 40%, then centrifuged at 10,000
X £min together with the supernatant of the first centrif
ugation. The sediments of the latter centrifugation were
washed once at 6,000 X £m¡n.
From the collected supernatants, the heavy mitochondria were sedimented at 35,000
X gmjn and washed twice with the isolation medium free of
EOT A.
Through this isolation procedure, which represents an
adaptation of the method used by De Duve et al. (9) for
liver mitochondria, we obtained tumor mitochondria of the
same degree of purity as liver mitochondria.
This was
ascertained by measuring the activity of cytochrome oxidase,
NADPHicytochrome
c reducÃ-ase, and acid phosphatase as
markers for, respectively, mitochondria, microsomes, and
lysosomes (9, 22).
AUGUST 1970
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2223
Francesco Feo and Antonio Matli
The metabolic-dependent
swelling induced by phosphate
(P., Tris salt) and the metabolic-independent
swelling
induced by DGT2 were measured at 25°in 2 ml of reaction
mixture containing 0.29 M sucrose-0.02 M Tris-Ci, pH 7.4.
For determination of the degree of the P¡-inducedswelling, 5
mM Tris-succinate and 2 pM rotenone were added to the
basal medium. For the determination of the DGT-induced
swelling, the substrate was omitted and antimycin A, 1
Mg/ml, was added together with the rotenone. The effect of
various concentrations of KC1 on the swelling as well as on
the shrinkage were studied by substituting osmotically
equivalent amounts of KC1 to sucrose in the reaction
mixture.
The spectrophotometric
measurements
were
performed at 520 HIMin a Beckman DB spectrophotometer
connected with a recorder and a scale expander.
For the determination of the total and extramitochondrial
water content, the mitochondria (5 to 7 mg of protein) were
suspended in 2.5 ml of the aforementioned reaction mixture
containing, together with the substrate or inhibitors, 274 Mg
of dextran-carboxyl-14C
(New England Nuclear Corp.,
Boston, Mass., 2.0 X IO6 dpm/mg) and 25 pi of 3H20 (New
England Nuclear, 7.2 X IO4 apm/pì). The mixture was
stratified on 1.6 ml of silicone oil (p = 1.06) in small plastic
tubes, on the bottom of which 0.4 ml of 1.6 M HC104 was
placed (17). For determination of the swelling, at zero time
5 mM PJ or 200 MM DGT were rapidly and carefully mixed
into the mitochondrial suspension, with the aid of a plastic,
flat-bottomed
rod. After 20 min at 25°, shrinkage was
induced by adding to some samples 20 mM Mg++.
Immediately after the last addition, the tubes were centrifuged in a Spinco-Beckman Model L centrifuge (50SW rotor)
by running the rotor to 30,000 rpm and then stopping. Once
the mitochondria were separated from the reaction mixture,
the aqueous layer was removed by a Pasteur pipet, the walls
were dried, and the silicone layer was removed by hexane
(30). The total and extramitochondrial
water content were
determined on the basis of the specific radioactivities of the
2 isotopes in the supernatant and of the total radioactivity
in the acid extracts of the sediments. The radioactivity
measurements
were performed
in the Mark-1 liquid
scintillating computer (Nuclear-Chicago Model 6860) with
the use of the Bray's scintillating fluid (6).
3 times with
the same medium.
Mg"1"1"was determined,
according to the method of Orange and Rhein (21), in the
acid extracts of the pellets; corrections were made for
extramitochondrial
Mg"1"1"in unwashed samples. In some
experiments, the determination of Mg"1"1"
uptake after short
incubation was performed in a system analogous to that used
for the water content measurements. For incubation times
shorter than 60 sec, an adaptation of the "layer filtration
method" (17) was adopted as in a previous research (12).
For corresponding amounts of added Mg"1"1",the Mg"1"1"
uptake, corrected for the extramitochondrial bivalent cation,
was the same in all procedures used.
Proteins were determined by the biuret procedure (13).
RESULTS
Correspondence between Absorbance and Water Content
Changes. It has been previously observed (12) that, in
mitochondria isolated from the ascites hepatoma cells, the
absorbance changes induced by P¡or Mg"1"1"
correspond to
changes of the intramitochondrial
water content. These
results have now been extended
to the metabolicindependent swelling induced by DGT by using the double
labeling technique for evaluating the intra- and extramito
chondrial water content, in place of the less accurate method
of the wet-dry weights measurements. The results are com
pared to those of the packed volume determinations.
As is shown in Table 1, the Mg"1"1"
addition to mitochondria
incubated with the swelling agent induces, independently of
the presence of metabolism, evident shrinkage, as is proved
by the absorbance increments on one hand and by the water
content and packed volume decrements on the other. In
general, there is a good agreement between the 3 parameters
of the mitochondrial volume. We have observed, however, a
discrepancy regarding the packed volume of the mito
chondria incubated with DGT. In fact, the packed volume
determinations seem to indicate a greater amount of DGT
swelling than that obtained with P¡.This does not accord with
the results of both water content and spectrophotometric
determinations. This could depend, however, on a difference
between the sedimentation patterns of DGT-treated mito
Packed volumes were measured by centrifuging at 270,000
chondria and of those swollen in the presence of P¡.
X £min,in small cytocrits; the mitochondria suspension was
The ascites hepatoma mitochondria, freshly isolated and
incubated under the same conditions used for the spectro
suspended in medium free from swelling and shrinking
photometric and water content experiments.
agents, contain 3.6 pi of water per mg of protein (Table 1).
For determination
of the metabolic-independent
Mg"1"1"
The dry weight/protein ratio for these mitochondria is about
uptake, the mitochondria (5 mg of protein) were incubated
1.7 (1.5 to 1.8). This value, corrected for the weight of
at 25° in 3 ml of reaction mixture containing 0.0016 to
solutes in the adherent supernatant fluid, falls to 1.55. It
0.038 M MgCl2, 0.02 M Tris-Cl, pH 7.4; 2 MM rotenone;
may be calculated, on the basis of the corrected value, that a
antimycin A, 1 Mg/ml; and sufficient amounts of sucrose to water content of 3.6 Ml/mg of protein corresponds to 2.32
bring the osmolarity to 0.33. After 3 min of incubation, the Ml/mg of dry weight, a figure slightly higher than that
suspension was diluted 3 times with Mg+"l"-containing
obtained in previous gravimetric determinations (2.05 Ml/mg
medium or Mg"l"l"-freemedium and centrifuged at 150,000 X
dry weight, cf. Réf.12). This water content of the tumor
£min.Samples diluted with Mg"l"l"-freemedium were washed
mitochondria is about 1.5 times higher than the one found,
2The abbreviation used is: DGT, digitonin.
2224
CANCER RESEARCH VOL. 30
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Volume Changes and Mg++ Binding in Mitochondria
Table 1
Mg++-induced reversal of the metabolic-dependent swelling supported by P¡
and the metabolic-indepent swelling supported by DOT
"Metabolizing" conditions were obtained by adding to the basal medium (0.29 M sucrose plus 0.02 M
Tris-Cl, pH 7.4) 5 mM Tris-succinate and 2 fiM rotenone. "Inhibited" conditions were obtained by adding
antimycin A, 1 Mg/ml, in place of the substrate. The spectrophotometric determinations were made at 520
mM in 2 ml of mitochondrial suspension (500 to 600 Mgprotein/ml). Water content was determined by
suspending the mitochondria (2.8 mg protein/ml) in 2.5 ml of doubly labeled basal medium containing
substrate and inhibitors, as in the spectrophotometric experiments. The double labeling was obtained by
the addition of 3H2U and dextran-carboxy-14C. After incubation with the swelling agent and Mg"1"1",
the
reaction was stopped by centrifugation through silicone. Water content was determined on the acid
extracts of the pellets by isotopie measurements, as described under "Materials and Methods." Packed
volumes were measured by centrifuging in small cytocrits the mitochondria (4 mg protein) at the end of
incubation in 1 ml of medium containing the swelling agents and Mg++. The swelling started in all
instances by the addition of P¡or DGT to the reaction mixture. After 20 min. in some samples Mg++ was
added. At the end of an additional 2 min of incubation, absorbance, water content, and packed volumes
(PK) were determined. Temperature, 25°C.
volume
(cu mm/mg)5.856.803.335.807.623.24A/>K+0.95-3.47+1.82-4.3
/Metabolizing
Conditions
mitochondria5
P¡5
mM
Mg++5
mM P¡+ 5 mM
Mg"1""1"Inhibited
mM P¡+ 20 mM
mitochondria200
DGT200
MM
Mg"1""1"200
MMDGT + 5 mM
MMDGT + 20 mM Mg++l/mg.33.12.65.72.33.121.581.75AA-0.21+0.53+0.60-0.21+0.46+0.63H20(Ml/mg)3.604.001.943.603.802.32AH20+0.40
in the same experimental conditions, in liver mitochondria
(12).
Once tumor mitochondria have undergone Mg++-linked
shrinkage, the lowering of concentration
of the bivalent
cation in the medium does not readily release the shrinkage.
As shown in Table 2, the Mg++ addition to DGT-swollen
mitochondria
induces a nearly complete reversal of the
swelling. When the Mg++ concentration
becomes, respec
tively, 20, 33, and 44% lower, as a consequence of successive
additions of Mg++-free medium, the resulting absorbance
decrements are in no way more extended than those which
may be predicted on the basis of the dilution of the
mitochondrial suspension. A 44% decrease of Mg++ con
centration would induce at least a 25% shrinkage reversal, as
ascertained by titration with Mg"1"1"of DGT-swollen mito
chondria.
In an analogous manner, when DGT-swollen mitochondria
from ascites hepatoma are incubated for 2 min with 20 mM
Mg++ and then washed twice with Mg++-free medium, a
water content of 2.32 ¿d/mgof protein is found, which is
indicative of a shrunken condition.
Metabolic-independent
Swelling and Shrinkage in Media of
Different KC1 and Sucrose Content. In a previous paper (11),
it has been observed that the phosphate-induced swelling of
tumor mitochondria, suspended in a reaction mixture con
taining 0.130 M KC1, is completely reversed by 20 to 30
mM Mg++.The data from Table 1 in the present paper clearly
Table 2
Effect of the dilution ofMg++ in the medium on the
volume of ascites hepatoma mitochondria
Mitochondria (700 Mg protein) suspended at 25°in 1.5 ml of 0.145
M KO; 0.02 M Tris-Cl, pH 7.4; 2MMrotenone; 1 Mgof antimycin A
per ml. The swelling was started by addition of DGT, and the
absorbance decreases was measured at 520 mM. In Experiment A, 30
min of incubation with the swelling agent were followed by addition of
1.5 ml of Mg++-free medium. In Experiment B, shrinkage of swollen
mitochiondria was induced by Mg++ addition followed, 2 min later, by
3 successive additions of 0.5 ml of Mg++-free medium. The observed
AA's represent differences between absorbances recorded before and
after every addition. Theoretical AA's are calculated on the basis of
dilution of the mitochondrial suspension.
ATheoretical-0.409-0.161
AdditionsExperiment
A
100 MMDGT
mediumExperiment
1.5 ml of
B
100 MMDGT
5.15 mM Mg++
0.5 ml of medium
0.5 ml of medium
0.5 ml of mediumA
-0.398-0.188
+0.170
-0.157
-0.108
-0.106
-0.055
-0.080Observed-0.196
AUGUST 1970
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2225
Francesco Feo and Antonio Modi
show that these mitochondria, swollen in a sucrose medium,
may shrink, as a consequence of the Mg++ addition, to a great
extent: the absorbance increments overcome the decrease
induced by the swelling.
These results raise the question of the effect of the
composition of the reaction mixture on the extent of both
the swelling and the shrinkage. As a first approach to the
question, the osmotic properties of ascites hepatoma mito
chondria in KC1 and in sucrose media have been investigated
to ascertain whether in the 2 media tumor mitochondria
behave,
like those from normal tissues, as perfect
osmometers.
The reciprocal plots of the osmolarity versus the initial
absorbance of tumor mitochondria behave linearly in both
KC1 and sucrose media (Chart 1), showing a typical
osmometer response. The slope of the straight line showing
Table 3
Effect of the composition of the reaction mixture on the initial
absorbance, DGT-induced swelling, and Mg**-induced shrinkage in
ascites hepatoma mitochondria
Mitochondria (500 to 600 Mg protein/ml) suspended at 25°in 2 ml
of medium containing, in addition to the indicated amounts of KCl or
sucrose, 0.02 M Tris-Cl, pH 7.5; 2 MMrotenone; antimycin A, 1 mg/ml.
The swelling started by DGT addition and was followed at 520 mMfor
20 min. The shrinkage started by adding 20 mM Mg++ and was
recorded until completion. Ao = absorbance at zero time.
KCl
(jumoles/ml)50100(¿imoles/ml)290
145Sucrose
(A
A/mg)-0.215 (A
A/mg)+0.435
-0.165
+0.232
190
1.48
-0.183
+0.203
90A0/mg1.38 1.57
+0.104
1.62Swelling-0.150Shrinkage
2
3.0
S
S
«r
SUCROSE
10mMMg
25
I 2.0
O.
g"
50
100
150
I/ OSMOLARITY
Ì-1.5
C_3
Chart 1. Reciprocal plots of absorbance versus osmolarity in ascites
hepatoma mitochondria. Mitochondria (800 Mg protein/ml) were
suspended at 25 in 2 ml of reaction mixture containing 0.004 M
Tris-Cl, pH 7.4, and different amounts of KCl or sucrose to obtain the
desired osmolarity. The swelling was started by rapid mixing into the
medium of 10 to 20 M! of mitochondria! suspension, placed on the
flattened bottom of a plastic rod. This procedure took less than 2 sec;
during this time the recorder tracer was not stopped. After 2 sec, the
recorder "noise" disappeared and the absorbance was recorded with an
<C
CO
i
1.0
NOKCl
OD
7.5mMMg
0.5
0L,
0
5
accuracy of ±0.005.
Chart
the osmotic behavior of mitochondria in the KCl medium is
steeper than in sucrose, indicating that a larger osmotic
compartment is responding to the KCl. Thus, in both the
reaction mixtures used to study the metabolic-independent
swelling and shrinkage of the ascites hepatoma mitochondria,
the morphological integrity of the osmotically active com
partment is fully preserved.
The effect of the KCl and sucrose content of the medium
on tumor mitochondria volume changes, is shown in Chart 2
and Table 3. In Chart 2, data obtained with liver mito
chondria are included for comparison. It appears that the
DGT induces in normal rat liver mitochondria suspended in
KCl medium a more pronounced swelling than in sucrose
medium. The Mg++ addition does not induce, according to
previous observations (11), significant shrinkage, even after
the mitochondria have undergone extensive swelling. The
KCl content of the medium does not significantly influence
the extent of the DGT-supported swelling of the tumor
mitochondria.
On the contrary,
it affects the initial
absorbance of the mitochondrial suspension as well as the
2226
10 15 20 25 30
TIME (min)
2. DGT-induced swelling and Mg++-induced shrinkage in
mitochondria from ascites hepatoma and normal liver. Mitochondria
(500 to 600 Mgprotein/ml) were suspended at 25°in 2 ml of reaction
mixture containing 0.29 M sucrose or 0.145 M KCl; 0.02 M Tris-Cl, pH
7.4; 2 MMrotenone; antimycin A, 1 Mg/ml. The swelling was started by
the addition of 200 MM DGT. Absorbance changes were recorded at
520 mM.
extent
of the
shrinkage
induced
by Mg"1"1".The initial
absorbance increases with the concentration of the KCl, the
shrinkage decreases as the KCl content increases (Table 3).
From the data in Table 3, it may be calculated that
half-maximal inhibition of the shrinkage is obtained in a
reaction mixture containing 0.05 M KCl. In 0.29 M sucrose,
the initial absorbance is decreased by 15% with respect to
the absorbance in the sucrose-free medium, in the latter the
inhibition of the shrinkage is 76%.
The changes in the initial absorbances found in the mito
chondrial suspensions, according to the media used, may
CANCER RESEARCH VOL. 30
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Volume Changes and Mg++ Binding in Mitochondria
reflect differences of the refractive index of the media,
rather than true volume differences (29). It is not possible to
ascertain, on the basis of the water content, whether a slight
volume difference really exists between freshly isolated
mitochondria suspended in KC1 or sucrose media. However,
slight differences in the final volume of DGT-swollen mito
chondria would have little effect on the extent of the
Mg+"l"-supported shrinkage and, consequently, on the marked
inhibition induced by K+.
The effect of increasing the concentration of Mg"1"1"
on the
K+ inhibitory effect of the shrinkage is shown in Chart 3.
10
20
30
[Mg"] (mM)
Chart 4. Effect of the external Mg++ concentration
40
on the Mg++
uptake by ascites hepatoma mitochondria. Mitochondria
protein) were incubated at 25 in 3 ml of reaction mixture
0.02 M Tris-Cl, pH 7.4; 2 >iM rotenone; antimycin A, l
indicated Mg++ concentrations; and sufficient amounts of
(5 mg of
containing
¿ig/ml;the
sucrose to
bring the osmolarity to 0.33. After 3 min of incubation, the samples
were diluted with Mg++-containing medium (unwashed mitochondria)
or Mg++-free medium (washed mitochondria) and then centrifuged as
described under "Materials and Methods." The pellets of samples
diluted with Mg++-free medium were washed twice with the same
medium. The Mg++ was determined in the acid extracts of the pellet.
Data indicate the Mg++ taken up corrected for endogenous Mg'1"1"
(40
OJD5
0.10.1502
1/Amóles
Mg*ADDED
Chart 3. Reciprocal plots of the inhibitory effect of KC1 on the
Mg++-induced shrinkage,
in ascites hepatoma
mitochondria.
Mitochondria (500 to 600 Mgprotein/ml) were suspended at 25 in 2
ml of medium containing, together with 0.02 M Tris-Cl, pH 7.4; 2 nM
rotenone; antimycin A, 1 ¿ig/ml;the indicated concentrations of KC1;
and sucrose in sufficient amounts to bring the osmolarity to 0.33.
The swelling started by the addition of 200 ¡MDGT. Mg++ was added
after 10 min of incubation in the presence of the swelling agent; the
shrinkage induced by the bivalent cation was recorded until
completion. Absorbance changes were recorded at 520 m/j.
nmoles/mg protein).
is shown. It may be seen that, at Mg++ concentrations
higher
than 19 nmoles/ml, the amount of bivalent cation firmly
bound does not increase any further, but these "saturating"
conditions are not reached in unwashed mitochondria for the
same range of external Mg"1"1"concentrations. These results
are in agreement with the observations made by O'Brien and
Brierley (20) on heart mitochondria, except for a higher
passive Mg"1"1"uptake in tumor mitochondria, than in those
The reciprocal plots of the Mg"1"1"
concentration
versus the
absorbance changes in sucrose and in KC1 media, show that
the K"1"inhibition of the shrinkage is greatly affected by the
concentration
of Mg++. The slope of the straight lines
showing the mitochondrial volume changes in KC1 media is
steeper than that in sucrose. At infinite Mg"1"1"
concentration,
corresponding to the theoretical, maximal shrinkage which
may be induced by the bivalent cation, the inhibitory effect
of the K+ disappears. These features could indicate that a
mechanism unrelated to the mitochondrial volume before the
addition of Mg++ is involved in the K+ inhibitory effect of
the shrinkage. Perhaps, a competitive phenomenon between
K+ and Mg++ for the same mitochondrial receptors occurs.
Metabolic-independent Uptake of Mg++. Mitochondria from
ascites hepatoma cells, incubated in isotonic reaction mixture
containing 0.02 M KC1 and 0.02 M MgCl2, take up 170
nmoles of Mg++ per mg of protein, in the complete absence
of metabolism (12). About one-half of the Mg"1"1"
uptake may
be washed out by centrifuging in a Mg++-free reaction
mixture. The degree of Mg++ uptake is independent from
the existence of a swelling condition (12).
In Chart 4, the effect of increasing concentrations
of Mg"1"1"
in the reaction mixture on the uptake of the bivalent cation
isolated from the heart. Furthermore, the mitochondria from
the hepatoma attain saturating conditions at higher Mg"1"1"
concentrations
than heart mitochondria.
Using the same
criteria as O'Brien and Brierley (20) for defining the 2
fractions of Mg++ taken up, we may state that, as a
"plateau" is reached in the washed mitochondria curve, the
saturation of the Mg"1"1"-bindingsites is complete. Any further
increase of the Mg"1"1"uptake in unwashed mitochondria,
after this saturation, represents only the Mg"1"1"
in solution in
an accessible mitochondrial space which is in equilibrium
with the external bivalent cation.
The passive uptake of Mg"*"1"is influenced by the com
position of the reaction mixture in a similar manner as is the
shrinkage. The data from Table 4 show that in isotonic KC1
the Mg++ binding is lower than in isotonic sucrose. In the
presence of 5, 10, and 15 mM Mg++, the K+ inhibition is,
respectively, 82.4, 76, and 56%. It appears that the
inhibition regards almost exclusively the Mg"1"1"
binding, the
soluble fraction of the bivalent cation not being extensively
affected. The data from Table 4 show, moreover, that the
degree of Mg"1"1"
binding is not related to the swelling state of
the mitochondria,
as it appears from the comparison
between the uptakes in isotonic and hypotonie conditions.
AUGUST 1970
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2227
Francesco Feo and Antonio Matli
Table 4
Effect of the composition of the reaction mixture on the Mg++uptake
by ascites hepatoma mitochondria
Mitochondria (5 to 7 mg protein/ml) syspended at 25°in 3 ml of
medium containing, in addition to the indicated amounts of sucrose,
KC1, and MgCl2, 0.02 M Tris-Cl, pH 7.4; 2 MM rotenone and
antimycin A, 1 mg/ml. Other conditions as indicated in Chart 4.
Bound Mg++ represents the bivalent cation found in washed samples.
The soluble Mg"1"1"
was calculated by subtracting the bound bivalent
not change further by prolonging the time of incubation to
10 min.
The passive uptake of Mg"1"1"
does not significantly change
for incubation times between 30 sec and 10 min. Unfor
tunately, the adopted experimental conditions do not permit
determinations of Mg++ uptake for times shorter than 25 to
30 sec. It is apparent from Chart 6, however, that after 30
sec both the shrinkage and the uptake are nearly complete.
cation from the total uptake.
uptake(nmoles/mg)Bound
mediumSucrose
Suspending
mM)Sucrose
(275 mM), MgCh
mM)Sucrose
(260 mM), MgCh
mM)Sucrose
(245 mM), MgCh
mM)KC1( 30 mM), MgCh
Soluble51.265.085.486.09.015.637.570.0148.0128.7
(5
(10
(15
(15
i
mM)KC1
(130 mM), MgCh (5
mM)KC1
(115 mM), MgCh (10
mM)KC1
(100 mM), MgCh (15
( 15 mM), MgCh (15 mM)Mg++
The reciprocal plots of the Mg"1"1"
uptake resistant to washes
versus the Mg++concentration show that the inhibitory effect
of the K1"on the Mg++ binding behaves with similar features
as the inhibitory effect on the shrinkage (Chart 5).
6
8
10
TIME(min)
Chart 6. Kinetics of the Mg++-induced shrinkage and of the Mg++
uptake in ascites hepatoma mitochondria. For the spectrophotometric
determination of the shrinkage, the same conditions as in Table 1 were
adopted. For determination of the passive Mg"1"1"
uptake, mitochondria
(5 mg of protein) suspended at 25°in 2.5 ml of 0.29 M sucrose; 0.02 M
Tris-Cl, pH 7.4; 2 MM rotenone; and antimycin A, 1 Mg/ml, were
centrifugea through silicone 3, 5, and 10 min after the addition of 15
nmoles of Mg++/ml. For incubation times of 30 and 60 sec, 0.5 ml of
mitochondrial suspension (5 mg of protein) in Mg++-free medium was
pushed down by centrifugation in a layer of medium containing 15 mM
Mg++. The time of incubation was determined by the thickness of this
20
layer. The reaction was stopped when the mitochondria reached the
silicone on which the "incubation" layer was stratified. Mg++ was
determined in acid extracts of the mitochondrial pellets. Points with
vertical bars represent average values of 5 experiments ±S.D.; other
points are average values of 2 experiments.
DISCUSSION
0.1 02 0.3
1/pmolesMg*'ADDED
Chart 5. Reciprocal plots of the inhibitory effect of KC1on the Mg++
binding by ascites hepatoma mitochondria. Mitochondria (5 mg of
protein) were suspended at 25°in 3 ml of medium containing, together
with 0.02 M Tris-Cl, pH 7.4; 2 MM rotenone; antimycin A, 1 Mg/ml;
and the indicated concentrations of Mg"1""1",
0.29 M sucrose, or 0.145 M
KC1. After 3 min of incubation, the suspensions were diluted with
Mg++-free medium, centrifuged, and washed twice. Mg"1""1"
was deter
mined in the acid extracts of the pellets.
Kinetics of the Shrinkage and the Mg"1"1"
Uptake. A striking
feature of the Mg"1"1"-inducedshrinkage is its rapidity. After 2
sec, there occurs about an 80% rise of the absorbance. As is
shown in Chart 6, after 60 to 90 sec of incubation, the
phenomenon is practically complete, the absorbance does
2228
It has been well established that mitochondria
from
normal tissues accumulate bivalent cations by a metabolicdependent
mechanism (for review, see Ref. 19). Mito
chondria also take up some amounts of bivalent cations in
the absence of metabolism (20, 23, 24). As a consequence of
the bivalent metal ion uptake, mitochondrial swelling has
been observed in some conditions (4, 8). This swelling has
been interpreted as an attempt to maintain an equal osmotic
pressure on both sides of mitochondrial membrane. In other
experimental conditions, certain bivalent cations may prevent
the mitochondrial swelling if added before the swelling agent
(12, 28); however, when added to swollen mitochondria
together with ATP, they enhance the shrinking effect of the
nucleotide (7, 18).
In ascites hepatoma mitochondria, the Mg"*"1"
effect differs
in some respects from that of this bivalent cation on
mitochondria from normal tissues. The Mg"1"1"induces, in
swollen tumor mitochondria,
unrelated
to the presence
shrinkage which appears
of exogenous
ATP and
CANCER RESEARCH VOL. 30
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Volume Changes and Mg++ Binding in Mitochondria
metabolism. An analogous bivalent cation-induced shrinkage
has been described in metabolically inhibited chloroplasts
and chloroplast grana (10, 14, 27). In mitochondria from
normal tissues a metabolic-independent
shrinkage has been
observed with Mn++ (5) and Mg++ (11), but the volume
decrease induced by the 2 cations is not higher than 20 to
25%.
Concurrently with the shrinkage, a metabolic-independent
uptake of relatively large amounts of Mg"1"1"occurs. The
mitochondria isolated from liver during the early stages of
tumorigenesis, and later in well-growing tumors, suggest the
occurrence of functional and probably structural changes in
tumor mitochondria. However, the role of these changes in
the determination of the tumor mitochondria response to
environmental conditions, as for instance the bivalent cation
addition, is not yet understood.
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CANCER RESEARCH VOL. 30
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Metabolic-independent Volume Changes and Mg++ Binding in
Mitochondria Isolated from AH-130 Yoshida Ascites Hepatoma
Francesco Feo and Antonio Matlí
Cancer Res 1970;30:2223-2230.
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