Download Differences in Total Mitochondrial Proteins and

(CANCER RESEARCH 38, 1584-1588, June 1978]
0008-5472/78/0038-OOOOS02.00
Differences in Total Mitochondrial Proteins and Proteins Synthesized
by Mitochondria from Rat Liver and Morris Hepatomas 9618A, 5123C,
and 5123tc1
Carol C. Irwin,2 Leonard I. Malkin,
and Harold P. Morris
Department ot Biochemistry,
Wayne State University School ol Medicine, Detroit,
Howard University School of Medicine, Washington, D. C. 20001 ¡H.P. M.]
ABSTRACT
Mitochondria were isolated from a slow-growing (9618A)
and two intermediate-to-fast-growing
(5123C, 5123tc) Mor
ris hepatomas and host livers. The mitochondria! proteins
were solubilized and fractionated on sodium dodecyl sulfate:polyacrylamide
slab gels. One Coomassie
bluestained band was absent or reduced in amount in all
tumors relative to host livers. In addition, a major mito
chondria! enzyme present in normal liver, carbamyl phos
phate synthetase, was missing or greatly reduced in the
slow-growing, highly differentiated
hepatoma 9618A, a
tumor that is considered to be similar to normal liver in
many biochemical and morphological respects. Incuba
tion of mitochondria with [35S]methionine and a suitable
amino acid incorporation system resulted in labeling of
specific mitochondria! proteins. Autoradiography of the
slab gels disclosed four prominently labeled fractions and
a number of minor fractions. Preparations from hepatoma
5123tc demonstrated two labeled bands that were absent
or greatly reduced in host liver. Host liver preparations
displayed a minor band that was absent or greatly re
duced in hepatoma 5123C. However, no single change in
labeling pattern was common to all three tumors, sug
gesting the absence of a causal relationship between
carcinogenesis and mutations in mitochondria! DMA.
INTRODUCTION
The DNA of mammalian mitochondria has a molecular
weight of approximately 107 and codes for at least 2 rRNA
molecules, several tRNA molecules, and several proteins,
all of which are unique to mitochondria (13). Only 5 to 10%
of total mitochondrial proteins are synthesized by mitochondrial ribosomes. Evidence suggests that these proteins
are coded for by mitochondrial DNA. These proteins are
closely associated with the inner membrane of mitochon
dria, and they are insoluble except in detergents such as
sodium dodecyl sulfate, so that their identification has been
difficult. Only 8 proteins have been positively identified as
belonging to specific mitochondrial membrane:enzyme
complexes (13).
Malignant transformation of cells is accompanied, to a
' Supported by the National Cancer Institute (CA 15539), the National
Science Foundation (BMS73-02031), the Research Corporation (BH512), and
in part by USPHS Grant CA 10729.
2 Fellow of the National Cancer Institute (CA 05109). Present address:
Eunice Kennedy Shriver Center for Mental Retardation, Waltham, Mass.
02154.
3To whom requests for reprints should be addressed.
Received August 26. 1977; accepted March 7, 1978.
1584
Michigan
48201 [C. C. I., L. I. M.¡,and Department
oÃ-Biochemistry,
greater or lesser degree, by altered enzyme activities and
ultrastructure of mitochondria (16). These alterations could
arise from mutations in either nuclear or mitochondrial DNA
or both. Chang ef a/. (2) have demonstrated modified
electrophoretic properties of 1 inner membrane protein in
mitochondria from several Morris hepatomas. We have
examined mitochondria from 3 hepatomas different from
those studied by Chang ef a/. (2), and we have found 1
Coomassie blue-stained band absent or greatly reduced in
all 3 tumors. We have established that this is not a mitochondrially synthesized protein and probably not coded for
by mitochondrial DNA. Hepatoma 9618A, a very-slow-grow
ing minimal-deviation tumor, lacks a major Coomassie
blue-stained band that is present in host liver and the other
hepatomas that have been studied. Based on the work of
Clarke (3), this is almost certainly carbamyl phosphate syn
thetase, an enzyme that is present in large amounts in the
livers of ureotelic organisms. This protein is also not coded
for by mitochondrial DNA.
We have used a sensitive autoradiographic method for
detecting proteins synthesized specifically by mitochondria
in an in vitro amino acid incorporation system, and we have
observed a larger number of labeled proteins than has
previously been reported by most workers with gel slicing
techniques. This observation agrees with recent reports on
the labeling of mitochondrial proteins by yeast (4) and by
animal tissue culture cells (6). These proteins presumably
are coded for by mitochondrial DNA. Hepatoma 5123tc, a
fast-growing tumor, yielded 2 labeled bands that were
absent or much less conspicuous in all the other tumors
and host livers. The other fast-growing hepatoma, 5123C,
and a slow-growing hepatoma, 9618A, did not differ from
their host livers in major respects.
MATERIALS
AND METHODS
Animals and Hepatomas. Male tumor-bearing Buffalo
rats were obtained from the colony of Dr. Harold P. Morris
and kept under standard conditions. All tumors were car
ried i.m. by bilateral inoculation into thigh muscle. Hepa
toma 9618A (generation 10) was a highly differentiated,
slow-growing tumor (transplantation time, approximately
12 months) that has been reported to have a normal chro
mosome number and karyotype (12). Hepatomas 5123C
(generation 120) and 5123tc (generation 150) were fast
growing (transplantation time, 1 to 2 months) with a lesser
degree of differentiation. Previous generations of 5123C
and 5123tc have been reported to have abnormal chromo
some numbers but minimal chromosome changes (12).
CANCER
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Mitochondria! Protein Synthesis in Rat Liver and Hepatoma
Hepatomas from 1 to 3 animals were pooled for each
experiment.
Isolation and Purification of Mitochondria. All solutions
were prepared 1 to 2 days before each experiment and were
autoclaved.
Glassware was also sterilized.
Procedures
throughout
the isolation of mitochondria
were performed
at 0-4°. Rats were killed by decapitation,
and host livers
were quickly removed and placed into approximately
3
volumes of 10 mM Bicine:4 2 mM EDTA:0.3 M sucrose buffer
(pH 7.5). The liver was cut into small pieces with scissors
and washed with fresh buffer 3 times by settling and dé
cantation. Buffer was then added to 2 volumes of liver wet
weight and homogenized with a motor-driven, Teflon:glass
homogenizer with a clearance of about 0.25 mm. The motor
was driven at 500 rpm and only 2 up-and-down
strokes
were used. The homogenate was diluted with another 6
volumes of buffer and centrifuged at 1400 x g for 6 min
in 50-ml polycarbonate
tubes, with caps, in the No. 276
swinging bucket rotor of an EC refrigerated centrifuge.
The supernatant
solution was separated by décantation
from the light brown loose layer of the pellet, then passed
through 2 layers of cheesecloth,
and recentrifuged
as
above. The supernatant solution from the second centrifugation was placed in clean 50-ml polycarbonate tubes and
centrifuged at 6000 x g for 10 min in a Sorvall refrigerated
centrifuge. The supernatant solution was decanted off of
the loose brown layer, and the pellet was partially resuspended with a "cold finger" (sterile 15-ml test tube filled
with ice). Resuspension was completed by adding a small
amount of buffer and drawing up the suspension into a
cotton-plugged,
sterile 10-ml blow-out pipet, followed by
gravity out-flow, a total of 2 to 3 times. The suspension was
diluted to a final volume of one-half of the original volume
and recentrifuged
for 10 min at 6000 x g. The pellet was
resuspended
into one-fourth
of the original volume of
buffer prior to the final centrifugation,
after which all the
supernatant solution was discarded, including any loose
material. The final pellet was resuspended to a final concen
tration of approximately
25 mg mitochondrial
protein per
ml.
In the case of hepatomas, tumors were removed from the
thigh muscle of the rats after they had attained a diameter
of approximately 2 to 3 cm. Tumors of this size were seldom
necrotic. Nontumorous tissue or, rarely, necrotic tissue was
carefully trimmed away prior to isolation of mitochondria,
as described for host liver. The only difference in procedure
was the increase in homogenization
from 2 strokes to 10.
The characterization
of these mitochondria
preparations
has been reported previously (11).
Amino Acid Incorporation. The complete incubation sys
tem was based on that of Malkin (11) and contained 10 mM
Bicine, 10 mM K2HPO4, 2 mM EDTA, 0.154 M KCI, 10 mM
MgCI2, 1 mM ATP, 5 mM phosphoenolpyruvate,
20 /xg
pyruvate kinase, 19 amino acids (minus u-methionine) (2 /xg
each), 0.5 mg cycloheximide,
1 to 2 mg mitochondrial
protein, and 0.14to 0.25 mCi L-[35S]methionine (Amersham/
Searle Corp., Arlington Heights, III.). Specific activity of the
L-[35S]methionine was 5 to 6 Ci/mole. Final volume was 1
ml and the final pH was 7.4. Chemicals
were purchased
* The abbreviation used is: Bicine, W,/V-bis(2-hydroxyethyl)glycine.
from Sigma Chemical Co., St. Louis, Mo. Stock solutions
of Bicine, K,HPO4, EDTA, KCI, and MgCI, were freshly
prepared and autoclaved 1 to 2 days before use. Other
stock solutions were prepared with sterile water. The basic
incorporation
mix, before addition of mitochondria
and L[35S]methionine, was filtered immediately before use with a
Millipore bacteriological
filter.
After incubation for 1 hr at 37°in glass Sorvall centrifuge
tubes, the incorporation
mixtures were placed on ice and
diluted with 5 ml wash buffer [0.25 M sucrose, 0.01 M Tris
(pH 6.7), 0.025 M KCI, and 0.005 M MgCL] containing cold
L-methionine
(1 mg/ml). Mitochondria
were pelleted by
centrifugation
at 12,000 x g for 10 min in the SS34 rotor in
a Sorvall refrigerated centrifuge. Pellets were washed twice
and then dissolved in 0.5 ml of a dissociating
medium,
modified from that of Douglas and Butow (4), containing
2% sodium dodecyl sulfate, 0.002 M EDTA, 0.2% (v/v) 2mercaptoethanol,
0.05 M Tris (pH 6.8), and 10% (v/v) glycerol. The pellets were heated for 2 min at 90°and then
either stored overnight at room temperature or used imme
diately for electrophoresis.
Overnight incubation
did not
alter either stained or labeled banding patterns.
Protein Assay. Protein was estimated by the method of
Lowry ef al. (10). In the case of mitochondrial
protein
dissolved in electrophoresis
medium, correction was made
for the artifactual contribution
of 2-mercaptoethanol.
Bacterial Counting. Control tubes that contained incor
poration mix, mitochondria,
but no isotopes were incu
bated in parallel with expérimentais. One-tenth-ml aliquots
were plated on blood agar and incubated for 72 hr at 37°,
and the number of colonies was determined.
Slab Gel Electrophoresis
and Autoradiography.
The
general procedures for electrophoresis
and autoradiography were those of Douglas and Butow (4). Approximately
50 /xg of mitochondrial
protein per well were layered on the
15-cm slab gels described by Studier (14). The discontin
uous buffer system of Laemmli (8) was used, except that
0.002 M EDTA was included in the stacking and resolving
gels, and 0.5% polyacrylamide
(BDH Laboratories, GallardSchlesinger Chemical Mfg. Corp., Carle Place, N. Y.) was
included in the resolving gel to ensure even drying of the
gel for autoradiography
(4). The resolving gel (7.5 to 12%
linear acrylamide gradient) was prepared 1 to 2 days before
hand, and the 5% stacking gel was prepared immediately
prior to electrophoresis.
Up to 13 samples of mitochondrial
or marker protein could be subjected to electrophoresis
at
1 time. Electrophoresis
was performed at 30 ma constant
current until the bromphenol blue tracking dye reached 1
cm from the bottom of the slab (about 5 hr).
The gels were dried either immediately after electropho
resis, or after staining, on Whatman No. 3MM paper over a
steaming water bath under reduced pressure. For staining,
gels were soaked in 10% trichloroacetic
acid at 70°for 20
min, stained in methanohacetic
tained 0.05% (w/v) Coomassie
acid:water (5:1:5) that con
blue at 37°overnight, and
destained in 7.5% acetic acid that contained 5% methanol.
If stained gels were to be dried, they were soaked for 2 hr in
1% glycerol. Dried gels were exposed from 3 weeks to 2
months to Kodak RP X-omat medical X-ray film that was
then developed in Kodak liquid X-ray developer for 5 min at
22°.
JUNE 1978
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1585
C. C. Irwin et al.
Marker Proteins. Molecular weight estimations on gels the host liver.
were made with marker proteins: /3-lactoglobulin (M.W.
Because of the variation in tumor growth, mitochondrial
18,000); chymotrypsinogen A (M.W. 26,000); ovalbumin (M. proteins from the various hepatomas had to be subjected to
W. 43,000); and serum albumin (M.W. 67,000). ß-Lactoglob- electrophoresis separately, resulting in slight variations in
ulin migrates somewhat anomalously in our gel system, at length of run and gel composition. In addition, there was
an apparent molecular weight of 15,000. All marker pro
an apparent variation in the extent of labeling (note the
teins were purchased from Sigma Chemical Co., St. Louis, largar number of bands detected in mitochondria from
Mo., except for the chymotrypsinogen A (Worthington Bio
9618A and 5123C). We believe that these experimental
chemical Corp., Freehold, N. J.).
variations account for the small apparent differences seen
among host livers, although the possibility of actual varia
tions in individual host livers cannot be completely ex
RESULTS
cluded. We have consistently observed, however, that la
Coomassie Blue-stained Gels: Total Proteins. The band beled proteins from several normal livers yield identical
ing patterns for all 3 hepatomas and their host livers are patterns when run on the same gel.
shown in Fig. 1. When allowance is made for slight differ
Bacterial contamination was negligible, as shown by the
ences due to separate gel runs, the banding patterns are results of blood agar plating. The mitochondrial incubation
identical among host livers. In general, mitochondrial pro
mixture contained less than 200 bacteria/ml.
teins from each hepatoma bear great similarity to those of
their host livers. There are several exceptions to this, DISCUSSION
however. One prominent difference between all 3 hepato
The incorporation of [35S]methionine into mitochondrial
mas and their respective host livers is the absence or
proteins of both normal liver and hepatomas was carried
reduction of a polypeptide with an approximate molecular
out in the presence of cycloheximide, an inhibitor of cytoweight of 36,000 in tumor mitochondria (dashed arrow).
Other differences can be detected between tumor and host plasmic but not mitochondrial protein synthesis (11). There
fore cytoplasmic protein synthesis does not contribute to
liver mitochondria, but these are minor and are not com
labeling of the proteins in the system used (Fig. 2) although
mon to all of the hepatomas that are studied.
The most striking difference is the absence in hepatoma minor bands might not have been detected in these experi
9168A of the high-molecular-weight polypeptide that ap
ments. In addition, the level of bacterial contamination in
pears to be the most prominent band in host liver (so//'d
these experiments is much lower than that needed (30,000
arrow). Although we have not definitively identified this bacteria/ml) to show significant bacterial amino acid incor
polypeptide by biochemical means, the position of the band poration (11).
and the absence or reduction of the band in non-liver tissue
Autoradiographic studies of mitochondrially synthesized
such as brain, kidney, and heart, as well as fetal liver proteins indicate that there are 2 additional (or more highly
labeled) bands in Morris hepatoma 5123tc compared with
(unpublished results), indicate that this polypeptide is prob
ably the protein with the molecular weight of 165,000, host liver mitochondria. Except for the absence or reduc
carbamyl phosphate synthetase, observed by Clarke (3) to tion in label of a minor band in mitochondria from hepa
occur in liver of ureotelic organisms. This protein makes up toma 5123C, no other differences between tumor and host
15 to 20% of the total mitochondrial protein in normal liver livers were detectable. Apparently no single alteration in
mitochondrial translation products is common to all hepa
(3).
Mitochondrially Synthesized Proteins. Incubation of iso tomas; therefore the role of mitochondrial DNA in tumorilated mitochondria with [35S]methionine of very high spe genesis remains uncertain. It has been reported that mito
cific activity, along with an amino acid incorporation sys chondria do preferentially accumulate certain carcinogenic
tem, resulted in sufficient labeling of mitochondrially syn
hydrocarbons (5) and also that mitochondrial DNA may be
thesized proteins to allow autoradiography. The results of 3 particularly sensitive to mutagenesis since it lacks tightly
such experiments are shown in Fig. 2. When mitochondrial
bound proteins (15). Certain carcinogenic hydrocarbons do
proteins from each hepatoma are compared with those of in fact preferentially interact with mitochondrial DNA as
its host liver, hepatoma 5123tc is the only one of the 3 opposed to nuclear DNA (17). It would be of interest to
tumors that differs greatly from the host. Culture cells from establish whether the band pattern of hepatoma 5123tc is
which tumor 5123tc was derived came from generation 20 due to an alteration of mitochondrial DNA itself or to a
of tumor line 5123C, and the lines have not been mixed in subsequent step in the translation process. It is possible
subsequent generations.
that the changes seen are the indirect effect of cytoplasmic
There are 4 major labeled bands (marked with solid changes that are known to affect in large degree such
arrows) in all the gels. In 5123tc appear 2 labeled bands mitochondrial functions as the proper assembly of enzyme
(dashed arrows) that are absent or greatly reduced in host complexes (e.g., cytochrome oxidase) that originate both
from nuclear and mitochondrial gene products (13).
liver. These bands have molecular weights of approximately
15,000 and 31,000 (based on molecular weight standards
The autoradiographic method used in this study allows
that were run on the same gel). Hepatoma 5123C also much better resolution than has previously been obtained
differs from its host control by the absence or reduction in with the widely used gel slicing technique (e.g., Refs. 1 and
9). Our results substantially agree with another radiolabeling of a minor band of approximately 60,000 (dotted
arrow). By contrast, the minimal-deviation hepatoma 9618A graphic study by Jeffreys and Craig (6), who distinguished
apparently has no labeled proteins that differ from those of 7 to 11 labeled major bands and a number of minor bands
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Mitochondria! Protein Synthesis in Rat Liver and Hepatoma
from mitochondria of animal culture cells. Douglas and
Butow (4) have resolved 21 labeled bands from yeast mito
chondria that were labeled under conditions that excluded
cytoplasmic protein synthesis. Whether all 21 proteins in
yeast, or in the animal mitochondria that have been studied,
are unique gene products as opposed to aggregates and/or
cleavage products remains to be seen.
Kuzela ef al. (7) conducted a study in which mitochondria
from rat liver and Zajdela hepatoma were labeled in vivo
with [14C]leucine in the presence of cycloheximide. The
distribution of label in sodium dodecyl sulfate:polyacrylamide gels was determined by the gel slicing technique
and was the same in both cases. The study was limited,
however, by the low resolution of gel slicing and the low
level of radioactivity in the labeled bands. While it is
possible that in fact no actual differences existed between
the hepatoma in question and normal liver, as we found in
1 of our hepatomas, the difference that we detected with
the autoradiographic procedure would have gone unno
ticed by the method of Kuzela ef a/. (7).
When total mitochondrial proteins by means of Coomassie blue staining rather than specific mitochondrial transla
tion products were considered, we found 1 difference that
was common to all the hepatomas studied, namely, the
absence or reduction of a band with a molecular weight of
approximately 36,000. This agrees with the report of Chang
ef a/. (2), who analyzed mitochondrial membrane proteins
from another series of hepatomas of varying degrees of
differentiation. However, the molecular weight of their pro
tein was not determined, and therefore the identity of the
protein with ours cannot be established. The polypeptide
that we observed to be missing is apparently not a mitochondrially synthesized protein, inasmuch as it did not
coincide with any of the bands in the autoradiographs.
The absence in hepatoma 9168A of the major band near
the top of the gel seen in normal liver is somewhat surpris
ing because this tumor is highly differentiated and very
slow growing, a type of tumor that differs little in many
respects from normal tissue. The work of Clarke (3) sug
gests that this band corresponds to carbamyl phosphate
synthetase, a polypeptide with a molecular weight of
165,000. Clarke found that this band is reduced in fetal liver
(3), and we have established the same phenomenon with
the band in question (unpublished data). The enzyme pat
terns in many transformed cells conform to fetal patterns,
but the observation of such a relationship in a highly differ
JUNE
entiated tumor, especially when such a pattern is not seen
in faster-growing, less differentiated tumors, is striking.
In summary, the absence of a common difference in
mitochondrially synthesized proteins (comparing 3 hepato
mas to host liver) would seen to argue against a causal
relationship between carcinogen induction of hepatomas
and specific mutations in mitochondrial DNA.
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1587
C. C. Irwin et al.
9618A
T
L
—165
1-165
-67
-43
x
*
-26
-15
-15
I
2
Fig. 1. Comparison of electrophoretic properties of total Coomassie blue-stained mitochondrial proteins from hepatomas (7")and host livers (L). The cath
ode is at the fop.
Fig. 2. Comparison of autoradiographs of 35S-labeled mitochondrially synthesized proteins from hepatomas (7) and host livers (i.). The cathode is at the
top.
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Differences in Total Mitochondrial Proteins and Proteins
Synthesized by Mitochondria from Rat Liver and Morris
Hepatomas 9618A, 5123C, and 5123tc
Carol C. Irwin, Leonard I. Malkin and Harold P. Morris
Cancer Res 1978;38:1584-1588.
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