Download Mitochondria! Protein Content and Enzyme

[CANCER RESEARCH 34. 1989-1994, August 1974]
Mitochondria! Protein Content and Enzyme Activity of Reuber
Hepatoma H-351
Marjorie W. Myers2 and H. Bruce Bosmann '
Department of Pharmacology and Toxicology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
and neoplastic cells on the basis of specific activity of
protein content of the mitochondrial fraction. Lipid content
of several malignant cell mitochondria has been reported to
Mitochondria were isolated by differential centrifugation
from ACI rat liver, Reuber hepatoma H-35, and host rat be altered from the normal cell type mitochondria (1, 17).
liver. Hepatoma mitochondria were isolated in 0.3 M The possibility of altered lipid-to-protein ratio between
sucrose-2 mM ethylenediaminetetraacetic acid-30 mM nico- normal and malignant mitochondria makes it necessary to
tinamide-0.7% bovine serum albumin, pH 7.4. Normal and determine whether protein is an adequate basis for expres
host liver mitochondria were isolated in 0.3 Msucrose-2 mM sion of specific activity of mitochondrial functions. The
ethylenediaminetetraacetic acid-30 mM nicotinamide-0.7% experiments reported below are designed to determine
bovine serum albumin, pH 7.4, or 0.3 M sucrose-2 mM whether protein content or enzyme activity is altered
ethylenediaminetetraacetic acid-30 mM nicotinamide, pH between the mitochondria of Reuber hepatoma H-35 and
normal or host liver.
7.4 and were compared for sedimentation properties, pro
tein content, and enzyme activity. Normal and host liver
prepared in 0.3 M sucrose-2 mM ethylenediaminetetraacetic
acid-30 mM nicotinamide, pH 7.4, and 0.3 M sucrose-2 mM MATERIALS AND METHODS
ethylenediaminetetraacetic acid-30 mM nicotinamide-0.7%
Tumor Passage
bovine serum albumin, pH 7.4, were equivalent in protein
content per mitochondrion, separation from plasma mem
The tumor used was Reuber H-35. It was serially
brane and microsomal membrane enzymes, and cytochrome
transplanted bilaterally in ACI rat thigh muscle (rectus
oxidase and succinic dehydrogenase activities. Hepatoma
dorsalis). The tumor was obtained from Dr. Harold P.
mitochondria contained equivalent amounts of protein per
Morris, Howard University, Washington, D. C. Generations
mitochondrion and cytochrome oxidase activity as liver
83 through 88 were passed by excision of the tumor from
mitochondria but significantly less succinic dehydrogenase
activity. These data indicate that Reuber hepatoma H-35 the thigh muscle, mincing in sterile 0.9% NaCl solution, and
i.m. injection of 0.3 ml (approximately 100 to 150 mg of
mitochondrial membranes may have an altered enzyme
tumor protein). The tumors were usually palpable within 4
activity but have similar protein content per mitochondrion
weeks and, by 7 to 8 weeks, were 2 to 3 cm in diameter. For
to normal or host liver mitochondria.
use, tumors were excised, rinsed, and homogenized. Any
necrotic tissue present was discarded.
SUMMARY
INTRODUCTION
Mitochondrial involvement in the neoplastic status of the
cell has been implicated by disruptions in malignant cell
respiratory controls (29), by altered mitochondrial enzyme
activities (13, 23), by differences in mitochondrial mem
brane components (6), by mitochondrial DNA abnormali
ties in malignant cells (7, 26), and by the effects of
oncogenic viruses on mitochondrial DNA, RNA, protein,
and glycoprotein synthesis (5, 18, 20, 21). Many of these
experiments compare mitochondrial activities of normal
'This work was supported in part by USPHS Grants GM-00032 and
CA-13320.
2Present address: Department of Pharmacology, Yale University
School of Medicine, 333 Cedar St., New Haven, Conn.
'Career Development Awardee of the National Institute of General
Medical Sciences.
Received January 21, 1974; accepted April 23, 1974.
Preparation of Rat Liver and Hepatoma Mitochondria
Rat liver mitochondria were originally prepared by a
modification of the Schneider and Hogeboom (25) method
of differential centrifugation (4). Coote and Work (8)
recommended preparing mitochondria in the buffer solu
tion, SEN.4 Mitochondria so prepared form pellets in the
same way as those prepared in 0.25 Msucrose alone, but are
more active with respect to protein synthesis.
When attempts were made to prepare mitochondria from
excised hepatoma material, mitochondrial pellets were
opaque and scant. A method was found for preparation of
' The abbreviations used are: SEN, 0.3 Msucrose-2 mM EDTA-30 mM
nicotinamide, pH 7.4; SENA, 0.3 M sucrose-2 mM EDTA-30 mM nicotin
amide-0.7% bovine serum albumin. pH 7.4; SDH, succinic dehydrogenase;
BSA, bovine serum albumin.
AUGUST 1974
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1989
M. W. Myers and H. B. Bosmann
tumor mitochondria in which albumin is incorporated into
the isolation medium (27). Thus SENA was the medium
used to prepare hepatoma mitochondria. Mitochondria so
prepared form pellets that appear and behave like those of
rat liver. Rat liver mitochondria were prepared in both SEN
and SENA and were compared for purity of isolation and
activity in the assays to be used.
Counting Mitochondria
Mitochondria were resuspended in isolation medium
(SEN) and pipetted onto an improved Neubauer platelet
counting chamber. Ten counts of each sample were made,
and each sample was done in duplicate. Six preparations of
normal liver mitochondria and 5 preparations of hepatoma
and host liver mitochondria were counted.
Preparation of Samples for Enzyme Assay
Three to 5 g of rat liver or hepatoma, rinsed free of blood,
were minced and homogenized in 10 ml of cold 0.1% Triton
X-100 in a Potter-Elvehjem homogenizer, followed by 30
strokes in a TenBroeck homogenizer. The crude homogenate was centrifuged at 40,000 x g for 15 min and the
supernatant was used as enzyme source.
Mitochondria! pellets, 500 to 1000 mg, were suspended in
2 to 3 ml of 0.1% Triton X-100 and homogenized by 30
strokes of a TenBroeck homogenizer. This extract was used
as enzyme source. All mitochondria were utilized immedi
ately after isolation; frozen samples were not used.
Enzyme Assays
In all instances of enzyme assay, the reaction was linear
with respect to time for the period of time of assay and for
all tissue sources utilized; in all instances initial rates of
reaction were studied.
SDH (EC 1.3.99.1). SDH was assayed according to the
method of Pennington (22). Data are reported as /umoles of
formazan reduced per hr per mg of protein.
5'-Nucleotidase (EC 3.1.3.5). 5'-Nucleotidase was mea
sured by the release of inorganic phosphate from 5'-AMP
(11). Data are reported as /umoles of phosphate released.
Arylesterase (EC 3.1.1.2). Arylesterase activity was mea
sured by the formation of p-nitrophenol from the substrate
p-nitrophenyl-acetate (3). Data are reported as Amólesof
p-nitrophenol formed per hr per mg of enzyme.
Cytochrome Oxidase (EC 9.1.3.1). The activity of cytochrome oxidase was measured as the ability of a mitochondrial fraction suspended in ice-cold 5 mM potassium phos
phate buffer, pH 7.4, to oxidize reduced cytochrome c. Data
are reported as the increase in absorbance per min:
j, —¿
Ablank)—¿
(A(l —¿
Ab
Õ2- fi
1990
= A units decreased/min
(method of Appelmans el al. [2].)
Protein. Protein was determined by the method of Lowry
et al. (14).
RESULTS
The marker enzymes 5'-nucleotidase (plasma mem
brane), esterase (microsomes) (12), succinic dehydrogenase
(mitochondria), and acid phosphatase (lysosomes) were
assayed in whole-cell homogenates, crude mitochondria,
and 5-times washed mitochondria of normal liver, host liver,
and hepatoma. Mitochondria of normal liver were isolated
in both SEN and SENA to compare the effects of albumin
on isolation of mitochondria. The results of membrane
marker enzyme assays on these fractions are presented in
Charts 1 and 2. Data presented are nmoles of product
formed per hr per mg of sample protein. Homogenates of all
cell types in SEN and SENA contained equivalent amounts
of 5'-nucleotidase and esterase activity. Loss of these
membrane markers in the first mitochondria! pellet and in
the subsequent washes was marked, and was similar for all
cell types and for both media used. The final mitochondrial
pellets had little contaminating plasma membrane or microsomal enzyme activities. Succinic dehydrogenase activity
was present to the same extent in liver homogenized in both
SEN and SENA and in host liver. However, hepatoma
tissue homogenized in SENA had less activity per mg of
protein. In all tissues, mitochondrial pellets increased in
specific activity of SDH with subsequent washes by about
10-fold, indicating enrichment of mitochondria. Acid phos
phatase activity was also not affected in normal liver tissue
by homogenization in SENA as opposed to SEN. However,
both host liver and hepatoma in SENA exhibited higher
activity of this lysosomal enzyme than did normal liver. In
all cases, very little purification of mitochondria from
lysosomes occurred. Lysosomal enzyme activity was neither
enriched nor depleted in the final mitochondrial pellet, as
would be expected since density gradient separations were
not used. Thus it is possible to conclude that the addition of
BSA to the homogenization and isolation medium does not
alter the separation of mitochondria from other membra
nous organelles by differential centrifugation, while in some
way it protects the more fragile hepatoma mitochondria
against lysis during preparation. The mitochondria of
normal liver in both SEN and SENA, of host liver in
SENA, and of hepatoma in SENA are purified from
5'-nucleotidase and esterase activity but not from acid
phosphatase activity.
The question was raised whether comparison of enzyme
activities based on the amount of protein in a sample of
mitochondria from different tissues was a true reflection of
those activities. In order to answer this question, another
mitochondrial enzyme, cytochrome oxidase, was assayed.
Table 1 presents data showing activity in normal and host
liver and hepatoma mitochondria. The data are expressed as
the change in absorbance of the reduced cytochrome c with
time, as explained in "Materials and Methods," and are
CANCER
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Mitochondria and Enzyme Activity of Reuber Hepatoma
9-Nucleotidase
Chart 1. 5'-Nucleotidase and esterase activity
Esterase
SI»
in subcellular fractions of normal liver (NL), host
liver (HL), and hepatoma (HP). The fractions are
labeled as homogenate (//), crude mitochondria
(A/,), and mitochondria washed 5 times (A/6).The
isolation media SEN and SENA are as given in
the text. Data are reported as nmoles or Amólesof
product formed per hr of assay per mg of enzyme
source protein.
311
HM)M|
NL
SEN
HAy*,
NL
SENA
H M|M_
H NY*,
HL
SENA
HP
SENA
NI
SEN
Succinte Dehydrogenase
4i
NL
SENA
Acid
H rn.rn~
HI
SENA
H "**"•
HP
SENA
Phosphatase
3-
Chart 2. Succinic dehydrogenase and acid
phosphatase activity in fractions of normal
liver (NL), host liver (HL), and hepatoma
(HP). The fractions are labeled as homogenate
(//), crude mitochondria (A/,), and mitochon
dria washed 5 times (Me). The isolation media
SEN and SENA are as explained in the text.
Data are reported as /imoles of product
formed per hr of incubation per mg of enzyme
source protein.
o
o.
o>
TI
E
o
I
H M. M_
NL
SEN
H MJn_
NI
SENA
H "<|'*V
HL
SENA
•¿"
H fVi|M_
HP
SENA
NL
NL
HL
HP
SEN
SENA
SENA
SENA
H W_W_
H
AUGUST 1974
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1991
M. W. Myers and H. B. Bosmann
were deduced to have lysed. Several reports of preparation
of mitochondria from various tumor sources mentioned the
fragility of these mitochondria and specified the use of
Specific
activity"
Mitochondria
serum albumin in the isolation medium (9, 10, 15, 16, 27). In
±0.12"1.59
all of these cases, 0.5 to 1.5% BSA was reported to protect
Normal
liverHost
liverHepatoma5441.73
±0.091.48
fragile tumor mitochondria from lysis during preparative
±0.09
manipulations. Some reports of normal tissue mitochondria
isolated in the presence of BSA also appeared in the
" Decrease in A/min/mg protein.
" Mean ±S.E.
literature (24, 28). The reports indicated that differential
centrifugation in such media produced mitochondria tightly
taken from the 1st min of incubation, as initial velocity of coupled and morphologically intact. Thus an attempt to
prepare functional tumor mitochondria in media contain
the reaction was faster than at later times. Measurements
reported were taken during the early linear portions of the ing BSA was made. The mitochondria so isolated behaved
as liver mitochondria during centrifugation; pellets were
curve of decreasing absorbance. Normal liver mitochondria
firm and light tan, and a fluffy pink layer (microsomes) was
showed cytochrome oxidase activity such that the absorb
ance of reduced cytochrome c decreased 1.73 units per min easily washed off the early pellets. The lowest concentration
of BSA that would allow isolation of tumor mitochondria
per mg of protein. Host liver and hepatoma mitochondria
produced a reduction in absorbance of 1.59 and 1.48 units was found to be 0.7%. Thus the medium SENA was used
throughout in the preparation of tumor mitochondria.
per min per mg protein. These data show no significant
In order to avoid comparing possible effects of albumin
differences between normal, host, and hepatoma mitochon
dria with respect to oxidation of cytochrome c when based on mitochondrial sedimentation, the characteristics of nor
mal liver mitochondria prepared in SEN and SENA were
on amount of protein in the enzyme sample.
Enzyme activity and protein content were also calculated
determined. Both preparations were compared for en
on a per mitochondrion basis. Mitochondria were counted
zymatic purity and for protein and enzyme content. Purifi
cation of normal liver mitochondria from 5'-nucleotidase
in a Neubauer platelet-counting chamber, as described
above. Aliquots of the same sample were assayed for and esterase activity proceeded in parallel throughout the
succinic dehydrogenase activity and for total protein con
progressive washes. Final mitochondrial pellets of both
tent. Table 2 presents data of protein and enzyme activity preparations were equally free of plasma membrane and
calculated per mitochondrion. Data are reported as mean ± microsomal contamination. The sedimentation of lysoS.E.; n is 6 for normal liver and 5 for host liver and somes in both media proved to be the same, with no
hepatoma. There is no significant difference in mg protein
enrichment or depletion of lysosomes from mitochondrial
detected by the Lowry method per mitochondrion isolated
pellets, and the increases in specific activity of succinic
from either normal liver, host liver, or hepatoma. Isolation
dehydrogenase by the final pellets were comparable in both
in SEN or SENA makes no difference in the amount of media. Thus it was concluded that the presence of albumin
protein per mitochondrion. SDH activity (reported as in the isolation medium does not alter the sedimentation
characteristics of normal rat liver organdÃ-es.
nmoles product formed per hr), when expressed per mito
chondrion, is not significantly different for host or normal
The protein and succinic dehydrogenase per mitochon
drion were determined for normal liver mitochondria pre
liver in either SEN or SENA. However, hepatoma mito
chondria contain significantly less succinic dehydrogenase
pared with or without albumin. No difference was found in
activity than do liver mitochondria.
amount of protein per mitochondrion or in enzyme activity
per mitochondrion. Cytochrome oxidase activity expressed
as per mg of mitochondrial protein was equivalent in both
DISCUSSION
mitochondrial preparations. This indicated that mitochonTable 1
Cytochrome oxidase activity
The isolation of rat liver mitochondria by differential
centrifugation is a classical method (2). However, several
modifications in the technique have been made. SEN, the
medium used here, was developed by Coote and Work (8)
and provides mitochondria capable of supporting protein
synthesis for longer time periods than the simple 0.25 M
sucrose medium used before. An increase in the number of
washes of mitochondria! pellets (4) serves to remove
successively more of the contaminating enzyme activity of
other cellular membranes. It was expected that this method
would suffice for the preparation of hepatoma mitochon
dria. However, both the yield and purification of mitochon
dria prepared in this medium from hepatoma were very
poor. The mitochondria would not support protein synthe
sis, would not resuspend without clumping in SEN, and
1992
Table 2
SDH activity of normal, host, and hepaloma mitochondria
mitochondrion
lo)2.78
(mgx 10±0.319°
Normal liver isolated in
SEN
Normal liver isolated in
2.91
±0.3512.74
SENA
Host liver isolated in SENA
±0.297
Hepatoma isolated in SENAProtein/3.03 ±0.349SDH
activity:
Formazan/hr/
mitochondrion
(nmoies
IO'7)9.37
x
±1.05"
9.67
0.99*9.57
±
±1.12"
3. 18 ±0.486
1Mean ±S.E.
'Significantly different from hepatoma (p < 0.01).
CANCER
RESEARCH
VOL.
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34
Mitochondria and Enzyme Activity of Reuber Hepatoma
dria from normal liver prepared in the medium modified by
the addition of BSA were equivalent to mitochondria
prepared by more classical means and that no albumin or
cellular material had adhered to the mitochondria thereby
altering their enzymatic characteristics or protein content.
The enzymatic purification of hepatoma and host liver
mitochondria prepared in SENA was also examined. En
zymatic data indicated that these mitochondria were sepa
rated from plasma membrane and microsomes, but not
lysosomes. These data demonstrate that preparation of
mitochondria by differential centrifugation of host liver and
hepatoma in SENA medium is equivalent to that of normal
liver in SEN or SENA.
The mitochondria of hepatoma, host, and normal liver
prepared in SENA have the same amount of protein per
mitochondrion and cytochrome oxidase activity per mg of
protein. One enzyme, succinic dehydrogenase, exhibits less
activity per mitochondrion in hepatoma than in normal or
host liver and thus less activity per mg of mitochondrial
protein. These data answer the question of the validity of
expressing the specific activity of various mitochondrial
functions on a mg protein basis. Mitochondrial activities
will not be affected either by inherent differences in the
mitochondrial protein content or by isolation of the mito
chondria in an albumin-containing medium.
The data presented here, which show no difference in
amount of protein per mitochondrion of hepatoma, host, or
normal liver, agree with those of White and Tewari (30).
These investigators examined the protein and enzymatic
content of Novikoff hepatoma, and of host and normal
livers of female Sprague-Dawley rats. The amount of
protein per mitochondrion can be calculated from their data
to be 2.01 x 10~'°mg/mitochondrion for hepatoma, and
2.4 x 10 10and 2.1 x 10~10mg/mitochondrion for normal
and host liver, with no significant differences. Therefore,
they conclude that the several enzymes they find to be lower
in activity in hepatoma (for example, succinic dehydrogen
ase) are not due to changes in gross protein content but to
specific enzymatic activity alterations. Cytochrome oxidase
was among the enzymes they found not to be different on a
protein specific activity basis among the 3 types of mito
chondria. They propose, on the basis of which enzyme
activities are different in hepatoma mitochondria from
those of liver, that the malignant state alters the outer
membrane and intramembrane space enzymes but not the
inner membrane. The enzyme data of Reuber H-35 hepa
toma would tend to support that hypothesis, based only on
succinic dehydrogenase and cytochrome oxidase activity.
Further work investigates the proteins and glycoproteins of
hepatoma mitochondrial membranes, compared with nor
mal and host liver (18, 19).
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CANCER
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Mitochondrial Protein Content and Enzyme Activity of Reuber
Hepatoma H-35
Marjorie W. Myers and H. Bruce Bosmann
Cancer Res 1974;34:1989-1994.
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