Download Studies on Liver Plasma Membranes of Rats Fed

[CANCER RESEARCH 30, 2876—2880,December 1970]
Studies on Liver Plasma Membranes of Rats Fed
N-2-Fluorenylacetamide'
N.
Chandrasekhara2
and
K.
Ananth
Narayan3
TheBurnsidesResearchLaboratory, University oflllinois,
Urbana,Illinois 61801
SUMMARY
study was to investigate the lipid composition of membranes
at 2 different stages of carcinogenesis. In this article, we report
Plasma membranes were prepared from livers of normal and
N-2-fluorenylacetamide-ingesting
rats. Of the two marker
enzymes studied, the activity of 5'-nucleotidase was markedly
decreased, whereas that of Mg2@-Na3-r-ATPase was unaffected
striking
in the membranes
from animals
fed the carcinogen.
normal liver. Unlike in preparations from normal livers, a large
amount of floating fraction (d < 1.16) was invariably observed
from preneoplastic
and neoplastic
livers.
INTRODUCTION
The probable involvement of the cell surface in the neo
plastic process has been indicated by several studies. Among
these may be cited the work of Abercrombie et aL (1) regard
ing contact inhibition; studies on the altered electrophoretic
mobility of tumor cells (2), the decreased intercellular com
munication
between
cancer
cells (15),
and
the
decreased
incidence of the tight junction (12); and the more recent ob
servation by Burger (4) of a difference in the architecture of
the surface membrane of virally transformed cells.
As a result of the development of techniques for the isola
tion of plasma membranes from rat liver (18, 23), hepatomas
(13), mouse fibroblast cells (26), Ehrlich ascites carcinoma
(25), and HeLa cells (3), it has been possible to study the
properties of the cell surface more directly. Wallach (24) has
hypothesized that neoplasms would appear as a result of
alterations critically involving the plasma membrane.
There has been no study regarding changes in lipid composi
tion and the characterization of the proteins of rat liver plasma
membranes during carcinogenesis. The main object of this
1 Supported
by
a grant
from
the
American
Cancer
Society
and
Grant
CA-01932 from the USPHS. A preliminary report has been published
(6).
2 On
leave
from
the
Biochemistry
Division,
Central
Food
in the phospholipid:cholesterol
ratios and in
The per
centage of phospholipids in the preneoplastic and neoplastic
liver membranes was very much higher than in the controls,
and the phospholipid :cholesterol ratio was also significantly
enhanced. The percentage of polyunsaturated fatty acids was
higher in these membranes than in the plasma membranes of
in membrane preparations
changes
the relative levels of total phospholipids in membranes from
both preneoplastic and neoplastic livers, in comparison with
the ratios and levels in membranes from normal liver.
MATERIALS
AND METHODS
One group of male weanling rats (60 g, Holtzman Co.,
Madison, Wis.) was fed a corn oil diet (10 g oil/lOO g diet), and
2 other groups were fed the corn oil diet, containing 0.05 and
0.3 g FAA4/kg (16) (Eastman Organic Chemicals, Rochester,
N. Y.). At the end of 20 weeks on the diet, the livers of the
rats fed the high level of FAA were composed of large tumors,
whereas livers from the rats fed the lower level of the car
cinogen were in the preneoplastic stage (1 6). The average liver
weight of the rats in the present experiment was: normal,
11.3 ; low FAA, 14.2; and high FAA, 20.4. The animals (not
fasted) were sacrificed under Nembutal anethesia and bled
through
the abdominal
aorta. Sera from 2 animals were pooled
and constituted 1 sample. Livers were similarly pooled, and 20
g were used for the preparation of plasma membranes. In brief,
the procedure was as follows. The 1,500 X g pellet obtained
according to the procedure of Neville (18) was homogenized
with 3 to 4 strokes of the loose-fitting pestle of a Dounce
homogenizer. The density of this homogenate was adjusted to
1.197 with sucrose of density 134. Twenty-eight to 30 ml of
this solution formed the bottom layer in a centrifuge tube for
the SW 25.2 rotor. Twenty ml of sucrose of density 1.18 and
10 ml ofsucrose ofdensity 1.16 were layered and centrifuged at
98,500
X g for 150 mm. After collection,
the floating layer (d
< 1.16) and the plasma membranes (d 1J6 to 1.18) Were
diluted with 4 volumes of 1 mM NaHCO3 and centrifuged at
10,000
X g for 30 mm. The pellets were dispersed
in 1 mM
NaHCO3 .
Lipid, phospholipid, and protein determinations; gas chro
matographic analyses of fatty acids; and the assays for
5'-nucleotidase (EC 3.1 .3.5), ATPase (EC 3.6.1 .4), and glucose
6-phosphatase (EC 3.1 .3.9) were all carried out as reported
before (5). Succinic dehydrogenase (EC 1.3 .99. 1) was assayed
according to the method of Shelton and Rice (22). All sub
Techno
logical Research Institute, Mysore, India.
3 Recipient
of
a
Research
Career
Development
31,063 from the National Cancer Institute.
Received June 29, 1970; accepted August 21, 1970.
2876
Award,
5K3-CA
4The abbreviations used are: FAA,N-2-fluorenylacetamide;ATPase,
Mg@-Na@-K@-adenosine
triphophatase, all 3 ions included in the incuba
tion medium; EFA, essential fatty acid.
CANCER RESEARCH VOL. 30
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Hepatoma Plasma Membranes
strates
were purchased
from Sigma Chemical
Co., St. Louis,
among
Mo. All enzyme assays were done at 2 enzyme concentrations,
and the incubation time and concentration of enzyme were
adjusted to conform to zero-order kinetics.
the 3 groups of livers in the present
experiments
with
respect to the ease ofhomogenization. The yield was about 5 mg
membrane protein from 20 g normal liver and nearly twice as
much from livers of animals fed the carcinogen.
As indicated in Table 1, the specific activities of
5'-nucleotidase and ATPase were lower in liver homogenates of
FAA-fed rats than in normal liver homogenates. The specific
activities of these 2 marker enzymes in membranes from
RESULTS
normal livers were enhanced about 15- and 5-fold as compared
with the homogenate.
These enzyme activities were in the
Unlike EFA-deficient livers, which were more easily
homogenizable than normal livers (5), there was no difference
range reported in the literature (5, 13). An important observa
tion in the present experiments was that membranes from
both preneoplastic
5'-nucleotidase
Table 1
However,
Enzyme activities ofliver and plasmamembranes
Results are means ±S.D. of 6 preparations (each preparation was
obtained from 2 rats).Inorganic
Sample
LH°
3.1 ±0.6
7.4 ±1.3
2.3 ±
0.4―
5.9 ±
0.3―
0.6cControl
High FAA
LH
2.0 ±O.3@
5.5 ±
PM
45.1 ±6.1
36.7 ±7.9
Low FAA
PM
26.9 ±
6.3'@
37.0 ±3.6
High FAA
PM
19.6 ±7•6d
31.8 ±6.9
PM, plasma membrane.
and high FAA,
0.05
and 0.3 g FAA/kg
diet,
there was no change in the ATPase activity.
Similar
animals (5), succinic dehydrogenase activity of membranes
from animals fed FAA was higher than that of membranes
S'-Nucleotidase
LH
b i.ow
lower
from normal livers.
suggested that there was very little contamination
with micro
somes. As in the case of membranes
from EFA-deficient
Low FAAb
a LH, liver homogenate;
livers had a markedly
changes in enzyme activities were observed by Emmelot and
Bos (13) in hepatoma membranes. Glucose 6-phosphatase
activity was quite low in membranes from all groups, and this
phosphate formed
(jsmoles/mg protein/hr)
ATPaseControl
Diet
and neoplastic
activity than membranes
respectively.
from normal
animals.
On the basis of experiments
with iso
lated mitochondria under identical conditions, such con
tamination was estimated to be less than 10% (N. Chandra
sekhara and K. A. Narayan, manuscript in preparation).
As shown in Table 2, the lipid composition of plasma
membranes from normal liver was similar to the values re
ported by Dod and Gray. (1 1), Pfleger et a!. (20), and Ray et
a!. (21). One of the significant fmdings of this study was a
“p
<0.05.
dp <0.005.
marked
increase
membranes
in the percentage
of total
from both preneoplastic
phospholipids
in
and neoplastic livers in
Table 2
Lipid composition ofserum, liver, and plasmamembranes
Results
are means ±S.D. of 6 preparations
(each preparation
was obtained
from 2 rats), except in the
case of serum from the high FAA group, in which the results were obtained from 4 preparations.
as
ratiomg/mg
% of total lipidTotal
lipidPhospholipids
DietSampleTotal
[email protected]
0.1Low
o.2eHigh
FAAbPM0.45
±0.0359.3
±0.04―71.4
[email protected]
FAAPM0.53
±0.1679.3
0.5Low
±0.0357.7
0.2cHigh
FAALH0.21
±ControlSerummg/100
FAALH0.22
1.5High
FAASerum361.6
FAASerum499.2
a PM,
plasma
membrane;
b Low and high FAA,
Cp
@
±2.414.4
±2•3d14.2
±65d13.7
±1.42.1
±1.12.5
±2.53.0
±4.65.5
±2.26.8
±
±
o.ole60.3
±0.02e66.5
±[email protected]
ml
289.7 ±32.741.2
1.6Low
LH,
0.05
liver
cholesterol,
cholesterol as
mole
% of total lipidPhospholipid:
±
±
±
±
±2.324.7
±
±[email protected]
±1.9e22.7
±
±291d41.6
±0.227.4
±2.1
homogenate.
and 0.3 g FAA/kg
diet,
respectively.
<0.01.
d@ <
ep < 0.05.
DECEMBER
1970
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2877
N. Chandrasekhara and K. Ananth Narayan
comparison with the percentage in membranes from normal
livers. However, the percentages of phospholipid in livers of
animals fed the carcinogen were only nominally increased in
comparison with those in animals with normal livers. On the
other hand, there was no change in the percentage of choles
terol in the membranes during carcinogenesis, although there
was an increase in the percentage of cholesterol in the liver.
The above changes were also reflected in a higher mole ratio of
phospholipid:cholesterol
in these membranes. The lipid:
protein ratio decreased significantly in the livers of animals fed
the carcinogen. There was a marked increase in the total lipids
of serum in FAA-fed animals, but little change was seen in the
percentage of phospholipid or cholesterol.
The fatty acid composition of the total lipids of plasma
membranes, livers, and sera is given in Table 3. No marked
difference was observed in the fatty acid composition of livers
from the 3 groups of animals. However, there was a significant
increase (p < 0.05) in the total polyunsaturated fatty acids and
a corresponding decrease in total saturated fatty acids in mem
branes from FAA-fed rats in comparison with the fatty acid
composition in membranes from normal rats. An increase in
18: 1 and 18:2 acids and a decrease in 18:0 and 20:4 acids
were observed in the sera of animals receiving the carcinogen.
However, there was no difference between the sera of the
control and experimental groups of animals when the satu
rated and the polyunsaturated acids were taken into considera
tion.
In studies with plasma membranes from EFA-deficient rats
Table 3
Fatty acid composition ofthe total lipids ofserum, liver, and plasmamembranes
Results are means ±S.D. of 6 preparations (each preparation was obtained from 2 rats), except in the case of serum from
preparations.DietSampleFatty
the high
FAAgroup, in which the results wereobtained from4
total16:016:118:018:118:220:4Control
1.8Control
High FAAPM@
PM
PM25.4
±4.0
±1.5
±0.8
±1.4
±0.8
±3.0
25.5 ±
18.9 ±2.5
2.7 ±O.8@
18.8 ±1.3
11.3 ±0.8
20.7 ±0.6c
21.8 ±[email protected] 2.3 ±0•8d19.416.8 ±[email protected] 11.1 ±1.517.6 21.8 ±2.0'@20.323.7 ±
±1.2
±0.5
Low FAA
LH
22.3 ±
2.5
3.8 ±1.6
2.9Control
High FAALH
LH23.0
21.7 ±3.03.3
3.2 ±0.711.6
Low FAA
High FAASerum
Serum
Serum19.0
±1.7
19.4 ±0.8
18.8 ±1.02.8
±0.5
3.5 ±O.S@
2.2 ±0.48.2
Low FAAb
@
acid as% of
a PM, plasma membrane;
b Low and high FAA,
Cp
±1.8
12.3 ±0.6
13.6 ±0.914.8
±2.0
21.3 ±4.0
16.4 ±0.8―25.224.2 ±2.320.5
±1.1
±2.0
16.5 ±1.3
20.8 ±4.3
±1.4
±1.3
±0.6
16.4 ±0.8'@
32.2 ±2.l@
6.9 ±O.6@
1.2e26.6
6.8 ±0.le13.4 15.5 ±[email protected]
19.1 ±
±3.1
20.7 ±13d
22.3 ±1.8
LH, liver homogenate.
0.05
and 0.3 g FAA/kg
diet,
respectively.
<0.05.
dp < 0.01.
ep < 0.001.
Table 4
Propertiesof the floating fraction
Results are means ±S.D. of 6 preparations (each preparation was obtained from 2 rats). The results for the floating fraction from EFA-deficient
rats were obtained from 5 preparations.
formedSample(jsmoles/mgInorganic phosphate
protein/hr)Total
lipid
ATPasePlasma
0.10fedmembranes from
±
5.2
34.5 ±3.80.59
±2.3
20.9 ±1.51.4
20.9 ±7.9
39.7 ±9.10.64
rats21.2
as
as
cholesterol,
% of total lipidPhospholipid:
% of total lipidCholesterol
mole
(mg/mg protein)Phospholipid
ratio5'-Nucleotidase
±0.0653.9
±
6.718.3
±2.31.5
±
±
2.416.5
±1.70.5
±
diet(17
0.3 g FAA/kg
diet)Floating
weeks on
0.10fed fraction from rats15.0
diet(17
0.3 g FAA/kg
diet)Floating
weeks on
0.77deficient
fraction from EFA
rats'@
a These data are from a previous experiment
2878
± 0.1016.5
±
0.0732.3
±1 1.77.3
±1.02.2
±
(5).
CANCER RESEARCH
VOL. 30
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Hepatoma Plasma Membranes
(5)
as well
as in the
a considerable
present
amount
experiments,
of material
it was observed
floated
that
on top of sucrose
with a density of 1.16. In comparison, 5 to 10 mg protein
collected as float in the case of preparations from livers of
both EFA-deficient and FAA-fed rats, whereas about 1 mg
protein floated at this density in normal preparations. The
properties of the floating fraction (obtained in a subsequent
experiment) are summarized in Table 4. The floating layer was
characterized by fairly high activities of 5'-nucleotidase and
ATPase and low activities of glucose 6-phophatase and succinic
dehydrogenase. However, the lipid:protein ratio was much
higher, and the percentage of phospholipid was much lower in
this fraction than in the corresponding plasma membranes.
The increased yield of plasma membranes and the presence
fraction
in preparations
from preneoplastic
and
neoplastic livers, in comparison with the yields in preparations
from normal livers, may perhaps be due to the presence of
increased quantities, of membrane precursors. Whether similar
increased yields would be obtained with rapidly growing
normal
tissue,
like the regenerating
liver, needs investigation.
It is fairly well established that 5'-nucleotidase is a useful
plasma membrane marker (12, 13). Although the total activity
in the membrane isolated under our experimental conditions is
only a small percentage of the total 5'-nucleotidase activity of
the liver, its presence at the cell surface is important in view of
the regulatory role of AMP in key enzyme systems. The ob
served decrease in 5'-nucleotidase activity of membranes from
FAA-fed rats may therefore be of greater significance than is
recognized at the present time.
In agreement with the earlier observations of Narayan (17),
we observed an increase in the total lipid content of serum in
the FFA-fed animals. However, the lipid content of preneo
plastic and neoplastic livers was significantly less than that of
normal liver. We also observed a marked change in the phos
pholipid: cholesterol ratio and a shift towards polyunsaturated
fatty acids in the total lipids of membranes from livers of rats
fed FAA. It has been shown that the permeability of lipo
somes is influenced by the cholesterol content (7, 8) and the
degree of unsaturation of the fatty acid moiety of the lecithin
(9). The importance of cholesterol and phospholipids in mem
branes
has also been demonstrated
in studies concerning
the
mode of action of polyene antibiotics (10). Therefore, it is
possible that the changes observed in the lipid composition of
hepatoma membranes may have a bearing on the increased
leakiness of the plasma membranes of cancer cells (24).
Emmelot and Bos (13) reported that hepatoma plasma
membranes had a lower density than normal liver plasma mem
branes, but we have observed that membranes from livers of
FAA-fed rats, as well as those from normal rats, floated be
tween d 1.16 andd 1.18. However, a floating fraction(d< 1.16)
was always observed in preparations from animals fed the car
cinogen. On the basis of the density of this fraction (d < 1.16),
the reported
density of 1.13 for the outer mito
chondrial membrane (19), and our similar results from the
DECEMBER
total
lipid:protein
and
a considerably
lower
content
distinguished
the floating
fraction
membranes.
Recently,
pholipid
plasma
ratio,
Evans (14)
1970
reported
phos
from
the sub
fractionation of normal plasma membranes (d 1.18) into a
low-density (d 1.12) vesicular fraction and a high-density (d
1.2 1) sheet-like fraction, which differed from each other in
chemical composition and enzyme activities. Whether plasma
membranes from livers of FAA-fed rats are more susceptible to
subfractionation is worthy of consideration.
In conclusion, it may be stated that important alterations
take place in the lipid composition
DISCUSSION
of the floating
preparation of plasma membranes from EFA-deficient rats (5),
the possibility that the fraction was the outer mitochondrial
membrane was considered. Assay for monoamine oxidase
ruled out this suggestion . A lower ATPase activity, a higher
of rat liver plasma mem
branes during carcinogenesis, and these lead to subtle changes
in the properties of the plasma membrane. Whether membrane
proteins are also similarly altered in neoplasia deserves to be
investigated.
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CANCER RESEARCH VOL. 30
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1970 American Association for Cancer Research.
Studies on Liver Plasma Membranes of Rats Fed N
-2-Fluorenylacetamide
N. Chandrasekhara and K. Ananth Narayan
Cancer Res 1970;30:2876-2880.
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