J. Cell Sri. 57, 1-13 (1982)
Printed in Great Britain © Company of Biologists Limited 1982
FRACTIONATION OF ISOLATED LIVER
CELLS AFTER DISRUPTION WITH A
NITROGEN BOMB AND SONICATION
F. AUTUORI, U. BRUNK, E. PETERSON AND G. DALLNER
Department of Biochemistry, Arrhenius Laboratory, University of Stockholm,
Department of Pathology at Huddinge Hospital, Karolinska Institutet, Stockholm,
and Department of Pathology, University of Linkoping, Linkoping, Stveden
SUMMARY
Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and
smooth microsomes and mitochondria were prepared after cell disruption. By applying
1000 lb/in ! (1 lb/in 1 = 69 kPa) in a nitrogen bomb followed by decompression, 75 % of the
cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and
smooth microsomes with little contamination were prepared from the homogenate. A more
rapid disruption was attained by a short sonication with a low output, thus increasing the
efficiency of homogenization. The microsomal subtractions prepared from this homogenate
were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These,
however, were partly removed by additional centrifugation with a discontinuous sucrose
gradient containing divalent cations.
INTRODUCTION
Fractionation of isolated cells often entails significant problems. While disruption
of the cell membrane of solid tissue can easily be achieved by normal homogenization
techniques, breakage of the cell membrane of isolated cells is often a difficult task.
Obviously, reorganization of membrane components occurs when cells exist as
individual entities without cooperation with neighbouring cells.
It is easy to break up hepatocytes with a standard teflon-glass homogenizer when
pieces of liver tissue are used. On the other hand, when hepatocytes are first isolated
and then homogenized, only a very small number of cells are broken up. Presumably,
the application of a large mechanical force would break up the cell membrane, but
such a procedure would most probably damage the cytoplasmic organelles. In
previous eexperiments various types of bacteria and animal and plant cells were
broken up by the application of high pressure with an N 2 bomb (Fraser, 1951;
Wallach, Soderberg & Bricker, i960; Hunter & Commerford, 1961; Dowben,
Gaffey & Lynch, 1968; Loewus & Loewus, 1971; Short, Maines & Davis, 1972).
In some cells, such as ascites tumour cells, the homogenate obtained was fractionated
and chemical and enzymic analyses of the isolated membranes were performed
(Wallach et al. i960; Wallach & Ullrey, 1964). Disruption of hepatocytes by sonication has also been reported (Gellerfors & Nelson, 1979). Hence the possibility arose
2
F. Autuori, U. Brunk, E. Peterson and G. Dallner
that these procedures, under controlled conditions, might also be useful for hepatocyte
fractionation.
In order to isolate unbroken microsomal vesicles with similar permeability properties to those of microsomes prepared from liver tissue, we applied both a nitrogen
bomb and short sonication as homogenization procedures for isolated hepatocytes.
These experiments demonstrate that homogenization of collagenase-isolated hepatocytes can provide reasonable starting material for isolation of intact intracellular
organelles.
MATERIALS AND METHODS
The rats were anaesthesized by injection of o-2 ml nembutal intraperitoneally and the portal
vein was canulated. Perfusion was performed as previously described (Mokteus, Hflgberg &
Orrenius, 1978). The first perfusion fluid was 150 ml Hanks' buffer containing 0-5 mM-EGTA
and 2% albumin. The second perfusion fluid was 100 ml Hanks' buffer containing 0-12%
collagenase (type V, Boehringer) and 2 mM-CaClj. The solutions were bubbled with carbogen
Isolated cells in sucrose (washed in buffer, — Mg1+, — Caa+)
I
I
N | treatment for 5 min, slow decompression
Centrifugation —r supernatant 1
Pellet, N , treatment
L
Centrifugation —r supernatant 2
Pellet, N , treatment
I
Centrifugation —r supernatant 3
Pellet, Nj treatment
I
Centrifugation —r supernatant 4
Supernatants 1 + 2 + 3 + 4 = broken cells (75 % of total)
Fig. 1. Schematic representation of hepatocyte disruption using a nitrogen bomb
(see Materials and Methods for details).
gas (95 % O,, 5 % CO,) and heated to 37 CC prior to use. The time of perfusion was 10 min
and 15 min for the first and second perfusion media, respectively. In order to decrease the
intracellular concentration of divalent cation the livers were shaken in Krebs-Henseleit buffer
without Mg'+ and Ca2+ to dissociate the hepatocytes. The isolated cell suspension was washed
first with the same Krebs-Henseleit buffer and then with 0-25 M-sucrose by centrifugation at
80 g for 5 min. The yield of hepatocytes from one rat liver was about 250 x io 6 cells. In our
experiments io 8 cells corresponded to about 2-5 mg protein. In a typical case, a 6 ml suspension
(70 x io 6 cells/ml) was diluted to 15 ml with 0-25 M-sucrose and placed in a cell disruption bomb
(Parr Instrument, Moline, 111.). The water phase was saturated with N s under continuous
magnetic stirring, pressure was kept at 1000 lb/in* (1 lb/in 1 = 6-9 kPa) for 5 min and, after
decompression, the non-broken cells were sedimented by centrifugation. The supernatant
was removed with a Pasteur pipette and retained (supernatant 1). The pellet was resuspended
in 15 times its volume of 0-25 M-sucrose, and treated with N , as before. The Whole procedure
was repeated twice. The four supernatants were mixed and used in further fractionations. The
disruption procedure is summarized in Fig. 1.
The suspension of disrupted hepatocytes was used to prepare both mitochondria and
microsomes. Mitochondria were prepared by first removing debris and nuclei by centrifugation
at 480 g for 10 min. The supernatant was decanted and the mitochondria were pelleted at
Fractionation of isolated liver cells
3
4300 g for 20 min. The pellet was resuspended in 0-25 M-sucrose and washed by centrifugation
at 4300 g for 15 min. This washing procedure was repeated and the final mitochondrial pellet
was resuspended in 0-25 M-sucrose (5 mg protein/ml). For preparation of total microsomes
the suspension of the disrupted cells was centrifuged at 10000 g for 20 min and the supernatant
(10000 g supernatant) was used to pellet the total microsomal fraction by centrifugation at
105000 g for 60 min. Rough and smooth microsomes were isolated by layering 3-5 ml of the
10000 g supernatant over 2 ml 1-3 M and 0-5 ml 06 M-sucrose solutions, both containing
15 mM-CsCl (Dallner, 1974). This gradient was centrifuged in a 40-2 rotor (Beckman) at
102000 g for 90 min. The smooth microsomes at the o-6 M/I-3 M-sucrose interface were
recentrifuged and the pellet, as well as the rough microsomal pellet, were resuspended in
0-25 M-sucrose. Disruption of hepatocytes in 35-ml samples of washed cell suspension (250 x
io 8 cells) was carried out by sonication with the fine tip of a Branson sonifier (model S-I25,
Branson Instruments Inc., Stamford, Conn.) at a setting of 0-5 A. Sonication was performed
in a cooling bath for 20 s. Subfractionations were performed as described above.
When contaminating membranes had been removed from the smooth microsomes isolated
from the sonicated hepatocyte suspension, the ioooog supernatant was supplemented with
7 mM-MgCl, and 4-5 ml of this was layered over 2 ml 1-15 M-sucrose-7 mM-MgCl| and
centrifuged at 105000 g for 30 min in a 40-2 rotor (Beckman). The pellet was resuspended
and used for measurements.
To determine contamination in isolated mitochondria and microsomes, various membrane
fractions of liver homogenates from starved rats were prepared. Lysosomes (Leighton et al.
1968), peroxisomes (Baudhuin, 1974), Golgi (Ehrenreich, Bergeron, Siekevitz & Palade, 1973)
and plasma membranes (Coleman, Michell, Finean & Hawthorne, 1967) were isolated using
established procedures and used for determination of specific marker enzymes. The values
obtained were, for acid phosphatase (lysosomes) i'i2 ftmo\ P|/min per mg protein, for urate
oxidase (peroxisomes) 0-36 fimol urate oxidized/min per mg protein, for UDP-galactosyl
transferase (Golgi) 1-67 nmol galactose transferred/30 min per mg protein and for AMPase
(plasma membrane) 0-83 fimol P|/min per mg protein. These specific activities were used to
calculate the percentage contamination (on a protein basis) in the isolated hepatocyte fractions
Activities of cytochrome c oxidase and monoamino oxidase in the mitochondria and NADPHcytochrome c reductase in the microsomes were identical in the fractions obtained either from
whole liver homogenate or from isolated hepatocytes.
Protein was measured according to Lowry, Rosebrough, Farr & Randall (1951). Both lipid
and RNA content were analysed as described previously (Ceriotti, 1951; Dallner, Siekevitz &
Palade, 1966). The various enzyme activities were determined using previously described
procedures (Sottocasa, Kuylenstierna, Ernster & Bergstrand, 1967; Eriksson, 1973; Beaufay
et al. 1974). All data in the Tables show representative results chosen from five to nine identical
and consecutive experiments.
Tissue samples used for electron-microscopic observations were fixed in 3 % glutaraldehyde
in o-i M-Na cacodylate-HCl buffer with o-i M-sucrose (pH 7-2), at +4°C overnight. They
were finally fixed in 1 % osmium tetroxide in 0-15 M-Na cacodylate-HCl buffer, (pH 7-2),
for 90 min at room temperature. The pellets were fixed in 1 % osmium tetroxide in 0-15 MNa cacodylate-HCl buffer (pH 7-2), for 60 min at +4 °C. The tissue samples and pellets were
dehydrated and embedded in Epoxy resin.
RESULTS
A prerequisite for the isolation of well-preserved cellular fractions is the availability
of isolated cells of high quality. Fig. 2 verifies that our procedure using calcium
depletion and collagenase is a suitable way to obtain unchanged liver cells. The
hepatocyte is well preserved, the membrane structures are delimited, the mitochondria are mostly in the condensed state, the endoplasmic channels are narrow
and most of the ribosomes appear to be membrane-attached. Clearly, the cells
isolated in these experiments do not show any sign of morphological damage and are
F. Autuori, U. Brunk, E. Peterson and G. Dallner
Fig. 2. Hepatocyte isolated by perfusion of rat liver with collagenase. Well-delimited
organelles with distinct membranes were noted. Neither mitochondrial swelling,
lysosomal rupture, dilatation, nor degranulation of endoplasmic reticulum was
recorded, x 21000.
Fractionation of isolated liver cells
5
2+
consequently suitable for subfractionation studies. The decrease in Ca concentrations during perfusion with collagenase and the elimination of divalent cations from
the Krebs buffer was not deleterious to the cell morphology or cell function, but
decreased the yield of cells and also the yield of fractions obtained after disruption.
If the Ca 2+ concentration was increased in the collagenase perfusion and divalent
cations were included in the Krebs buffer, separation of rough and smooth microsomes could not be achieved with the procedure employed.
The pressure used in a French press to break most cells varies from 20 to 60000 lb/
2
in (x 6-9 kPa) and for this reason cannot be used when isolating most intracellular
particles. By using a pressure of 1000 lb/in J (x 69 kPa), hepatocyte disruption is
only partial (Table 1). Saturation of the water phase with nitrogen followed by a
Table 1. Effect of nitrogen-bomb disruption on isolated liver cells
Fraction
Protein
(mg)
Released protein
(% of total)
—
Washed cells
310
ist disruption
—
210
Pellet
Supernatant
84
27
2nd disruption
—
Pellet
147
20
62
Supernatant
3rd disruption
—
Pellet
97
Supernatant
IS
47
4th disruption
—
Pellet
52
40
Supernatant
13
The preparation of cells and treatment with the nitrogen-bomb system were performed as
described in Materials and Methods. The values given for the released protein are expressed
as the ratio between the value for the supernatant after centrifugation and that of the washed
cells.
relatively slow release of pressure breaks up about 0-25 of the cells, since 27% of
the total protein is not sedimented after short centrifugation. When the pellet, after
the first disruption, is homogenized again and the procedure repeated three more
times, about 75 % of the protein is found in the supernatant, indicating an effective
disruption of the cells. The yield obtained by both disruption bomb and sonication
is about the same as the yield after homogenization of whole liver (Table 2). Chemically, the mitochondria and the microsomal fractions isolated from hepatocytes by
both procedures exhibit a composition similar to that found in liver tissues. The
lipid/protein ratio for mitochondria and rough and smooth microsomes does not
differ from those given in the literature for particles isolated by homogenization
of the liver. The RNA/lipid ratio indicates that a successful separation of rough and
smooth microsomes was attained.
F. Autuori, U. Brunk, E. Peterson and G. Dallner
Table 2. Chemical composition of nritochondrial and microsomalfractions
Preparation
Protein
(mg)
Lipid
(mg)
RNA
(mg)
Lipid/protein
RNA/lipid
Nitrogen bomb
—
—
—
222
—Homogenate
—
015
630
42
—
Mitochondria
0-29
O'3O
27
Rough micro8omes
7-83
2-34
0-41
369
0-09
Smooth microsomes
9
o-33
Sonication
—
—
—
Homogenate
—
31O9
—
0-16
Mitochondria
5-28
—
33
027
21
Rough microsomes
5-67
i-93
o-34
10
038
3-80
0-09
Smooth microsomes
°'34
Cells were broken up either with the nitrogen bomb or by sonication, and fractions were
prepared. Homogenate denotes the total released protein after four disruption cycles (supernatants 1-4 in Table 1) when the N t bomb was used in homogenization, and it denotes the
600 g supernatant (supernatant after centrifugation of the sonicated cell suspension at 600 g
for 10 min) when sonication was applied for homogenization.
Fig. 3. The mitochondrial fraction, isolated from hepatocytes treated with a nitrogen
bomb. Mitochondria in either the condensed or the orthodox state and with intact
outer membranes, and only a few contaminating smooth vesicles could be seen,
x 15000.
Fractionation of isolated liver cells
y
The mitochondrial fraction isolated by the nitrogen-bomb treatment consists of
intact mitochondria partly in the condensed and partly in the orthodox state (Fig. 3);
morphologically, they are not damaged and have not lost their outer membranes.
Electron-microscopic analysis of fractions from cells disrupted by the nitrogen bomb
showed rough microsomes as ribosome-covered intact vesicles and smooth microsomes as intact smooth vesicles of varying sizes (Fig. 4A,B). A few vesicles in the
rough microsomal fraction were devoid of ribosomes and some of the ribosomes
were in the free form. The vesicles in this fraction contain fewer bound ribosomes
than the rough microsomes isolated from liver tissue. Free ribosomes are also present
in the smooth microsomal fraction, which also contains Golgi elements and larger
vesicles, which probably originated from plasma membranes.
Table 3. Oxidation of NADH by isolated mitochondria prepared from hepatocytes
disrupted by nitrogen bomb or sonication
Preparation
Addition
1000 lb/in1
None
KCN
None
KCN
o-i % deoxycholate
o-i% deoxycholate + KCN
None
1500 lb/in1
1000 lb/in1
Sonication
KCN
NADH oxidized
(/tmol/min per mg protein)
O-II
O-0O2
054
0-003
I-2I
O-OO2
O-I4
0-002
Cells were treated with the nitrogen bomb four times, as described in Materials and Methods.
The value in lb/in 1 (1 lb/in 1 — 6-9 kPa) given in the table was applied during the whole
procedure. Sonication was performed as given in Materials and Methods. The concentration
of KCN was 1 nw.
The nitrogen-bomb treatment employed is a sufficiently mild procedure for
preparation of mitochondria from hepatocytes. If the mitochondria are prepared as
described in Materials and Methods by using 1000 lb/in 2 (x 6-9 kPa), penetration
by NADH is very limited, since the oxidation rate of the reduced co-enzyme is only
o-i of that obtained after the addition of deoxycholate (Table 3). The relatively
moderate increase of the pressure to 1500 (lb/in2) damages permeability seriously,
as demonstrated by the elevation of the NADH oxidation from o-n to 0-54/imol/
min per mg protein. The sonication used in these experiments also results in mitochondria that are non-permeable to NADH.
The situation is very similar with microsomes. These particles display high
nucleoside diphosphatase activity when the cells are disrupted with 1000 lb/in a
pressure, while the enzyme activity in the supernatant, representing the amount of
liberated nucleoside diphosphatase, is low (Table 4). Increasing the pressure to
1700 lb/in 2 interferes with membrane integrity and solubilizes about 0-25 of the
microsomal enzyme activity. Short sonication also causes a similar release of the
enzyme.
F. Autuori, U. Brunk, E. Peterson and G. Dallner
Fig. 4. Microsomal subfractions prepared from hepatocytes disrupted by a nitrogen
bomb. A. Rough microsomes; intact vesicles with attached ribosomes and some
contamination with smooth vesicles as well as groups of free ribosomes were observed.
B. Smooth microsomes; intact smooth vesicles with varying sizes, contaminating Golgi
elements and groups of free ribosomes could be seen, x 40000.
Fractionation of isolated liver cells
9
Table 4. Nucleoside diphosphatase activity in microsomes and supernatant of hepatocytes
after cell disruption using varying techniques
Expt
Preparation
Activity
{jimo\ P,/min per mg protein)
1000 lb/in 1
Microsomes
Supernatant
1700 lb/in !
Microsomes
Supernatant
Sonication (20 s)
Microsomes
Supernatant
086
0044
o-6i
013
0-63
O'I2
Cells were broken up either with the N , bomb (1000 lb/in 1 in Expt 1 and 1700 lb/in* in
Expt 2 or by sonication (20 s, Expt 3). Nucleoside diphosphate activity was measured in the
microsomal fraction and in the remaining supernatant after centrifugation at 105000 g for
60 min.
Table 5. Distribution of various enzymes in the mitochondrialfraction isolated by nitrogenbomb treatment of hepatocytes
Mitochondria
Cytochrome c oxidase"
Monoamino oxidase6
NADPH-cytochrome c reductase*
Acid phosphatase11
Urate oxidase"
UDP-galactosyl transferase'
AMPase"
Specific
activity
Calculated
contamination
(% of total protein)
i'3S
15-2
—
—
0002
0-07
004
005
001
3
6
11
3
1
Enzyme activities were measured in lysosomes, peroxisomes, Golgi and plasma membranes
isolated from liver tissue from starved rats. These values were used to calculate the contamination in the mitochondrial fraction of the isolated hepatocytes.
"/imol cytochrome c oxidase/min per mg protein; 6nmol bensaldehyde/min per mg protein;
c
/tmol NADPH oxidized/min per mg protein; d/J.mo\ P,/min per mg protein; 'fimo\ urate
oxidized/min per mg protein; 'nmol transferred/30 min per mg protein.
The mitochondrial fraction exhibits a high specific activity of both cytochrome
oxidase and monoamino oxidase, which tallies with the results of electron microscopy
showing the presence of predominantly mitochondrial elements (Table 5). Contamination of the mitochondrial fraction with other intracellular membranes is limited
and was calculated by measurements of the marker enzyme activities both in this
fraction and in organelles isolated from liver tissue. Microsomes (NADPH-cytochrome
c reductase), lysosomes (acid phosphatase), Golgi membranes (UDP-galactosyl
transferase) and plasma membranes (AMPase) contribute to the total protein by 3,
io
F. Autuori, U. Brunk, E. Peterson and G. Dallner
6, 3 and i%, respectively. The greatest contamination is due to peroxisomes (urate
oxidase), which make up n % of the total protein. Calculation of contamination in
mitochondria and in microsomes is based on the assumption that the appropriate
marker enzyme is present exclusively in one subcellular organelle. This is not quite
true since most of the marker enzymes are known to be present at several locations
(DePierre & Dallner, 1976). On the other hand, this fact influences the conclusion
only to a moderate extent and gives an overestimate of the degree of contamination.
Rough and smooth microsomes can be separated with a sucrose gradient containing monovalent cations if the perfusion medium used for isolation of the hepatocyte'
contains a relatively low concentration of divalent cations. As expected, rough
Table 6. Distribution of various enzymes in the microsomal fractions isolated by nitrogen
bomb treatment of hepatocytes
Rough microsomes
Smooth microsomes
A
Calculated
contamination
Specific
(% of total
activity
protein)
Specific
activity
Calculated
contamination
(% of total
protein)
—
—
NADPH-cytochrome c reductase"
0044
0-041
4-01
—
—
Glucose-6-phosphataseb
4-52
05
0
003
0
Cytochrome c oxidase0
1
021
Monoamino oxidase d
0-56
4
008
Acid phosphataseb
0-12
5
9
1
2
0-006
Urate oxidase'
o-oi
O-O2
1
O-II
UDP-galactosyl transferase/
7
1
OO08
2
0015
AMPase"
Enzyme activities were measured in lysosomes, peroxisomes, Golgi and plasma membranes
isolated from liver tissue of starved rats. These values were used to calculate the contamination
in the microsomal fraction of isolated hepatocytes.
"fimol NADPH oxidized/min per mg protein; b/imo\ Pt/min per mg protein; "fimol cytochrome c oxidized/min per mg protein; dnmol bensaldehyde/min per mg protein; 'fimol
urate oxidized/min per mg protein; 'nmol transferred/30 min per mg protein.
microsomes isolated from the nitrogen-treated cells are less contaminated by other
membranes than are smooth microsomes (Table 6). Rough microsomal membranes
make up 90% of the fraction. The smooth microsomal fraction is contaminated with
a greater percentage of membrane proteins, as the sedimentation velocity for smooth
vesicles is close to that of other cy loplasmic membranes in the homogenate. Considerable amounts of Golgi membranes (7%) and lysosomes (9%) are distributed in the
fraction and must be taken into consideration in fractionation studies.
Rough and smooth microsomes can be prepared easily and rapidly from hepatocytes, even after sonication. As shown in Table 7, the rough microsomes obtained
were similar to those prepared in the nitrogen-bomb system. On the other hand,
smooth microsomes exhibit an increased contamination with outer mitochondrial and
plasma membranes, as is apparent from the considerably increased activity of monoamino oxidase and AMPase.
Fractionation of isolated liver cells
11
Table 7. Distribution of various enzymes in the microsomal fraction prepared by
sonication
Rough microsomes
Smooth microsomes
f
Specific
activity
NADPH-cytochrome c reductase"
Glucose-6-phosphatase6
Cytochrome c oxidase
Monoamino oxidase
Acid phosphatase
Urate oxidase
UDP-galactosyl transferase
AMPase
0037
C36
—
—
—
—
—
—
Calculated
contamination
(% of total
protein)
Specific
activity
—
—
0-044
0-40
1
—
—
3
6
2
1
1
Calculated
contamination
(% of total
protein)
—
—
—
—
o-5
9
9
2
6
8
Enzyme activities were measured in lysosomes, peroxisomes, Golgi and plasma membranes
isolated from liver tissue of starved rats. These values were used to calculate the contamination
in the microsomal fraction of isolated hepatocytes.
°/imol NADPH oxidized/min per mg protein; '/irnol P,/min per mg protein.
Table 8. Removal of plasma membrane fragments from smooth microsomes prepared after
sonication
Smooth microsomes
(calculated contamination)
as % of total protein
Control
Cytochrome c oxidase
Monoamino oxidase
Acid phosphatase
Urate oxidase
UDP-galactosyl transferase
AMPase
Recentrifuged on
Mg I+ gradient
9
9
o-5
6
3
2
2
6
8
5
05
2
The isolated smooth microsomes were recentrifuged on a discontinuous gradient containing
Mg'+ as described in Materials and Methods. Contaminations were calculated as described
in Table 5.
Some of the non-microsomal membrane material, in particular plasma membranes,
can be removed by placing the fraction on a second discontinuous sucrose gradient
containing MgCl2 (Table 8). Obviously, some of the cytoplasmic membranes are
insensitive to divalent cations and do not precipitate and sediment like smooth
microsomes when this system is used. In this way a smooth microsomal fraction can
be produced that is similar in composition to the fraction obtained by centrifugation
of the nitrogen bomb-disrupted hepatocyte.
12
F. Autuori, U. Brunk, E. Peterson and G. Dallner
DISCUSSION
Homogenization of the isolated hepatocytes, like homogenization of most individual
cells, is a difficult task. Breakage of cell membranes requires the application of considerable shearing forces and the same procedure may therefore cause damage, not
only to the plasma membrane itself, but also to most of the cytoplasmic organelles.
Consequently, homogenization of individual hepatocytes requires much more closely
controlled conditions than homogenization of the liver itself.
The use of a nitrogen-bomb system based on decompression in a pressure vessel
was found to be advantageous in several previous investigations. This principle is
used for the disruption of tissues such as liver and spleen, ascites tumour cells, L cells
and fibroblasts, and also the preparation of submitochondrial vesicles from beef
heart mitochondria (Wallach et al. i960; Hunter & Commerford, 1961; Molnar,
1967; Dowben et al. 1968; Short et al. 1972; Fleischer, Meissner, Smigel & Wood,
1974). The advantage of such a system is obvious for many reasons: the force applied
is well defined and repeatable, no heat is generated during disruption, oxygen is
excluded by the use of inert gas and there is a uniform effect throughout the solution.
A pressure of 1000 lb/in 2 was the maximum applicable, because at higher pressures
the mitochondrial membrane permeability for NADH was impaired and the microsomes lost some intraluminal protein components. However, it was necessary to
repeat the disruption procedure four times in order to break up 75 % of the cells.
In this way it was possible to obtain intact microsomes with reasonable recovery.
The nitrogen-bomb system has the disadvantage of requiring more time to obtain
microsomal fractions than is advisable when studying enzymes and enzyme systems.
The sonication procedure is rapid and easy to use even under varying experimental
conditions. The recovery of microsomal subfractions is similar to that seen in the
nitrogen-bomb system; however, there is an increased contamination of the smooth
microsomes with other membranes. This contamination can be partially reduced
by using a Mg2+-containing gradient. In most membrane studies sonication is the
method of choice since it is rapid, simple, reproducible and gives high recovery.
Clearly, the loss of some nucleoside diphosphatase during sonication does not mean
an irreversible change in membrane permeability, since substrate permeability is
not changed and the enzymes localized on the inner surface require the addition of
detergents to obtain full activity. It appears, therefore, that the microsomal vesicles
obtained by the sonication procedure exhibit, at least to a large extent, the same
properties as vesicles obtained by other procedures.
This work was supported by grants from the Swedish Medical Research Council and the
National Cancer Institute (grant no. 1 RO ICA 26261-01.). Dr F. Autuori was on leave of
absence from the Institute of Histology and Embryology, Faculty of Science, University of
Rome, Italy.
Fractionation of isolated liver cells
13
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(Received 22 October IQ8I - Revised 27 April iq82)