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. DALLNERDepartment 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

SUMMARYHepatocytes from rat liver were prepared by perfusion with collagenase, and rough and

smooth microsomes and mitochondria were prepared after cell disruption. By applying1000 lb/in! (1 lb/in1 = 69 kPa) in a nitrogen bomb followed by decompression, 75 % of thecells were disrupted after four consecutive treatments. Intact mitochondria, and rough andsmooth microsomes with little contamination were prepared from the homogenate. A morerapid disruption was attained by a short sonication with a low output, thus increasing theefficiency of homogenization. The microsomal subtractions prepared from this homogenatewere comparable to those obtained after decompression. Sonication resulted in smooth micro-somes, which exhibited a higher contamination with non-microsomal membranes. These,however, were partly removed by additional centrifugation with a discontinuous sucrosegradient containing divalent cations.

INTRODUCTION

Fractionation of isolated cells often entails significant problems. While disruptionof the cell membrane of solid tissue can easily be achieved by normal homogenizationtechniques, breakage of the cell membrane of isolated cells is often a difficult task.Obviously, reorganization of membrane components occurs when cells exist asindividual entities without cooperation with neighbouring cells.

It is easy to break up hepatocytes with a standard teflon-glass homogenizer whenpieces of liver tissue are used. On the other hand, when hepatocytes are first isolatedand 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, butsuch a procedure would most probably damage the cytoplasmic organelles. Inprevious eexperiments various types of bacteria and animal and plant cells werebroken up by the application of high pressure with an N2 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 fractionatedand chemical and enzymic analyses of the isolated membranes were performed(Wallach et al. i960; Wallach & Ullrey, 1964). Disruption of hepatocytes by soni-cation 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 hepatocytefractionation.

In order to isolate unbroken microsomal vesicles with similar permeability proper-ties to those of microsomes prepared from liver tissue, we applied both a nitrogenbomb and short sonication as homogenization procedures for isolated hepatocytes.These experiments demonstrate that homogenization of collagenase-isolated hepato-cytes can provide reasonable starting material for isolation of intact intracellularorganelles.

MATERIALS AND METHODS

The rats were anaesthesized by injection of o-2 ml nembutal intraperitoneally and the portalvein 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-EGTAand 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

N | treatment for 5 min, slow decompressionI

Centrifugation —r supernatant 1Pellet, N, treatment

LCentrifugation —r supernatant 2

Pellet, N, treatmentICentrifugation —r supernatant 3

Pellet, Nj treatmentI

Centrifugation —r supernatant 4Supernatants 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 minand 15 min for the first and second perfusion media, respectively. In order to decrease theintracellular concentration of divalent cation the livers were shaken in Krebs-Henseleit bufferwithout Mg'+ and Ca2+ to dissociate the hepatocytes. The isolated cell suspension was washedfirst with the same Krebs-Henseleit buffer and then with 0-25 M-sucrose by centrifugation at80 g for 5 min. The yield of hepatocytes from one rat liver was about 250 x io6 cells. In ourexperiments io8 cells corresponded to about 2-5 mg protein. In a typical case, a 6 ml suspension(70 x io6 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 Ns under continuousmagnetic stirring, pressure was kept at 1000 lb/in* (1 lb/in1 = 6-9 kPa) for 5 min and, afterdecompression, the non-broken cells were sedimented by centrifugation. The supernatantwas removed with a Pasteur pipette and retained (supernatant 1). The pellet was resuspendedin 15 times its volume of 0-25 M-sucrose, and treated with N, as before. The Whole procedurewas repeated twice. The four supernatants were mixed and used in further fractionations. Thedisruption procedure is summarized in Fig. 1.

The suspension of disrupted hepatocytes was used to prepare both mitochondria andmicrosomes. Mitochondria were prepared by first removing debris and nuclei by centrifugationat 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 centrifugationat 4300 g for 15 min. This washing procedure was repeated and the final mitochondrial pelletwas resuspended in 0-25 M-sucrose (5 mg protein/ml). For preparation of total microsomesthe 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 at105000 g for 60 min. Rough and smooth microsomes were isolated by layering 3-5 ml of the10000 g supernatant over 2 ml 1-3 M and 0-5 ml 06 M-sucrose solutions, both containing15 mM-CsCl (Dallner, 1974). This gradient was centrifuged in a 40-2 rotor (Beckman) at102000 g for 90 min. The smooth microsomes at the o-6 M/I-3 M-sucrose interface wererecentrifuged and the pellet, as well as the rough microsomal pellet, were resuspended in0-25 M-sucrose. Disruption of hepatocytes in 35-ml samples of washed cell suspension (250 xio8 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 performedin a cooling bath for 20 s. Subfractionations were performed as described above.

When contaminating membranes had been removed from the smooth microsomes isolatedfrom the sonicated hepatocyte suspension, the ioooog supernatant was supplemented with7 mM-MgCl, and 4-5 ml of this was layered over 2 ml 1-15 M-sucrose-7 mM-MgCl| andcentrifuged at 105000 g for 30 min in a 40-2 rotor (Beckman). The pellet was resuspendedand used for measurements.

To determine contamination in isolated mitochondria and microsomes, various membranefractions 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 usingestablished procedures and used for determination of specific marker enzymes. The valuesobtained were, for acid phosphatase (lysosomes) i'i2 ftmo\ P|/min per mg protein, for urateoxidase (peroxisomes) 0-36 fimol urate oxidized/min per mg protein, for UDP-galactosyltransferase (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 tocalculate the percentage contamination (on a protein basis) in the isolated hepatocyte fractionsActivities of cytochrome c oxidase and monoamino oxidase in the mitochondria and NADPH-cytochrome c reductase in the microsomes were identical in the fractions obtained either fromwhole liver homogenate or from isolated hepatocytes.

Protein was measured according to Lowry, Rosebrough, Farr & Randall (1951). Both lipidand RNA content were analysed as described previously (Ceriotti, 1951; Dallner, Siekevitz &Palade, 1966). The various enzyme activities were determined using previously describedprocedures (Sottocasa, Kuylenstierna, Ernster & Bergstrand, 1967; Eriksson, 1973; Beaufayet al. 1974). All data in the Tables show representative results chosen from five to nine identicaland consecutive experiments.

Tissue samples used for electron-microscopic observations were fixed in 3 % glutaraldehydein o-i M-Na cacodylate-HCl buffer with o-i M-sucrose (pH 7-2), at +4°C overnight. Theywere 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 M-Na cacodylate-HCl buffer (pH 7-2), for 60 min at +4 °C. The tissue samples and pellets weredehydrated and embedded in Epoxy resin.

RESULTS

A prerequisite for the isolation of well-preserved cellular fractions is the availabilityof isolated cells of high quality. Fig. 2 verifies that our procedure using calciumdepletion and collagenase is a suitable way to obtain unchanged liver cells. Thehepatocyte is well preserved, the membrane structures are delimited, the mito-chondria are mostly in the condensed state, the endoplasmic channels are narrowand most of the ribosomes appear to be membrane-attached. Clearly, the cellsisolated 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-delimitedorganelles with distinct membranes were noted. Neither mitochondrial swelling,lysosomal rupture, dilatation, nor degranulation of endoplasmic reticulum wasrecorded, x 21000.

Fractionation of isolated liver cells 5

consequently suitable for subfractionation studies. The decrease in Ca2+ concentra-tions during perfusion with collagenase and the elimination of divalent cations fromthe Krebs buffer was not deleterious to the cell morphology or cell function, butdecreased the yield of cells and also the yield of fractions obtained after disruption.If the Ca2+ concentration was increased in the collagenase perfusion and divalentcations were included in the Krebs buffer, separation of rough and smooth micro-somes 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/in2 (x 6-9 kPa) and for this reason cannot be used when isolating most intracellularparticles. By using a pressure of 1000 lb/inJ (x 69 kPa), hepatocyte disruption isonly 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

Washed cellsist disruption

PelletSupernatant

2nd disruptionPelletSupernatant

3rd disruptionPelletSupernatant

4th disruptionPelletSupernatant

Protein(mg)

3 1 0

2 1 0

84

14762

9747

5240

Released protein(% of total)

—

—27

—2 0

—

IS

—

13

The preparation of cells and treatment with the nitrogen-bomb system were performed asdescribed in Materials and Methods. The values given for the released protein are expressedas the ratio between the value for the supernatant after centrifugation and that of the washedcells.

relatively slow release of pressure breaks up about 0-25 of the cells, since 27% ofthe total protein is not sedimented after short centrifugation. When the pellet, afterthe first disruption, is homogenized again and the procedure repeated three moretimes, about 75 % of the protein is found in the supernatant, indicating an effectivedisruption of the cells. The yield obtained by both disruption bomb and sonicationis about the same as the yield after homogenization of whole liver (Table 2). Chemic-ally, the mitochondria and the microsomal fractions isolated from hepatocytes byboth procedures exhibit a composition similar to that found in liver tissues. Thelipid/protein ratio for mitochondria and rough and smooth microsomes does notdiffer from those given in the literature for particles isolated by homogenizationof the liver. The RNA/lipid ratio indicates that a successful separation of rough andsmooth microsomes was attained.

F. Autuori, U. Brunk, E. Peterson and G. Dallner

Table 2. Chemical composition of nritochondrial and microsomalfractions

Preparation

Nitrogen bombHomogenateMitochondriaRough micro8omesSmooth microsomes

SonicationHomogenateMitochondriaRough microsomesSmooth microsomes

Protein(mg)

2 2 2

4227

9

31O9332110

Lipid(mg)

—6 3 0

7-83369

—5-285-673-80

RNA(mg)

——

2-34o-33

——i-93°'34

Lipid/protein

—0 1 5

0-290-41

—

0-160 2 7

0 3 8

RNA/lipid

—-—

O'3O0-09

——

o-340-09

Cells were broken up either with the nitrogen bomb or by sonication, and fractions wereprepared. Homogenate denotes the total released protein after four disruption cycles (super-natants 1-4 in Table 1) when the N t bomb was used in homogenization, and it denotes the600 g supernatant (supernatant after centrifugation of the sonicated cell suspension at 600 gfor 10 min) when sonication was applied for homogenization.

Fig. 3. The mitochondrial fraction, isolated from hepatocytes treated with a nitrogenbomb. Mitochondria in either the condensed or the orthodox state and with intactouter 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 ofintact 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 bombshowed rough microsomes as ribosome-covered intact vesicles and smooth micro-somes as intact smooth vesicles of varying sizes (Fig. 4A,B). A few vesicles in therough microsomal fraction were devoid of ribosomes and some of the ribosomeswere in the free form. The vesicles in this fraction contain fewer bound ribosomesthan the rough microsomes isolated from liver tissue. Free ribosomes are also presentin the smooth microsomal fraction, which also contains Golgi elements and largervesicles, which probably originated from plasma membranes.

Table 3. Oxidation of NADH by isolated mitochondria prepared from hepatocytesdisrupted by nitrogen bomb or sonication

Preparation

1000 lb/in1

1500 lb/in1

1000 lb/in1

Sonication

Addition

NoneKCNNoneKCNo-i % deoxycholateo-i% deoxycholate + KCNNoneKCN

NADH oxidized(/tmol/min per mg protein)

O-II

O-0O2

0 5 40-003

I-2IO-OO2

O-I4

0-002

Cells were treated with the nitrogen bomb four times, as described in Materials and Methods.The value in lb/in1 (1 lb/in1 — 6-9 kPa) given in the table was applied during the wholeprocedure. Sonication was performed as given in Materials and Methods. The concentrationof KCN was 1 nw.

The nitrogen-bomb treatment employed is a sufficiently mild procedure forpreparation of mitochondria from hepatocytes. If the mitochondria are prepared asdescribed in Materials and Methods by using 1000 lb/in2 (x 6-9 kPa), penetrationby NADH is very limited, since the oxidation rate of the reduced co-enzyme is onlyo-i of that obtained after the addition of deoxycholate (Table 3). The relativelymoderate 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 mito-chondria that are non-permeable to NADH.

The situation is very similar with microsomes. These particles display highnucleoside diphosphatase activity when the cells are disrupted with 1000 lb/ina

pressure, while the enzyme activity in the supernatant, representing the amount ofliberated nucleoside diphosphatase, is low (Table 4). Increasing the pressure to1700 lb/in2 interferes with membrane integrity and solubilizes about 0-25 of themicrosomal enzyme activity. Short sonication also causes a similar release of theenzyme.

F. Autuori, U. Brunk, E. Peterson and G. Dallner

Fig. 4. Microsomal subfractions prepared from hepatocytes disrupted by a nitrogenbomb. A. Rough microsomes; intact vesicles with attached ribosomes and somecontamination with smooth vesicles as well as groups of free ribosomes were observed.B. Smooth microsomes; intact smooth vesicles with varying sizes, contaminating Golgielements 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

ActivityExpt Preparation {jimo\ P,/min per mg protein)

1000 lb/in1

MicrosomesSupernatant

1700 lb/in!

MicrosomesSupernatant

Sonication (20 s)MicrosomesSupernatant

0 8 60044

o-6i0 1 3

0-63O'I2

Cells were broken up either with the N, bomb (1000 lb/in1 in Expt 1 and 1700 lb/in* inExpt 2 or by sonication (20 s, Expt 3). Nucleoside diphosphate activity was measured in themicrosomal fraction and in the remaining supernatant after centrifugation at 105000 g for60 min.

Table 5. Distribution of various enzymes in the mitochondrialfraction isolated by nitrogen-

bomb treatment of hepatocytes

Specificactivity

i'3S15-200020-070 0 40 0 50 0 1

Mitochondria

Calculatedcontamination

(% of total protein)

—

—

36

11

31

Cytochrome c oxidase"Monoamino oxidase6

NADPH-cytochrome c reductase*Acid phosphatase11

Urate oxidase"UDP-galactosyl transferase'AMPase"

Enzyme activities were measured in lysosomes, peroxisomes, Golgi and plasma membranesisolated from liver tissue from starved rats. These values were used to calculate the contami-nation 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\ urateoxidized/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). Contam-

ination 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 (urateoxidase), which make up n % of the total protein. Calculation of contamination inmitochondria and in microsomes is based on the assumption that the appropriatemarker enzyme is present exclusively in one subcellular organelle. This is not quitetrue 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 conclusiononly to a moderate extent and gives an overestimate of the degree of contamination.

Rough and smooth microsomes can be separated with a sucrose gradient contain-ing 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 nitrogenbomb treatment of hepatocytes

NADPH-cytochrome c reductase"Glucose-6-phosphataseb

Cytochrome c oxidase0

Monoamino oxidase d

Acid phosphataseb

Urate oxidase'UDP-galactosyl transferase/

AMPase"

Rough

Specificactivity

0-0414-010 0 3

0 2 1

0 0 8

o-oiO-O2

OO08

microsomes

Calculatedcontamination

(% of totalprotein)

——0 51

52

1

1

Smooth

Specificactivity

00444-520

0-560-120-006O - I I

0 0 1 5

microsomesA

Calculatedcontamination

(% of totalprotein)

——0

491

72

Enzyme activities were measured in lysosomes, peroxisomes, Golgi and plasma membranesisolated from liver tissue of starved rats. These values were used to calculate the contaminationin the microsomal fraction of isolated hepatocytes.

"fimol NADPH oxidized/min per mg protein; b/imo\ Pt/min per mg protein; "fimol cyto-chrome c oxidized/min per mg protein; dnmol bensaldehyde/min per mg protein; 'fimolurate oxidized/min per mg protein; 'nmol transferred/30 min per mg protein.

microsomes isolated from the nitrogen-treated cells are less contaminated by othermembranes than are smooth microsomes (Table 6). Rough microsomal membranesmake up 90% of the fraction. The smooth microsomal fraction is contaminated witha greater percentage of membrane proteins, as the sedimentation velocity for smoothvesicles is close to that of other cy loplasmic membranes in the homogenate. Consider-able amounts of Golgi membranes (7%) and lysosomes (9%) are distributed in thefraction and must be taken into consideration in fractionation studies.

Rough and smooth microsomes can be prepared easily and rapidly from hepato-cytes, even after sonication. As shown in Table 7, the rough microsomes obtainedwere similar to those prepared in the nitrogen-bomb system. On the other hand,smooth microsomes exhibit an increased contamination with outer mitochondrial andplasma membranes, as is apparent from the considerably increased activity of mono-amino oxidase and AMPase.

Fractionation of isolated liver cells

Table 7. Distribution of various enzymes in the microsomal fraction prepared bysonication

11

f

NADPH-cytochrome c reductase"Glucose-6-phosphatase6

Cytochrome c oxidaseMonoamino oxidaseAcid phosphataseUrate oxidaseUDP-galactosyl transferaseAMPase

Rough

Specificactivity

0037

C36——————

microsomes

Calculatedcontamination

(% of totalprotein)

——1

36211

Smooth

Specificactivity

0-0440-40

——

——

—

microsomes

Calculatedcontamination

(% of totalprotein)

—o-599268

Enzyme activities were measured in lysosomes, peroxisomes, Golgi and plasma membranesisolated from liver tissue of starved rats. These values were used to calculate the contaminationin 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 aftersonication

Smooth microsomes(calculated contamination)

as % of total protein

Cytochrome c oxidaseMonoamino oxidaseAcid phosphataseUrate oxidaseUDP-galactosyl transferaseAMPase

The isolated smooth microsomes were recentrifuged on a discontinuous gradient containingMg'+ as described in Materials and Methods. Contaminations were calculated as describedin Table 5.

Control

0 599268

Recentrifuged onMgI+ gradient

o-5632

52

Some of the non-microsomal membrane material, in particular plasma membranes,can be removed by placing the fraction on a second discontinuous sucrose gradientcontaining MgCl2 (Table 8). Obviously, some of the cytoplasmic membranes areinsensitive to divalent cations and do not precipitate and sediment like smoothmicrosomes when this system is used. In this way a smooth microsomal fraction canbe produced that is similar in composition to the fraction obtained by centrifugationof 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 individualcells, is a difficult task. Breakage of cell membranes requires the application of consi-derable shearing forces and the same procedure may therefore cause damage, notonly to the plasma membrane itself, but also to most of the cytoplasmic organelles.Consequently, homogenization of individual hepatocytes requires much more closelycontrolled conditions than homogenization of the liver itself.

The use of a nitrogen-bomb system based on decompression in a pressure vesselwas found to be advantageous in several previous investigations. This principle isused for the disruption of tissues such as liver and spleen, ascites tumour cells, L cellsand fibroblasts, and also the preparation of submitochondrial vesicles from beefheart 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 appliedis well defined and repeatable, no heat is generated during disruption, oxygen isexcluded by the use of inert gas and there is a uniform effect throughout the solution.A pressure of 1000 lb/in2 was the maximum applicable, because at higher pressuresthe mitochondrial membrane permeability for NADH was impaired and the micro-somes lost some intraluminal protein components. However, it was necessary torepeat 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 obtainmicrosomal fractions than is advisable when studying enzymes and enzyme systems.

The sonication procedure is rapid and easy to use even under varying experimentalconditions. The recovery of microsomal subfractions is similar to that seen in thenitrogen-bomb system; however, there is an increased contamination of the smoothmicrosomes with other membranes. This contamination can be partially reducedby using a Mg2+-containing gradient. In most membrane studies sonication is themethod of choice since it is rapid, simple, reproducible and gives high recovery.Clearly, the loss of some nucleoside diphosphatase during sonication does not meanan irreversible change in membrane permeability, since substrate permeability isnot changed and the enzymes localized on the inner surface require the addition ofdetergents to obtain full activity. It appears, therefore, that the microsomal vesiclesobtained by the sonication procedure exhibit, at least to a large extent, the sameproperties as vesicles obtained by other procedures.

This work was supported by grants from the Swedish Medical Research Council and theNational Cancer Institute (grant no. 1 RO ICA 26261-01.). Dr F. Autuori was on leave ofabsence from the Institute of Histology and Embryology, Faculty of Science, University ofRome, Italy.

Fractionation of isolated liver cells 13

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(Received 22 October IQ8I - Revised 27 April iq82)