FRACTIONATION OF ISOLATED LIVER CELLS AFTER DISRUPTION WITH A NITROGEN BOMB AND SONICATION

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1 J. Cell Sri. 57, -3 (982) Printed in Great Britain Company of Biologists Limited 982 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 000 lb/in! ( lb/in = 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, 95; Wallach, Soderberg & Bricker, i960; Hunter & Commerford, 96; Dowben, Gaffey & Lynch, 968; Loewus & Loewus, 97; Short, Maines & Davis, 972). 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, 964). Disruption of hepatocytes by sonication has also been reported (Gellerfors & Nelson, 979). Hence the possibility arose

2 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, 978). The first perfusion fluid was 50 ml Hanks' buffer containing 0-5 mm-egta and 2% albumin. The second perfusion fluid was 00 ml Hanks' buffer containing 0-2% collagenase (type V, Boehringer) and 2 mm-caclj. The solutions were bubbled with carbogen Isolated cells in sucrose (washed in buffer, Mg +, Ca a+ ) I N treatment for 5 min, slow decompression I Centrifugation r supernatant 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 = broken cells (75 % of total) Fig.. Schematic representation of hepatocyte disruption using a nitrogen bomb (see Materials and Methods for details). gas (95 % O,, 5 % CO,) and heated to 37 C C prior to use. The time of perfusion was 0 min and 5 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 Ca 2+ 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 5 ml with 0-25 M-sucrose and placed in a cell disruption bomb (Parr Instrument, Moline,.). The water phase was saturated with N s under continuous magnetic stirring, pressure was kept at 000 lb/in* ( lb/in = 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 ). The pellet was resuspended in 5 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.. 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 0 min. The supernatant was decanted and the mitochondria were pelleted at

3 Fractionation of isolated liver cells g for 20 min. The pellet was resuspended in 0-25 M-sucrose and washed by centrifugation at 4300 g for 5 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 0000 g for 20 min and the supernatant (0000 g supernatant) was used to pellet the total microsomal fraction by centrifugation at g for 60 min. Rough and smooth microsomes were isolated by layering 3-5 ml of the 0000 g supernatant over 2 ml -3 M and 0-5 ml 06 M-sucrose solutions, both containing 5 mm-cscl (Dallner, 974). This gradient was centrifuged in a 40-2 rotor (Beckman) at 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 -5 M-sucrose-7 mm-mgcl and centrifuged at 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. 968), peroxisomes (Baudhuin, 974), Golgi (Ehrenreich, Bergeron, Siekevitz & Palade, 973) and plasma membranes (Coleman, Michell, Finean & Hawthorne, 967) 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) -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 (95). Both lipid and RNA content were analysed as described previously (Ceriotti, 95; Dallner, Siekevitz & Palade, 966). The various enzyme activities were determined using previously described procedures (Sottocasa, Kuylenstierna, Ernster & Bergstrand, 967; Eriksson, 973; Beaufay et al. 974). 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 % osmium tetroxide in 0-5 M-Na cacodylate-hcl buffer, (ph 7-2), for 90 min at room temperature. The pellets were fixed in % osmium tetroxide in 0-5 M- Na 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

4 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 2000.

5 Fractionation of isolated liver cells 5 consequently suitable for subfractionation studies. The decrease in Ca 2+ 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 lb/ in 2 (x 6-9 kpa) and for this reason cannot be used when isolating most intracellular particles. By using a pressure of 000 lb/in J (x 69 kpa), hepatocyte disruption is only partial (Table ). Saturation of the water phase with nitrogen followed by a Table. Effect of nitrogen-bomb disruption on isolated liver cells Fraction Washed cells ist disruption Pellet Supernatant 2nd disruption Pellet Supernatant 3rd disruption Pellet Supernatant 4th disruption Pellet Supernatant Protein (mg) Released protein (% of total) 3 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 IS 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.

6 F. Autuori, U. Brunk, E. Peterson and G. Dallner Table 2. Chemical composition of nritochondrial and microsomalfractions Preparation Nitrogen bomb Homogenate Mitochondria Rough micro8omes Smooth microsomes Sonication Homogenate Mitochondria Rough microsomes Smooth microsomes Protein (mg) O Lipid (mg) RNA (mg) 2-34 o-33 i-93 '34 Lipid/protein RNA/lipid - O'3O 0-09 o 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 -4 in Table ) 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 0 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 5000.

7 Fractionation of isolated liver cells 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 y Preparation Addition NADH oxidized (/tmol/min per mg protein) 000 lb/in 500 lb/in 000 lb/in Sonication None KCN None KCN o-i % deoxycholate o-i% deoxycholate + KCN None KCN O-II O-0O I-2I O-OO2 O-I Cells were treated with the nitrogen bomb four times, as described in Materials and Methods. The value in lb/in ( lb/in 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 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 000 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 500 (lb/in 2 ) 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 000 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 700 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.

8 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

9 Fractionation of isolated liver cells 9 Table 4. Nucleoside diphosphatase activity in microsomes and supernatant of hepatocytes after cell disruption using varying techniques Activity Expt Preparation {jimo\ P,/min per mg protein) 000 lb/in Microsomes Supernatant 700 lb/in! Microsomes Supernatant Sonication (20 s) Microsomes Supernatant o-6i O'I2 Cells were broken up either with the N, bomb (000 lb/in in Expt and 700 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 g for 60 min. Table 5. Distribution of various enzymes in the mitochondrialfraction isolated by nitrogenbomb treatment of hepatocytes Mitochondria Specific activity Calculated contamination (% of total protein) Cytochrome c oxidase" Monoamino oxidase 6 NADPH-cytochrome c reductase* Acid phosphatase Urate oxidase" UDP-galactosyl transferase' AMPase" i'3s 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; 6 nmol 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,

10 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, 976). 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 NADPH-cytochrome c reductase" Glucose-6-phosphatase b Cytochrome c oxidase 0 Monoamino oxidase d Acid phosphatase b Urate oxidase' UDP-galactosyl transferase / AMPase" Rough microsomes Specific activity o-oi O-O2 OO08 Calculated contamination (% of total protein) Smooth microsomes Specific activity O-II 005 A Calculated contamination (% of total protein) 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\ P t /min per mg protein; "fimol cytochrome c oxidized/min per mg protein; d nmol 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

11 Fractionation of isolated liver cells Table 7. Distribution of various enzymes in the microsomal fraction prepared by sonication Rough microsomes Smooth microsomes NADPH-cytochrome c reductase" Glucose-6-phosphatase 6 Cytochrome c oxidase Monoamino oxidase Acid phosphatase Urate oxidase UDP-galactosyl transferase AMPase f Specific activity 0037 C36 Calculated contamination (% of total protein) Specific activity Calculated contamination (% of total protein) 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. o Table 8. Removal of plasma membrane fragments from smooth microsomes prepared after sonication Cytochrome c oxidase Monoamino oxidase Acid phosphatase Urate oxidase UDP-galactosyl transferase AMPase Smooth microsomes (calculated contamination) as % of total protein Control 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 Recentrifuged on Mg I+ gradient o 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 MgCl 2 (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 2 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, 96; Molnar, 967; Dowben et al. 968; Short et al. 972; Fleischer, Meissner, Smigel & Wood, 974). 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 000 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 Mg 2+ -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. RO ICA ). Dr F. Autuori was on leave of absence from the Institute of Histology and Embryology, Faculty of Science, University of Rome, Italy.

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