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[19] Preparation and assay of phosphorylating mitochondria from ascites tumor cells
[19]
MITOCHONDRIA FROM ASCITES TUMOR CELLS
[ 19] P r e p a r a t i o n
and Assay of Phosphorylating
from Ascites Tumor
105
Mitochondria
Cells
B y RAY W u 1 and LEONARDA. SAUER
A number of methods have been described for the preparation of mitochondrial fractions from mouse ascites tumor cells, la-ll This seems to be a result of the fact that ascites tumor cells are very resistant to homogenization. However, even with the variety of homogenization methods that have been used, the literature shows that mitochondria capable of oxidative phosphorylation, but with various degrees of efficiency, may be isolated from ascites cells. An adequate description of each published method would be beyond the scope of this section; therefore, we have chosen to describe in detail two quite different procedures with which we have had considerable experience. These methods involve homogenization by grinding with glass beads (R.W.) or with the Dounce homogenizer (L.A.S.). Both methods yield mitochondrial fractions with high P : O and respiratory control ratios. Materials T u m o r Transplantation and Harvesting. The procedures used are the same as previously described2,1° Reagents. Crystalline hexokinase (140 units/rag) was purchased from Boehringer Mannheim Co., New York. The enzyme was dialyzed overnight against 100 volumes of 50 m M triethanolamine buffer (pH 7.4) containing 1 m M glucose, and kept frozen at --20 °. Succinate was pur-
1This manuscript was prepared while the author was at the Department of Biochemistry, The Public Health Research Institute of the City of New York, Inc., New York. 1, E. Kun, P. Talalay, and H. G. Williams-Ashman, Cancer Res. 11, 855 (1951). O. Lindberg, M. Ljunggren, L. Ernster, and L. R~v~sz, Exptl. Cell Res. 4, 243 (1953). C. E. Wenner and S. Weinhouse, Cancer Res. 13~ 21 (1953). 4R. Wu and E. Racker, J. Biol. Chem. 234, 1029, 1036 (1959). ~B. Chance and B. Hess, J. Biol. Chem. 234, 2413 (1959). P. Emmelot, C. J. Bos, P. J. Brombacher, and J. F. Hampe, Brit. J. Cancer 13, 348 (1959). : W. Luehrs, G. Bacigalupo, B. Kadenbach, and E. Heise, Experientia 15, 376 (1959). s p. Borst, J. Biophys. Bioehem. Cytol. 7, 381 (1960). ~A. O. I-Iawtrey and M. H. Silk, Biochem. J. 74, 21 (1960). I°L. A. Saner, A. P. Martin, and E. Stotz, Cancer Res. 20, 251 (1960). 11L. A. Sauer, A. P. Martin, and E. Stotz, Cancer Res. 22, 632 (1962).
106
PREPARATION OF MITOCHONDRIA
[19]
chased from the same company or from Eastman Kodak Co. All other reagents used were analytical grade. Procedure for Measuring Oxidative Phosphorylation Both the principle and the procedure for this assay have been previously described. 12 Oxygen uptake is determined manometrically. Each Warburg vessel (15 ml capacity) contained the following in a total volume of 2 ml: substrate for respiration, 10 mM; potassium phosphate (pH 7.4), 15 mM; ATP, 2 mM; MgCl~, 5 mM; triethanolamine (or Tris-HC1) buffer (pH 7.4), 15 mM; DPN, 0.5 raM; glucose, 20 mM; KCI (recrystallized from 2 mM EDTA solution), 25 mM; mannitol or sucrose, 60 mM; EDTA, 1 mM; dialyzed crystalline hexokinase, 2.5 units per vessel (or Sigma Type III, 0.6 mg per vessel) ; and tumor mitochondria (1.2-4 mg of protein). In the center well are placed 0.15 ml of 5 N KOH and a roll of filter paper. The vessels are equilibrated at 30 ° for 5 minutes before the substrate is tipped in from the side arm of the vessel. The oxygen uptake is measured for the next 30 or 40 minutes. Usually 4-8 microatoms of oxygen and 6-20 micromoles of P~ are taken up. The reaction is stopped by removing the vessel from the bath and is immediately deproteinized with 2 ml of 10% trichloroacetic acid. After centrifugation, 0.05-0.1 ml aliquots of the supernatant solution are removed for P~ determination,is Procedure for Measuring Oxygen Uptake and ADP Respiratory Control Ratio Oxygen consumption was measured with the use of a Clark oxygen electrode. 14 The reaction vessel contained the following in a total volume of 1.0 ml (or 6.5 ml) : substrate for respiration, 5 or 10 mM; potassium phosphate (pH 7.4), 5 mM; MgC12, 10 mM; triethanolamine buffer (pH 7.4), 30 mM; KC1, 25 mM; serum albumin (dialyzed against 50 mM triethanolamine buffer for 2 days), 0.3 or 1%; KF, 5 mM; EDTA, 1 mM; mannitol (or sucrose), 50 mM; and tumor mitochondria, 0.7 to 1.4 mg of protein/ml (or 5-8 mg dry wt/6.5 ml). After 2 minutes at 25 °, l~See Vol. II [lOll; also see this volume [3]. i~K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926). Modified by using 5% (instead of 2.5%) ammonium molybdate in 5 N H~SO4.See W. D. Harris and P. Popat, J. Am. Oil Chem. Soc. 31, 124 (1954) as modified by T. Conover, Thesis, Univ. of Rochester, 1959. l~See Vol. VI [33]. The electrode used by one of us (R.W.) was modified by mounting the electrode in a horizontal position near the bottom of the reaction vessel (1 ml capacity), so that reagents can be conveniently added from the top of the reaction vessel. This modification was made by Dr. M. E. Pullman and Mr. M. Kandrach and is gratefully acknowledged.
[19]
MITOCHONDRIA FROM ASCITES TUMOR CELLS
107
A D P (final concentration 0.1-0.4 raM) was added and the oxygen consumption was recorded for approximately 10 more minutes. The respiratory control ratio (defined as the rate of respiration in the presence of A D P divided by the rate obtained following the expenditure of ADP)1~, 1~ was then calculated. Procedure for the Preparation of Ascites T u m o r Mitochondria
Method A. Grinding of the Tumor Cells with Mortar and Pestle. The Ehrlich ascites tumor cells are obtained by harvesting the ascites fluids from 4-6 mice (7-9 days tumor age). The fluids are immediately diluted in 2 volumes of ice-cold mannitoW (0.25 M) solution containing E D T A (2 m M ) , p H 7.5, and the cells are centrifuged at 600 g for 5 minutes. The packed cells are washed twice by resuspending the cells in approximately 10 volumes of the same solution and centrifuging as above. For homogenization, the packed tumor cells (approximately 5 ml) are mixed with 1 ml of the m a n n i t o l - E D T A solution and 0.1 ml of 1 M triethanolamine buffer (pH 7.4). The mixture is poured into a precooled mortar (7 cm diameter) ; 3 g of washed glass beads ~s are added, and the mixture is ground forcefully with a pestle for 6 minutes at 4 °. Then 2 5 m l of m a n n i t o l - E D T A solution is added to the mortar and the mixture is ground for 20 seconds to give an even suspension. The mixture is poured into a 40 ml polyethylene tube and centrifuged at 600 g for 10 minutes. The supernatant solution is decanted and stored at 4 °. The pellet is mixed with 2 ml of the m a n n i t o l - E D T A solution, 0.1 ml of 1 M triethanolamine buffer, 2 g of washed glass beads; the mixture is ground for another 4 minutes. Then 25 ml of m a n n i t o l - E D T A solution is added and the mixture is centrifuged as above. The supernatant solution from the second batch is combined with t h a t from the first, and the combined solution is divided among 2 polyethylene tubes and centrifuged at 9000 g for 15 minutes in the 9 RA head of the Lourdes refrigerated centrifuge. The supernatant solution and "fluffy layer" are decanted and discarded. The mitochondrial pellets are washed twice after their suspension in 25 ml of m a n n i t o l - E D T A solution with a Teflon pestle inserted directly into the centrifuge tube. The final pellets are suspended in 2 ml of the UB. Chance and M. Baltscheffsky, Biochem. J. 68, 283 (1958). 1~B. Chance, Ciba Found. Syrup. Regulation Cell Metabolism p. 91. Little, Brown, Boston, Massachusetts, 1959. ~ Sucrose (0.25 M) solution containing 1 or 2 mM EDTA may be used in place of mannitol-EDTA solution. All further steps were carried out at 4 °. 18Superbrite glass beads (0.1 mm diameter, type 130-5005, from Minnesota Mining and Manufacturing Company, St. Paul, Minnesota) were washed by soaking in 2 volumes of 5 N YIC1 overnight and washed with 10 volumes of deionized water about 6 times until the acidity of the washes approached pH 5.
108
PREPARATION OF MITOCHONDRIA
[19]
m a n n i t o l - E D T A solution and evenly dispersed with the use of a Teflon pestle. Method B. Dounce Homag~enizer Method. ~1,19 The ascitic fluid from 5 or 6 mice (8-11 days t u m o r age) is removed by syringe and transferred to two 40-ml graduated centrifuge tubes packed in ice. All further procedures are carried out in the cold room. The ascitie fluid is centrifuged for 5 minutes at 900 g, and the s u p e r n a t a n t fluid is decanted and discarded. The packed cell volume should be a b o u t 10-15 ml of bloodless cells. 2° T h e packed cells are washed twice with 30-ml portions of 0.25 M sucrose t h a t contains 0.001 M disodium E D T A , p H 4-5 (solution S). The twice washed tumor cells are then resuspended with 40 ml of solution S, to which has been added 0.1 ml of 0.1 M citric acid, and transferred to a Dounce homogenizer (40-ml size) packed in ice. For support the homogenizer is inserted into a hole in a large rubber stopper. TM Solution S is added until fluid fills the cylinder and lower portion of the reservoir of the homogenizer. Homogenization is accomplished b y 10-15 vigorous, directly vertical passes of the pestle. The homogenate should be examined microscopically for unbroken cells and more passes made if necessary. 2~ The resultant homogenate (approximately 60 ml, depending upon the size of the homogenizer used) is divided between two 40-ml centrifuge tubes and centrifuged at 900 g for 20 minutes. T h e top twothirds of the s u p e r n a t a n t solutions obtained are decanted and stored in ice. The sediments are combined and resuspended to 60 ml with solution A. L. Dounce, R. F. Witter, K. J. Monty, S. Pate, and M. A. Cottone, J. Biophys. and Biochem. Cylol. 1, 139 (1955). Douuee homogenizers may be purchased from Blaessig Glass Specialties, Rochester, New York, or from Kontes Glass, Vineland, New Jersey. The clearance found most useful for the fractionation of aseites cells is 0.0005 inch. This corresponds to the tight-fitting pestle as supplied by Blaessig or the loose-fitting pestle as supplied by Kontes. 20Ascites cell samples that are grossly bloody should not be used. Samples that are only slightly bloody are suitable for fractionation after removal of the erythrocytes (RBC). This may be accomplished in two ways. Low speed centrifugations (125g for 4 minutes) in isotonic sucrose will sediment most of the tumor cells while leaving the RBC in the supernatant fluid. The RBC are then decanted and the procedure is repeated until the number of RBC are reduced to acceptable levels. Erythrocytes may also be removed by "differential lysis." In this procedure the contaminated ascites cells are suspended to 4 or 5 times their volume with cold 1:4 diluted Ringer solution and centrifuged for 5 minutes at 960 {7. The swollen, intact ascites cells will sediment and the supernatant fluid which contains the hemoglobin is decanted. The ascites cells may then be washed in isotonic media. The latter method is recommended since there is little or no apparent damage to the tumor cells and the removal of RBC is quick and nearly complete. 21If on microscopic examination the cell breakage is less than 60% after at least 20 passes, it is likely that homogenization is either not vigorous enough or that the clearance of the homogenizer is too large or too small.
[19]
MITOCHONDRIA FROM ASCITES TUMOR CELLS
109
S; the homogenization and centrifugation (900 g for 20 minutes) are repeated. During the second homogenization, extremely vigorous passes are not necessary. The yield from the second homogenization is usually small. The upper portions of the supernatant solutions obtained from the centrifugation of the second homogenate are combined with those obtained from the first, and the mixture is distributed among 8 No. 40 tubes for the Spinco centrifuge and centrifuged for 15 minutes at 8000 g. Any refrigerated centrifuge capable of developing this centrifugal force is suitable. The supernatant solutions and fluffy layers are decanted and discarded. Each sediment is resuspended with 6 ml of solution S by inserting a small motor-drive Teflon pestle (rotating at slow speed) directly into each centrifuge tube. The suspended sediments are again centrifuged at 8000 q for 15 minutes, and then this washing procedure is repeated. The twice washed sediments are resuspended to 10 ml (the mitochondrial fraction), which contains about 12-15 mg dry weight per milliliter (8-10 mg protein). The mitochondrial fraction represents a 7-10% recovery of the total dry weight of the homogenate. Properties of the Ascites T u m o r Mitochondria The P : O ratio, the respiratory control ratio, the yield, and the specific activity (millimicromoles of P~ esterified per minute per milligram of protein) of mitochondria prepared by the two methods are shown in the table. OXIDATIVE PHOSPHORYLATION WITH ]~HRLICH ASCITES TUMOR MITOCHONDRIA
Mitochondria prepared by
ADP respiratory S p e c i f i c control activity ratio
Yield
Substrate
P: 0 ratio
Grinding
26"
Succinate a-Ketoglutarate Pyruvate -}- malate
1.8 2.4 2.4
270 260 260
5.6 5.3 5.0
Dounce homogenization
85b
Suceinate a-Ketoglutarate Pyruvate -5 malate
1.6 2.4 2.2
210 160 160
4.5 5.7 4.0
"Milligrams of mitochondrial protein obtained per gram of cell protein. The values given for the manometric assays represent averages of duplicate determinations for a single preparation. The ADP respiratory control ratio values, which were obtained polarographlcally, represent averages for 3 (pyruvate ~ malate), 4 (succinate), or 12 (a-ketoglutarate) preparations. Considerable variation among preparations is to be expected, for example, for the latter 12 preparations the range for the respiratory control ratios was 2.5-9.5. This variation has been noted by others [e.g., P. Borst, J. Biophys. Biochem. Cytol. 7, 381 (1960)].
110
PREPARATION OF MITOCHONDRIA
[20]
Comments
Grinding of ascites tumor cells with glass beads in the mortar and pestle (Method A) can be applied to small amounts of ceils, e.g., 1 ml of packed cells (140 mg of cell protein), if necessary. When more than 5 ml of packed cells is used, either a larger mortar can be used or the grinding procedure can be operated with 5 ml of packed cells in each run. Homogenization of tumor cells in a Dounce homogenizer (method B) is more applicable to experiments where larger amounts of mitochondria are needed. Mitochondria prepared by these two methods give high P: 0 ratio and respiratory control ratio, comparable to mitoehondria prepared from other tissue sources.
[ 20 ] Neoplastic Tissue Mitochondria
By THOMAS M. DEVLI~¢ The multiplicity of solid tumor types, originating from a variety of normal tissues, makes it impossible to generalize about the isolation, properties, and activities of mitoehondria from neoplasms. Even with one tumor type, variations in the amount of connective tissue, degree of necrosis, contamination by normal tissue, and lability of the mitochondria requires that each tumor line be considered as a different entity, with preparative and assay procedures tailored to the individual malignancy. H o m o g e n i z a t i o n and Isolation M e d i u m
With some tumors, isotonic sucrose (0.25 M) suffices for the isolation of active mitoehondria comparable to those from normal tissues. 1-3 This is particularly the case for soft tumors, such as hepatomas, where disruption of the cells occurs under mild homogenization2 Higher sucrose concentrations (e.g., 0.35 M) have been employed to yield mitochondria with latent ATPase from Murphy-Sturm rat lymphosarcoma. 4 The use of mannitol, polyvinylpyrrolidone, or raffinose has not been extensively tested, but may yield better preparations from some tumors. ~ Emmelot 1R. K. Kielley, Cancer Res. 12, 124 (1952). 2A. C. Aisenberg, Cancer Res. ~.1, 295 (1961); Cancer Res. 21, 304 (1961). 3G. E. Boxer and T. M. Devlin, Science 134, 1495 (1961). See also T. M. Devlin and M. P. Pruss, Proc. Am. Assoc. Cancer Res. 3, 315 (1962). M. Blecher and A. White, J. Biol. Chem. 235, 3404 (1960). 5p. Emmelot, C. J. Bos, P. J. Brombacher, and J. F. :Hampe, Brit. J. Cancer 13, 348 (1959).
MITOCHONDRIA FROM ASCITES TUMOR CELLS
[ 19] P r e p a r a t i o n
and Assay of Phosphorylating
from Ascites Tumor
105
Mitochondria
Cells
B y RAY W u 1 and LEONARDA. SAUER
A number of methods have been described for the preparation of mitochondrial fractions from mouse ascites tumor cells, la-ll This seems to be a result of the fact that ascites tumor cells are very resistant to homogenization. However, even with the variety of homogenization methods that have been used, the literature shows that mitochondria capable of oxidative phosphorylation, but with various degrees of efficiency, may be isolated from ascites cells. An adequate description of each published method would be beyond the scope of this section; therefore, we have chosen to describe in detail two quite different procedures with which we have had considerable experience. These methods involve homogenization by grinding with glass beads (R.W.) or with the Dounce homogenizer (L.A.S.). Both methods yield mitochondrial fractions with high P : O and respiratory control ratios. Materials T u m o r Transplantation and Harvesting. The procedures used are the same as previously described2,1° Reagents. Crystalline hexokinase (140 units/rag) was purchased from Boehringer Mannheim Co., New York. The enzyme was dialyzed overnight against 100 volumes of 50 m M triethanolamine buffer (pH 7.4) containing 1 m M glucose, and kept frozen at --20 °. Succinate was pur-
1This manuscript was prepared while the author was at the Department of Biochemistry, The Public Health Research Institute of the City of New York, Inc., New York. 1, E. Kun, P. Talalay, and H. G. Williams-Ashman, Cancer Res. 11, 855 (1951). O. Lindberg, M. Ljunggren, L. Ernster, and L. R~v~sz, Exptl. Cell Res. 4, 243 (1953). C. E. Wenner and S. Weinhouse, Cancer Res. 13~ 21 (1953). 4R. Wu and E. Racker, J. Biol. Chem. 234, 1029, 1036 (1959). ~B. Chance and B. Hess, J. Biol. Chem. 234, 2413 (1959). P. Emmelot, C. J. Bos, P. J. Brombacher, and J. F. Hampe, Brit. J. Cancer 13, 348 (1959). : W. Luehrs, G. Bacigalupo, B. Kadenbach, and E. Heise, Experientia 15, 376 (1959). s p. Borst, J. Biophys. Bioehem. Cytol. 7, 381 (1960). ~A. O. I-Iawtrey and M. H. Silk, Biochem. J. 74, 21 (1960). I°L. A. Saner, A. P. Martin, and E. Stotz, Cancer Res. 20, 251 (1960). 11L. A. Sauer, A. P. Martin, and E. Stotz, Cancer Res. 22, 632 (1962).
106
PREPARATION OF MITOCHONDRIA
[19]
chased from the same company or from Eastman Kodak Co. All other reagents used were analytical grade. Procedure for Measuring Oxidative Phosphorylation Both the principle and the procedure for this assay have been previously described. 12 Oxygen uptake is determined manometrically. Each Warburg vessel (15 ml capacity) contained the following in a total volume of 2 ml: substrate for respiration, 10 mM; potassium phosphate (pH 7.4), 15 mM; ATP, 2 mM; MgCl~, 5 mM; triethanolamine (or Tris-HC1) buffer (pH 7.4), 15 mM; DPN, 0.5 raM; glucose, 20 mM; KCI (recrystallized from 2 mM EDTA solution), 25 mM; mannitol or sucrose, 60 mM; EDTA, 1 mM; dialyzed crystalline hexokinase, 2.5 units per vessel (or Sigma Type III, 0.6 mg per vessel) ; and tumor mitochondria (1.2-4 mg of protein). In the center well are placed 0.15 ml of 5 N KOH and a roll of filter paper. The vessels are equilibrated at 30 ° for 5 minutes before the substrate is tipped in from the side arm of the vessel. The oxygen uptake is measured for the next 30 or 40 minutes. Usually 4-8 microatoms of oxygen and 6-20 micromoles of P~ are taken up. The reaction is stopped by removing the vessel from the bath and is immediately deproteinized with 2 ml of 10% trichloroacetic acid. After centrifugation, 0.05-0.1 ml aliquots of the supernatant solution are removed for P~ determination,is Procedure for Measuring Oxygen Uptake and ADP Respiratory Control Ratio Oxygen consumption was measured with the use of a Clark oxygen electrode. 14 The reaction vessel contained the following in a total volume of 1.0 ml (or 6.5 ml) : substrate for respiration, 5 or 10 mM; potassium phosphate (pH 7.4), 5 mM; MgC12, 10 mM; triethanolamine buffer (pH 7.4), 30 mM; KC1, 25 mM; serum albumin (dialyzed against 50 mM triethanolamine buffer for 2 days), 0.3 or 1%; KF, 5 mM; EDTA, 1 mM; mannitol (or sucrose), 50 mM; and tumor mitochondria, 0.7 to 1.4 mg of protein/ml (or 5-8 mg dry wt/6.5 ml). After 2 minutes at 25 °, l~See Vol. II [lOll; also see this volume [3]. i~K. Lohmann and L. Jendrassik, Biochem. Z. 178, 419 (1926). Modified by using 5% (instead of 2.5%) ammonium molybdate in 5 N H~SO4.See W. D. Harris and P. Popat, J. Am. Oil Chem. Soc. 31, 124 (1954) as modified by T. Conover, Thesis, Univ. of Rochester, 1959. l~See Vol. VI [33]. The electrode used by one of us (R.W.) was modified by mounting the electrode in a horizontal position near the bottom of the reaction vessel (1 ml capacity), so that reagents can be conveniently added from the top of the reaction vessel. This modification was made by Dr. M. E. Pullman and Mr. M. Kandrach and is gratefully acknowledged.
[19]
MITOCHONDRIA FROM ASCITES TUMOR CELLS
107
A D P (final concentration 0.1-0.4 raM) was added and the oxygen consumption was recorded for approximately 10 more minutes. The respiratory control ratio (defined as the rate of respiration in the presence of A D P divided by the rate obtained following the expenditure of ADP)1~, 1~ was then calculated. Procedure for the Preparation of Ascites T u m o r Mitochondria
Method A. Grinding of the Tumor Cells with Mortar and Pestle. The Ehrlich ascites tumor cells are obtained by harvesting the ascites fluids from 4-6 mice (7-9 days tumor age). The fluids are immediately diluted in 2 volumes of ice-cold mannitoW (0.25 M) solution containing E D T A (2 m M ) , p H 7.5, and the cells are centrifuged at 600 g for 5 minutes. The packed cells are washed twice by resuspending the cells in approximately 10 volumes of the same solution and centrifuging as above. For homogenization, the packed tumor cells (approximately 5 ml) are mixed with 1 ml of the m a n n i t o l - E D T A solution and 0.1 ml of 1 M triethanolamine buffer (pH 7.4). The mixture is poured into a precooled mortar (7 cm diameter) ; 3 g of washed glass beads ~s are added, and the mixture is ground forcefully with a pestle for 6 minutes at 4 °. Then 2 5 m l of m a n n i t o l - E D T A solution is added to the mortar and the mixture is ground for 20 seconds to give an even suspension. The mixture is poured into a 40 ml polyethylene tube and centrifuged at 600 g for 10 minutes. The supernatant solution is decanted and stored at 4 °. The pellet is mixed with 2 ml of the m a n n i t o l - E D T A solution, 0.1 ml of 1 M triethanolamine buffer, 2 g of washed glass beads; the mixture is ground for another 4 minutes. Then 25 ml of m a n n i t o l - E D T A solution is added and the mixture is centrifuged as above. The supernatant solution from the second batch is combined with t h a t from the first, and the combined solution is divided among 2 polyethylene tubes and centrifuged at 9000 g for 15 minutes in the 9 RA head of the Lourdes refrigerated centrifuge. The supernatant solution and "fluffy layer" are decanted and discarded. The mitochondrial pellets are washed twice after their suspension in 25 ml of m a n n i t o l - E D T A solution with a Teflon pestle inserted directly into the centrifuge tube. The final pellets are suspended in 2 ml of the UB. Chance and M. Baltscheffsky, Biochem. J. 68, 283 (1958). 1~B. Chance, Ciba Found. Syrup. Regulation Cell Metabolism p. 91. Little, Brown, Boston, Massachusetts, 1959. ~ Sucrose (0.25 M) solution containing 1 or 2 mM EDTA may be used in place of mannitol-EDTA solution. All further steps were carried out at 4 °. 18Superbrite glass beads (0.1 mm diameter, type 130-5005, from Minnesota Mining and Manufacturing Company, St. Paul, Minnesota) were washed by soaking in 2 volumes of 5 N YIC1 overnight and washed with 10 volumes of deionized water about 6 times until the acidity of the washes approached pH 5.
108
PREPARATION OF MITOCHONDRIA
[19]
m a n n i t o l - E D T A solution and evenly dispersed with the use of a Teflon pestle. Method B. Dounce Homag~enizer Method. ~1,19 The ascitic fluid from 5 or 6 mice (8-11 days t u m o r age) is removed by syringe and transferred to two 40-ml graduated centrifuge tubes packed in ice. All further procedures are carried out in the cold room. The ascitie fluid is centrifuged for 5 minutes at 900 g, and the s u p e r n a t a n t fluid is decanted and discarded. The packed cell volume should be a b o u t 10-15 ml of bloodless cells. 2° T h e packed cells are washed twice with 30-ml portions of 0.25 M sucrose t h a t contains 0.001 M disodium E D T A , p H 4-5 (solution S). The twice washed tumor cells are then resuspended with 40 ml of solution S, to which has been added 0.1 ml of 0.1 M citric acid, and transferred to a Dounce homogenizer (40-ml size) packed in ice. For support the homogenizer is inserted into a hole in a large rubber stopper. TM Solution S is added until fluid fills the cylinder and lower portion of the reservoir of the homogenizer. Homogenization is accomplished b y 10-15 vigorous, directly vertical passes of the pestle. The homogenate should be examined microscopically for unbroken cells and more passes made if necessary. 2~ The resultant homogenate (approximately 60 ml, depending upon the size of the homogenizer used) is divided between two 40-ml centrifuge tubes and centrifuged at 900 g for 20 minutes. T h e top twothirds of the s u p e r n a t a n t solutions obtained are decanted and stored in ice. The sediments are combined and resuspended to 60 ml with solution A. L. Dounce, R. F. Witter, K. J. Monty, S. Pate, and M. A. Cottone, J. Biophys. and Biochem. Cylol. 1, 139 (1955). Douuee homogenizers may be purchased from Blaessig Glass Specialties, Rochester, New York, or from Kontes Glass, Vineland, New Jersey. The clearance found most useful for the fractionation of aseites cells is 0.0005 inch. This corresponds to the tight-fitting pestle as supplied by Blaessig or the loose-fitting pestle as supplied by Kontes. 20Ascites cell samples that are grossly bloody should not be used. Samples that are only slightly bloody are suitable for fractionation after removal of the erythrocytes (RBC). This may be accomplished in two ways. Low speed centrifugations (125g for 4 minutes) in isotonic sucrose will sediment most of the tumor cells while leaving the RBC in the supernatant fluid. The RBC are then decanted and the procedure is repeated until the number of RBC are reduced to acceptable levels. Erythrocytes may also be removed by "differential lysis." In this procedure the contaminated ascites cells are suspended to 4 or 5 times their volume with cold 1:4 diluted Ringer solution and centrifuged for 5 minutes at 960 {7. The swollen, intact ascites cells will sediment and the supernatant fluid which contains the hemoglobin is decanted. The ascites cells may then be washed in isotonic media. The latter method is recommended since there is little or no apparent damage to the tumor cells and the removal of RBC is quick and nearly complete. 21If on microscopic examination the cell breakage is less than 60% after at least 20 passes, it is likely that homogenization is either not vigorous enough or that the clearance of the homogenizer is too large or too small.
[19]
MITOCHONDRIA FROM ASCITES TUMOR CELLS
109
S; the homogenization and centrifugation (900 g for 20 minutes) are repeated. During the second homogenization, extremely vigorous passes are not necessary. The yield from the second homogenization is usually small. The upper portions of the supernatant solutions obtained from the centrifugation of the second homogenate are combined with those obtained from the first, and the mixture is distributed among 8 No. 40 tubes for the Spinco centrifuge and centrifuged for 15 minutes at 8000 g. Any refrigerated centrifuge capable of developing this centrifugal force is suitable. The supernatant solutions and fluffy layers are decanted and discarded. Each sediment is resuspended with 6 ml of solution S by inserting a small motor-drive Teflon pestle (rotating at slow speed) directly into each centrifuge tube. The suspended sediments are again centrifuged at 8000 q for 15 minutes, and then this washing procedure is repeated. The twice washed sediments are resuspended to 10 ml (the mitochondrial fraction), which contains about 12-15 mg dry weight per milliliter (8-10 mg protein). The mitochondrial fraction represents a 7-10% recovery of the total dry weight of the homogenate. Properties of the Ascites T u m o r Mitochondria The P : O ratio, the respiratory control ratio, the yield, and the specific activity (millimicromoles of P~ esterified per minute per milligram of protein) of mitochondria prepared by the two methods are shown in the table. OXIDATIVE PHOSPHORYLATION WITH ]~HRLICH ASCITES TUMOR MITOCHONDRIA
Mitochondria prepared by
ADP respiratory S p e c i f i c control activity ratio
Yield
Substrate
P: 0 ratio
Grinding
26"
Succinate a-Ketoglutarate Pyruvate -}- malate
1.8 2.4 2.4
270 260 260
5.6 5.3 5.0
Dounce homogenization
85b
Suceinate a-Ketoglutarate Pyruvate -5 malate
1.6 2.4 2.2
210 160 160
4.5 5.7 4.0
"Milligrams of mitochondrial protein obtained per gram of cell protein. The values given for the manometric assays represent averages of duplicate determinations for a single preparation. The ADP respiratory control ratio values, which were obtained polarographlcally, represent averages for 3 (pyruvate ~ malate), 4 (succinate), or 12 (a-ketoglutarate) preparations. Considerable variation among preparations is to be expected, for example, for the latter 12 preparations the range for the respiratory control ratios was 2.5-9.5. This variation has been noted by others [e.g., P. Borst, J. Biophys. Biochem. Cytol. 7, 381 (1960)].
110
PREPARATION OF MITOCHONDRIA
[20]
Comments
Grinding of ascites tumor cells with glass beads in the mortar and pestle (Method A) can be applied to small amounts of ceils, e.g., 1 ml of packed cells (140 mg of cell protein), if necessary. When more than 5 ml of packed cells is used, either a larger mortar can be used or the grinding procedure can be operated with 5 ml of packed cells in each run. Homogenization of tumor cells in a Dounce homogenizer (method B) is more applicable to experiments where larger amounts of mitochondria are needed. Mitochondria prepared by these two methods give high P: 0 ratio and respiratory control ratio, comparable to mitoehondria prepared from other tissue sources.
[ 20 ] Neoplastic Tissue Mitochondria
By THOMAS M. DEVLI~¢ The multiplicity of solid tumor types, originating from a variety of normal tissues, makes it impossible to generalize about the isolation, properties, and activities of mitoehondria from neoplasms. Even with one tumor type, variations in the amount of connective tissue, degree of necrosis, contamination by normal tissue, and lability of the mitochondria requires that each tumor line be considered as a different entity, with preparative and assay procedures tailored to the individual malignancy. H o m o g e n i z a t i o n and Isolation M e d i u m
With some tumors, isotonic sucrose (0.25 M) suffices for the isolation of active mitoehondria comparable to those from normal tissues. 1-3 This is particularly the case for soft tumors, such as hepatomas, where disruption of the cells occurs under mild homogenization2 Higher sucrose concentrations (e.g., 0.35 M) have been employed to yield mitochondria with latent ATPase from Murphy-Sturm rat lymphosarcoma. 4 The use of mannitol, polyvinylpyrrolidone, or raffinose has not been extensively tested, but may yield better preparations from some tumors. ~ Emmelot 1R. K. Kielley, Cancer Res. 12, 124 (1952). 2A. C. Aisenberg, Cancer Res. ~.1, 295 (1961); Cancer Res. 21, 304 (1961). 3G. E. Boxer and T. M. Devlin, Science 134, 1495 (1961). See also T. M. Devlin and M. P. Pruss, Proc. Am. Assoc. Cancer Res. 3, 315 (1962). M. Blecher and A. White, J. Biol. Chem. 235, 3404 (1960). 5p. Emmelot, C. J. Bos, P. J. Brombacher, and J. F. :Hampe, Brit. J. Cancer 13, 348 (1959).