- Email: [email protected]
A simplified procedure for isolating plasma membranes from cultured mouse fibroblast cells: 3T3 and SV-3T3
ANALYTICAL
BIOCHEMISTRY
!%I,
98- 106 (1978)
A Simplified Procedure for Isolating Plasma Membranes from Cultured Mouse Fibroblast Cells: 3T3 and SV-3T3l S.HARSHMAN AND J. GRAHAMCONLIN Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received March 15. 1978 Plasma membranes have been isolated from 3T3 and SV-3T3 cells grown in culture. Cells were harvested mechanically and disrupted in simple isotonic buffered sait solutions without resorting to hypotonic swelling or chemical membrane “hardeners.” The method of storing collected cells, the cell concentration during disruption, and the method of mechanical disruption were found to be significant variables affecting the yield of plasma membranes. The plasma membranes were separated from mitochondria and other cellular organelles by a single centrifugation through a step sucrose gradient containing a viscosity barrier of Dextran T-500 (modified from A. S. Sun and B. Poole (1975) Anal. Biochem. 68, 260). The isolated plasma membranes were located by assay for the “marker” enzyme, alkaline phosphatase (EC 3.1.3.1). The isolated plasma membrane fraction was free of mitochondrial and essentially free of lysozymal and endoplasmic reticulum contamination, which were assayed by measuring cytochrome c reductase, arylsulfatase, and hydrolysis of cY-naphthol acetate, respectively. Of the enzymes tested, the phosphodiesterase activity was found to be the most specific assay for the plasma membrane from culture mouse fibroblast cells. The Y-nucleotidase (EC 3.1.3.5) activity, the other plasma membrane marker, was extremely low in activity and gave an additional peak of activity when 5’-adenilic acid was used as substrate as compared to the expected single peak obtained with 5’cytidilic acid as substrate. Overall recovery of isolated plasma membranes was greater than 75% as measured by the final recovery of phosphodiesterase activity.
Although there are a number of published procedures for cell fractionation and for isolation of various subcellular components (l-4), including plasma membranes (5-Q relatively few deal with cells containing high nuclear to cytoplasmic volume ratios, such as fibroblasts (9). Moreover, since it was our purpose to obtain plasma membranes as close to the native state as possible, we sought to avoid both “hardening” reagents such as ZnZ+ or fluorescein mercuric acetate (FMA)2 (lo), as well as r A preliminary report of this material was made at the 1975 Southeast-Southwest ACS meeting. This work was supported in part by NC1 Grant CA-17050. * Abbreviations used: FMA, fluorescein mercuric acetate; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; PBS, phosphate-buffered saline. 0003-2697/78/0901-0098$02.00/O Copyright 0 1978 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
98
PURIFICATION
OF MEMBRANES:
3T3 AND
SV-3T3
99
significant variations from isosmolar ionic conditions, which are known to selectively extract various membrane components, such as wheat germ agglutinin at low ionic strength (11) and histocompatibility antigen at high ionic strength (12). We describe below a simple procedure that permits the isolation of plasma membranes from mouse 3T3 and SV-3T3 fibroblast cells in 70 to 85% overall yields and approximately 90% purity. MATERIALS
AND METHODS
The cells (3T3) and the simian virus transformant derived from them (SV-3T3) were a gift of Dr. Howard Green. The cells were grown in roller flasks in modified Dulbecco’s high glucose medium. Additions to the medium included Tricine (3.58 g/liter) and Hepes (4.76 g/liter; Sigma Chemical Co., St. Louis, MO.), sodium bicarbonate (1.181 g/liter; Mallinckrodt Chemical Works, St. Louis, MO.), and 10% calf serum (Kansas City Biologicals, Lenexa, Kans.). The medium was sterilized by filtration. The homogenizers were obtained as follows: Dounce (B-14435, A. H. Thomas Co., Philadelphia, Pa.), power-driven Teflon (Tri-R-Instruments, Rockville Center, N. Y.), and the Tekmar (SDT-1OON Tekmar, Cincinnati, Ohio). PBS-K+ buffer consists of 0.14 M NaCl, 0.01 M KCl, and 0.01 M KH2POI, pH 7.4. Protein analysis was done by the method of Lowry et al. (13) using bovine serum albumin as the standard. Alkaline phosphodiesterase (EC 3.1.3.1) activity was measured as described by Touster et al. (5) except that the concentration of the stock, substrate p-nitrophenyl 5’-thymidilate, was reduced from 5 to 3 mg/ml. 5’-Nucleotidase (EC 3.1.3.5.) was assayed both spectrophotometrically as described by Chen et ul. (14) and by release of 32P04, as described by Glastris and Pfeiffer (15). Arylsulfatase (EC 3.1.6. l), a lysosomal marker, was measured using the Horvat and Touster (16) modification of the procedure reported by Roy (17). Hydrolysis of a-naphtholacetate, as described by Gomori (18) and glucose-6-phosphatase (EC 3.1.3.9) measurement using Swanson’s procedure (19) were used to assay for the presence of endoplasmic reticulum. Mitochondria were located by assaying for cytochrome c reductase (EC 1.6.99.3) by the method reported by Mahler (20). RESULTS
Collection
and Storage of Harvested
Cells
Since the cell mass available from the usual procedures of large scale tissue culture production is relatively small, we first investigated a variety of cell storage methods that would permit the convenient accumulation of harvested cells. Using the retention of “normal” morphology, as estimated by phase microscopy, and the retention of phosphodiesterase enzyme
100
HARSHMAN
AND CONLIN
activity as working criteria, we established the following collection routine for both 3T3 and SV-3T3 cells. The medium from the roller flasks to be harvested is decanted and the cells washed in situ with a 50-ml aliquot of PBS-K+ solution. Next, 20 ml of PBS-K+ is added and the cells are harvested by scraping. The cell suspension is decanted to a centrifuge tube and combined with a 20-ml rinse of the roller flask, and the cells are pelleted at 600g for 10 min. The cells are resuspended in 20 ml of PBS-K+ and an aliquot is taken for counting in a Model B Coulter cell counter. The cells are again centrifuged, the supernate is discarded totally, and the cell button is frozen in a -70°C freezer. Under these conditions, 95 to 100% of the original phosphodiesterase activity can be recovered after as much as 8 weeks of storage. In contrast, freezing the cells while suspended in 20 ml of PBS-K+ with or without added dimethylsulfoxide or glycerol (5-50%) led to a rapid near complete loss of enzymatic activity. Comparison
of Homogenization
Procedures
Our initial experiments using a Potter-Elvehjem homogenizer resulted in an unacceptably low recovery of phosphodiesterase activity which was below that of the original cell suspension. Therefore, we undertook a more detailed investigation of various homogenization procedures. Accordingly, we measured (a) the extent of cell disruption, as judged by microscopic examination, (b) the total recovery of phosphodiesterase activity, and (c) the percentage of total phosphodiesterase activity present in the supernate after removing the cell debris. In all cases, the total phosphodiesterase activity obtained after the initial homogenization was taken as 100%. The enzyme activity observed with the whole cell suspension, prior to homogenization, is approximately 70% of the post-homogenization activity. Examination of the results indicate that homogenization of fibroblast cells is best accomplished using the Tekmar homogenizer. Presumably local heating effects are not as readily controlled using the other homogenization methods tested. It is clear, too, that phase microscopy does not lend itself well to determining the extent of cell membrane rupture with these cell types. Thus, comparison of the percentage of cell rupture, which was obtained by examining the number of cells originally and after various times of homogenization using a hemocytometer and an inverted phase microscope, with the percentage of total phosphodiesterase activity, or more significantly, with the percentage of total phosphodiesterase recovered in the crude supernate, shows wide variations. The Effect of Cell Concentration An important variable in the homogenization procedure is the cell concentration itself. As shown in Fig. 1, maximum recovery of phosphodiesterase in the supernate obtained after low-speed centrifugation of the
PURIFICATION 100
OF MEMBRANES:
3T3 AND SV-3T3
101
r
1 40
CELL
1
I
80
120 NUMBER
I
160
,
200
X10-6/ml
FIG. 1. Effect of cell density on the recovery of phosphodiesterase activity in the crude supernate. The actual homogenization volume in each case was 3.0 ml. Each sample was homogenized by three 10-s bursts in a Tekmar homogenizer, and the crude debris was removed by centrifugation at 5°C at SOOg for 20 min. International centrifuge, Model UV. The percentage recovery of phosphodiesterase activity in the supemate was calculated as the ratio of enzyme activity of an aliquot from the supernate over the enzyme activity of an identical aliquot from the original homogenate times 100.
homogenate occurs at cell concentrations in the homogenization step of 80 x IO’Yml for both 3T3 and SV-3T3 cells. The unexpected drop in the fraction of “solubilized“ phosphodiesterase activity at high concentrations of SV-3T3 cells is not understood but is assumed to be related to spontaneous aggregation of these membranes. The configuration of the Tekmar homogenizer dictated a minimum volume of 3.0 ml. Thus, each homogenization batch was run with a total of about 2.5 x lOa cells in a volume of 3.0 ml of PBS-K+ buffer. This represents the combined yield of approximately 10 roller flasks for 3T3 cells and 3 flasks for SV-3T3 cells. Puri$cation of the Plasma Membrane Fraction
When the post-low-speed supernate material is centrifuged through a standard continuous sucrose gradient (5-25%), two bands of phosphodiesterase activity are obtained: a minor peak of activity in the density position expected for plasma membranes and a major peak at the density position expected for mitochondria. We interpreted this result as being indicative of a tight adsorption of plasma membrane onto the mitochondrial surface,
0
0
0
0
0
250 30 0
0
0
0
1
0
0I 2,035
0
0
296
1,055
0 0 0 0 I
[32P]AMP @pm)
8.76
0
6.36
0
AMP (pmol of Pi)
5’-Nucleotidase”
0
0
1 FRACTIONS
OF
0.264
2.8
0
0.227
0
0
0.7
0
0
10.5 12.9 15.2 5.4 5.0 6.0 0
0
0
0 0
0 0 0
0.133 0.063
Cytochrome c reductase (AOD/mg of proteinlmin at 550 nm)
0
36
35
234 274 103 65 60 56
Glucose-6phosphates 0.s of PJmg of protein/h)
Esterase (x 10 AODlmg of protein/l5 min at 540 nm)
SV-3T3 CELL HOMOGENATE
Enzyme activities assayed and units”
4.78
0
TABLE GRADIENT
CMP Cm01 of PiI
ASSAY OF VISCOSITY
0
0
0
0
0
2.1
21.0 18.0 17.2 22.8 3.8 1.9
Arylsulfatase (AOD/mg of protein/ 10 min at 520 nm)
ClFor details of procedure of each assay, see Materials and Methods section. Values in italics indicate fraction(s) with maximum activity. Samples below the interface with 64% sucrose were not taken. b 5’-Nucleotidase activity was so low that the fractions were pooled as indicated. [32P]AMP as substrate, cpm32Pi/mg ofprotein/h; AMP as substrate, Fmol of Pilmg of protein/h; CMP as substrate, pmol of P,/mg of protein/h.
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
I
Fraction (0.5 ml vol)
Phosphodiesterase (x 10m3 pm01 split/ mg of protein/h)
ENZYMATIC
PURIFICATION
OF MEMBRANES:
3T3 AND
SV-3T3
103
a common occurrence with homogenization of liver (4) and other tissues. Sun and Poole (21) reported a “viscosity barrier” gradient which was particularly effective in resolving plasma membranes from mitochondria. We have adapted their procedure for use in a stepwise gradient (see Table 2). From the top, the visible bands correspond to lipid-containing material at the surface, other membranous material next (which by enzyme analysis appears to be a mixture of lysosomal and endoplasmic reticulum membranes), followed closely by the plasma membrane fraction just below the interface with the Dextran T-500 fraction, and finally, the mitochondria band near the bottom of the centrifuge tube at the interface with the 64% sucrose fraction. The basis for the designation of the contents of each band is developed from the analysis of sequential samples of the gradient for selected marker enzyme activities (Table 1). It is clear that the phosphodiesterase activity centers over and is restricted to the lower of the two mid-tube bands, fractions 6 and 7. The discrete banding of the other marker enzymes is evident. The glucose-6-phosphatase activity appears to be distributed as would be expected for a soluble enyme. Calculations based on overlap of other enzymatic activities indicate a purity of the TABLE
2
SUMMARY OF PROCEDURE FOR ISOLATION OF PLASMA MEMBRANES FROM 3T3 AND SV-3T3 MOUSE FIBROBLAST CELLS A. Cell harvest 1. Rinse roller flasks with PBS-K’ 2. Harvest cells by scraping 3. Count cells 4. Pellet cell. decant buffer 5. Store -70°C B. Homogenate I. Rapidly thaw cells (2.5 x IO8 cells) 2. Suspend in 3.0 ml of PBS-K+ 3. Homogenize using Tekmar blender (3 x 10 s on, IO s off) C. Low speed centrifugation I. SOOT, 20 min, 5°C 2. Collect supemate fluid D. Viscosity gradient I. 2.0-m] sample 4.0-ml viscosity barrier 10% sucrose 13% Dextran T-500 3.0-ml 25% sucrose 3.0-ml 64% sucrose 2. SW-41 rotor 35.000 rpm. 30 min 3. Plasma membranes lower of the two mid-tube bands
104
HARSHMAN
AND
CONLIN
plasma membrane fraction of the order of 90%. For this calculation, the total protein content of the phosphodiesterase band is taken to be 100% plasma membranes, no activation of the phosphodiesterase is assumed, and the percentage contamination is obtained as that fraction of protein from the preceding band that overlaps the plasma membrane band. This value ranges from 2.8% using arylsulfatase to 10.3% using the esterase activity. DISCUSSION
We report here a procedure that permits the isolation of purified plasma membranes from 3T3 and SV-3T3 mouse fibroblast cells using only simple ionic buffers. The procedure is summarized in Table 2. The key features of the method, in addition to the isotonic salinephosphate buffer are (a) the rapid fragmentation of the cells using the Tekmar homogenizer, (b) the concentration of the cells during homog-
FIG. 2. Negative stained electron micrograph of mouse fibroblast membranes at various stages of purification. Plasma membrane fractions were examined by the negative staining procedure in the electron microscope. Drops of fragment suspensions were allowed to stand on carbon-coated grids to allow membrane fragments to adhere. The grids were then floated on distilled water to remove excess sucrose and drops of 2% sodium silicotungstate at neutral pH were placed on the grid. Excess stain was removed with filter paper and the grids were examined in the electron microscope. It is apparent that purification of the plasma membrane fragments involves removal of the larger components from the suspension. (A) after homogenization and low-speed centrifugation; (B) after sucrose viscosity barrier purification.
PURIFICATION
OF MEMBRANES:
3T3 AND SV-3T3
105
enization, and (c) the use of the “viscosity barrier” during the membrane isolation step. The method is rapid, and the plasma membranes are recovered as smaller fragments with most of the larger material being removed (Fig. 2). A novel feature that should be emphasized is the method of storing the harvested intact cells at -70°C as a pellet. This procedure should find general application in cell culture work and is superior in terms of enzyme recovery, to storing cell suspensions in a variety of combinations of media, dimethylsulfoxide, or glycerol that we tested. The curious observation of 5’-nucleotidase activity occurring in two positions in the gradient (Table 1) when 5’-adenilic acid is used as the substrate, has been reproduced many times and remains unexplained. A similar finding has been reported by Lipmann and Lee (22). When 5’-cytidilic acid is used as substrate, the 5’-nucleotidase activity is observed only at the gradient position corresponding to plasma membranes and is superimposable with the distribution of the phosphodiesterase activity. In agreement with the results reported by Sun et al. (23), the total activity of 5’-nucleotidase activity in 3T3 and SV-3T3 cells is very low and very large numbers of cells, such as we have employed here, are required to measure the enzyme activity. ACKNOWLEDGMENTS We thank K. Gale, S. Kaushal, and N. Derryberry for technical assistance. We are grateful to Dr. Sun for making available to us his data on 5’-nucleotidase activity in 3T3 and SV-3T3 cells (23) prior to publication and to Dr. John P. Robinson for preparing the negative stained electron micrograph of the plasma membrane fraction.
REFERENCES 1. 2. 3. 4. 5.
Yannarell, A., and Aronson, N. N., Jr. (1973) B&him. Biophys. Acta 31, 191. Dewald, B., and Touster, 0. (1973) J. Biol. Chem. 248, 7223. Robinson, D., and Willcox, P. (1%9) Biochim. Biophys. Acra 191, 183. Horvat, A., Baxandall, J., and Touster, 0. (1969) J. Cell Biol. 42, 469. Touster, O., Aronson, N. N., Jr., Dulaney, J. T., and Hendrickson, H. (1970) J. Biol.
6. 7. 8. 9.
Sheinin, R., and Onodera, K. (1972) Biochim. Biophys. Acra 274, 49. Shopsis, C., and Sheinin, R. (1976) Biochim. Biophys. Actu 433, 101. Carlsen, S. A., and Till, J. E. (1975) Canad. J. Biochem. 53, 106. Perdue, J. F. (1974) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 31, p. 162, Academic Press, New York. Warren, L. (1974) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 31, p. 156, Academic Press, New York. Burger, M. M. (1968) Nature (London) 219, 499. Prat, M., Tarone, G., and Conoglio, P. M. (1974) Immunochemistry 12, 9. Lowry, 0. H., Rosebrough, N. H., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 246, 710. Chen, P. S., Toribara, T. Y., and Warner, H. (1956) Anal. Chem. 28, 1756.
Chem.
10. 11. 12. 13. 14.
47, 604.
106
HARSHMAN
AND CONLIN
15. Glastris, B., andPfeifIer, S. E. (1974)in Methods in Enzymology(Colowick, S. P., and Kaplan, N. O., eds.), Vol. 32, p. 124, Academic Press, New York. 16. Horvat, A., and Touster, 0. (1968) Biochim. Biophys. Acfa 148, 725. 17. Roy, A. B. (1953) B&hem. .I. 53, 12. 18. Gomori, G. (1953) J. Lab. C/in. Med. 42, 445. 19. Swanson, M. A. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, p. 541, Academic Press, New York. 20. Mahler, H. R. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, p. 688, Academic Press, New York. 21. Sun, A. S., and Poole, B. (1975) Anal. Biochem. 68, 260. 22. Lipmann, F., and Lee, S. G. (1977) Proc. Nat. Acad. Sci. USA 74, 163. 23. Sun, A. S., Reinach, P. S., Gelman, S., and Rubin. E., in press.
BIOCHEMISTRY
!%I,
98- 106 (1978)
A Simplified Procedure for Isolating Plasma Membranes from Cultured Mouse Fibroblast Cells: 3T3 and SV-3T3l S.HARSHMAN AND J. GRAHAMCONLIN Department of Microbiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received March 15. 1978 Plasma membranes have been isolated from 3T3 and SV-3T3 cells grown in culture. Cells were harvested mechanically and disrupted in simple isotonic buffered sait solutions without resorting to hypotonic swelling or chemical membrane “hardeners.” The method of storing collected cells, the cell concentration during disruption, and the method of mechanical disruption were found to be significant variables affecting the yield of plasma membranes. The plasma membranes were separated from mitochondria and other cellular organelles by a single centrifugation through a step sucrose gradient containing a viscosity barrier of Dextran T-500 (modified from A. S. Sun and B. Poole (1975) Anal. Biochem. 68, 260). The isolated plasma membranes were located by assay for the “marker” enzyme, alkaline phosphatase (EC 3.1.3.1). The isolated plasma membrane fraction was free of mitochondrial and essentially free of lysozymal and endoplasmic reticulum contamination, which were assayed by measuring cytochrome c reductase, arylsulfatase, and hydrolysis of cY-naphthol acetate, respectively. Of the enzymes tested, the phosphodiesterase activity was found to be the most specific assay for the plasma membrane from culture mouse fibroblast cells. The Y-nucleotidase (EC 3.1.3.5) activity, the other plasma membrane marker, was extremely low in activity and gave an additional peak of activity when 5’-adenilic acid was used as substrate as compared to the expected single peak obtained with 5’cytidilic acid as substrate. Overall recovery of isolated plasma membranes was greater than 75% as measured by the final recovery of phosphodiesterase activity.
Although there are a number of published procedures for cell fractionation and for isolation of various subcellular components (l-4), including plasma membranes (5-Q relatively few deal with cells containing high nuclear to cytoplasmic volume ratios, such as fibroblasts (9). Moreover, since it was our purpose to obtain plasma membranes as close to the native state as possible, we sought to avoid both “hardening” reagents such as ZnZ+ or fluorescein mercuric acetate (FMA)2 (lo), as well as r A preliminary report of this material was made at the 1975 Southeast-Southwest ACS meeting. This work was supported in part by NC1 Grant CA-17050. * Abbreviations used: FMA, fluorescein mercuric acetate; Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; PBS, phosphate-buffered saline. 0003-2697/78/0901-0098$02.00/O Copyright 0 1978 by Academic F’ress, Inc. All rights of reproduction in any form reserved.
98
PURIFICATION
OF MEMBRANES:
3T3 AND
SV-3T3
99
significant variations from isosmolar ionic conditions, which are known to selectively extract various membrane components, such as wheat germ agglutinin at low ionic strength (11) and histocompatibility antigen at high ionic strength (12). We describe below a simple procedure that permits the isolation of plasma membranes from mouse 3T3 and SV-3T3 fibroblast cells in 70 to 85% overall yields and approximately 90% purity. MATERIALS
AND METHODS
The cells (3T3) and the simian virus transformant derived from them (SV-3T3) were a gift of Dr. Howard Green. The cells were grown in roller flasks in modified Dulbecco’s high glucose medium. Additions to the medium included Tricine (3.58 g/liter) and Hepes (4.76 g/liter; Sigma Chemical Co., St. Louis, MO.), sodium bicarbonate (1.181 g/liter; Mallinckrodt Chemical Works, St. Louis, MO.), and 10% calf serum (Kansas City Biologicals, Lenexa, Kans.). The medium was sterilized by filtration. The homogenizers were obtained as follows: Dounce (B-14435, A. H. Thomas Co., Philadelphia, Pa.), power-driven Teflon (Tri-R-Instruments, Rockville Center, N. Y.), and the Tekmar (SDT-1OON Tekmar, Cincinnati, Ohio). PBS-K+ buffer consists of 0.14 M NaCl, 0.01 M KCl, and 0.01 M KH2POI, pH 7.4. Protein analysis was done by the method of Lowry et al. (13) using bovine serum albumin as the standard. Alkaline phosphodiesterase (EC 3.1.3.1) activity was measured as described by Touster et al. (5) except that the concentration of the stock, substrate p-nitrophenyl 5’-thymidilate, was reduced from 5 to 3 mg/ml. 5’-Nucleotidase (EC 3.1.3.5.) was assayed both spectrophotometrically as described by Chen et ul. (14) and by release of 32P04, as described by Glastris and Pfeiffer (15). Arylsulfatase (EC 3.1.6. l), a lysosomal marker, was measured using the Horvat and Touster (16) modification of the procedure reported by Roy (17). Hydrolysis of a-naphtholacetate, as described by Gomori (18) and glucose-6-phosphatase (EC 3.1.3.9) measurement using Swanson’s procedure (19) were used to assay for the presence of endoplasmic reticulum. Mitochondria were located by assaying for cytochrome c reductase (EC 1.6.99.3) by the method reported by Mahler (20). RESULTS
Collection
and Storage of Harvested
Cells
Since the cell mass available from the usual procedures of large scale tissue culture production is relatively small, we first investigated a variety of cell storage methods that would permit the convenient accumulation of harvested cells. Using the retention of “normal” morphology, as estimated by phase microscopy, and the retention of phosphodiesterase enzyme
100
HARSHMAN
AND CONLIN
activity as working criteria, we established the following collection routine for both 3T3 and SV-3T3 cells. The medium from the roller flasks to be harvested is decanted and the cells washed in situ with a 50-ml aliquot of PBS-K+ solution. Next, 20 ml of PBS-K+ is added and the cells are harvested by scraping. The cell suspension is decanted to a centrifuge tube and combined with a 20-ml rinse of the roller flask, and the cells are pelleted at 600g for 10 min. The cells are resuspended in 20 ml of PBS-K+ and an aliquot is taken for counting in a Model B Coulter cell counter. The cells are again centrifuged, the supernate is discarded totally, and the cell button is frozen in a -70°C freezer. Under these conditions, 95 to 100% of the original phosphodiesterase activity can be recovered after as much as 8 weeks of storage. In contrast, freezing the cells while suspended in 20 ml of PBS-K+ with or without added dimethylsulfoxide or glycerol (5-50%) led to a rapid near complete loss of enzymatic activity. Comparison
of Homogenization
Procedures
Our initial experiments using a Potter-Elvehjem homogenizer resulted in an unacceptably low recovery of phosphodiesterase activity which was below that of the original cell suspension. Therefore, we undertook a more detailed investigation of various homogenization procedures. Accordingly, we measured (a) the extent of cell disruption, as judged by microscopic examination, (b) the total recovery of phosphodiesterase activity, and (c) the percentage of total phosphodiesterase activity present in the supernate after removing the cell debris. In all cases, the total phosphodiesterase activity obtained after the initial homogenization was taken as 100%. The enzyme activity observed with the whole cell suspension, prior to homogenization, is approximately 70% of the post-homogenization activity. Examination of the results indicate that homogenization of fibroblast cells is best accomplished using the Tekmar homogenizer. Presumably local heating effects are not as readily controlled using the other homogenization methods tested. It is clear, too, that phase microscopy does not lend itself well to determining the extent of cell membrane rupture with these cell types. Thus, comparison of the percentage of cell rupture, which was obtained by examining the number of cells originally and after various times of homogenization using a hemocytometer and an inverted phase microscope, with the percentage of total phosphodiesterase activity, or more significantly, with the percentage of total phosphodiesterase recovered in the crude supernate, shows wide variations. The Effect of Cell Concentration An important variable in the homogenization procedure is the cell concentration itself. As shown in Fig. 1, maximum recovery of phosphodiesterase in the supernate obtained after low-speed centrifugation of the
PURIFICATION 100
OF MEMBRANES:
3T3 AND SV-3T3
101
r
1 40
CELL
1
I
80
120 NUMBER
I
160
,
200
X10-6/ml
FIG. 1. Effect of cell density on the recovery of phosphodiesterase activity in the crude supernate. The actual homogenization volume in each case was 3.0 ml. Each sample was homogenized by three 10-s bursts in a Tekmar homogenizer, and the crude debris was removed by centrifugation at 5°C at SOOg for 20 min. International centrifuge, Model UV. The percentage recovery of phosphodiesterase activity in the supemate was calculated as the ratio of enzyme activity of an aliquot from the supernate over the enzyme activity of an identical aliquot from the original homogenate times 100.
homogenate occurs at cell concentrations in the homogenization step of 80 x IO’Yml for both 3T3 and SV-3T3 cells. The unexpected drop in the fraction of “solubilized“ phosphodiesterase activity at high concentrations of SV-3T3 cells is not understood but is assumed to be related to spontaneous aggregation of these membranes. The configuration of the Tekmar homogenizer dictated a minimum volume of 3.0 ml. Thus, each homogenization batch was run with a total of about 2.5 x lOa cells in a volume of 3.0 ml of PBS-K+ buffer. This represents the combined yield of approximately 10 roller flasks for 3T3 cells and 3 flasks for SV-3T3 cells. Puri$cation of the Plasma Membrane Fraction
When the post-low-speed supernate material is centrifuged through a standard continuous sucrose gradient (5-25%), two bands of phosphodiesterase activity are obtained: a minor peak of activity in the density position expected for plasma membranes and a major peak at the density position expected for mitochondria. We interpreted this result as being indicative of a tight adsorption of plasma membrane onto the mitochondrial surface,
0
0
0
0
0
250 30 0
0
0
0
1
0
0I 2,035
0
0
296
1,055
0 0 0 0 I
[32P]AMP @pm)
8.76
0
6.36
0
AMP (pmol of Pi)
5’-Nucleotidase”
0
0
1 FRACTIONS
OF
0.264
2.8
0
0.227
0
0
0.7
0
0
10.5 12.9 15.2 5.4 5.0 6.0 0
0
0
0 0
0 0 0
0.133 0.063
Cytochrome c reductase (AOD/mg of proteinlmin at 550 nm)
0
36
35
234 274 103 65 60 56
Glucose-6phosphates 0.s of PJmg of protein/h)
Esterase (x 10 AODlmg of protein/l5 min at 540 nm)
SV-3T3 CELL HOMOGENATE
Enzyme activities assayed and units”
4.78
0
TABLE GRADIENT
CMP Cm01 of PiI
ASSAY OF VISCOSITY
0
0
0
0
0
2.1
21.0 18.0 17.2 22.8 3.8 1.9
Arylsulfatase (AOD/mg of protein/ 10 min at 520 nm)
ClFor details of procedure of each assay, see Materials and Methods section. Values in italics indicate fraction(s) with maximum activity. Samples below the interface with 64% sucrose were not taken. b 5’-Nucleotidase activity was so low that the fractions were pooled as indicated. [32P]AMP as substrate, cpm32Pi/mg ofprotein/h; AMP as substrate, Fmol of Pilmg of protein/h; CMP as substrate, pmol of P,/mg of protein/h.
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
I
Fraction (0.5 ml vol)
Phosphodiesterase (x 10m3 pm01 split/ mg of protein/h)
ENZYMATIC
PURIFICATION
OF MEMBRANES:
3T3 AND
SV-3T3
103
a common occurrence with homogenization of liver (4) and other tissues. Sun and Poole (21) reported a “viscosity barrier” gradient which was particularly effective in resolving plasma membranes from mitochondria. We have adapted their procedure for use in a stepwise gradient (see Table 2). From the top, the visible bands correspond to lipid-containing material at the surface, other membranous material next (which by enzyme analysis appears to be a mixture of lysosomal and endoplasmic reticulum membranes), followed closely by the plasma membrane fraction just below the interface with the Dextran T-500 fraction, and finally, the mitochondria band near the bottom of the centrifuge tube at the interface with the 64% sucrose fraction. The basis for the designation of the contents of each band is developed from the analysis of sequential samples of the gradient for selected marker enzyme activities (Table 1). It is clear that the phosphodiesterase activity centers over and is restricted to the lower of the two mid-tube bands, fractions 6 and 7. The discrete banding of the other marker enzymes is evident. The glucose-6-phosphatase activity appears to be distributed as would be expected for a soluble enyme. Calculations based on overlap of other enzymatic activities indicate a purity of the TABLE
2
SUMMARY OF PROCEDURE FOR ISOLATION OF PLASMA MEMBRANES FROM 3T3 AND SV-3T3 MOUSE FIBROBLAST CELLS A. Cell harvest 1. Rinse roller flasks with PBS-K’ 2. Harvest cells by scraping 3. Count cells 4. Pellet cell. decant buffer 5. Store -70°C B. Homogenate I. Rapidly thaw cells (2.5 x IO8 cells) 2. Suspend in 3.0 ml of PBS-K+ 3. Homogenize using Tekmar blender (3 x 10 s on, IO s off) C. Low speed centrifugation I. SOOT, 20 min, 5°C 2. Collect supemate fluid D. Viscosity gradient I. 2.0-m] sample 4.0-ml viscosity barrier 10% sucrose 13% Dextran T-500 3.0-ml 25% sucrose 3.0-ml 64% sucrose 2. SW-41 rotor 35.000 rpm. 30 min 3. Plasma membranes lower of the two mid-tube bands
104
HARSHMAN
AND
CONLIN
plasma membrane fraction of the order of 90%. For this calculation, the total protein content of the phosphodiesterase band is taken to be 100% plasma membranes, no activation of the phosphodiesterase is assumed, and the percentage contamination is obtained as that fraction of protein from the preceding band that overlaps the plasma membrane band. This value ranges from 2.8% using arylsulfatase to 10.3% using the esterase activity. DISCUSSION
We report here a procedure that permits the isolation of purified plasma membranes from 3T3 and SV-3T3 mouse fibroblast cells using only simple ionic buffers. The procedure is summarized in Table 2. The key features of the method, in addition to the isotonic salinephosphate buffer are (a) the rapid fragmentation of the cells using the Tekmar homogenizer, (b) the concentration of the cells during homog-
FIG. 2. Negative stained electron micrograph of mouse fibroblast membranes at various stages of purification. Plasma membrane fractions were examined by the negative staining procedure in the electron microscope. Drops of fragment suspensions were allowed to stand on carbon-coated grids to allow membrane fragments to adhere. The grids were then floated on distilled water to remove excess sucrose and drops of 2% sodium silicotungstate at neutral pH were placed on the grid. Excess stain was removed with filter paper and the grids were examined in the electron microscope. It is apparent that purification of the plasma membrane fragments involves removal of the larger components from the suspension. (A) after homogenization and low-speed centrifugation; (B) after sucrose viscosity barrier purification.
PURIFICATION
OF MEMBRANES:
3T3 AND SV-3T3
105
enization, and (c) the use of the “viscosity barrier” during the membrane isolation step. The method is rapid, and the plasma membranes are recovered as smaller fragments with most of the larger material being removed (Fig. 2). A novel feature that should be emphasized is the method of storing the harvested intact cells at -70°C as a pellet. This procedure should find general application in cell culture work and is superior in terms of enzyme recovery, to storing cell suspensions in a variety of combinations of media, dimethylsulfoxide, or glycerol that we tested. The curious observation of 5’-nucleotidase activity occurring in two positions in the gradient (Table 1) when 5’-adenilic acid is used as the substrate, has been reproduced many times and remains unexplained. A similar finding has been reported by Lipmann and Lee (22). When 5’-cytidilic acid is used as substrate, the 5’-nucleotidase activity is observed only at the gradient position corresponding to plasma membranes and is superimposable with the distribution of the phosphodiesterase activity. In agreement with the results reported by Sun et al. (23), the total activity of 5’-nucleotidase activity in 3T3 and SV-3T3 cells is very low and very large numbers of cells, such as we have employed here, are required to measure the enzyme activity. ACKNOWLEDGMENTS We thank K. Gale, S. Kaushal, and N. Derryberry for technical assistance. We are grateful to Dr. Sun for making available to us his data on 5’-nucleotidase activity in 3T3 and SV-3T3 cells (23) prior to publication and to Dr. John P. Robinson for preparing the negative stained electron micrograph of the plasma membrane fraction.
REFERENCES 1. 2. 3. 4. 5.
Yannarell, A., and Aronson, N. N., Jr. (1973) B&him. Biophys. Acta 31, 191. Dewald, B., and Touster, 0. (1973) J. Biol. Chem. 248, 7223. Robinson, D., and Willcox, P. (1%9) Biochim. Biophys. Acra 191, 183. Horvat, A., Baxandall, J., and Touster, 0. (1969) J. Cell Biol. 42, 469. Touster, O., Aronson, N. N., Jr., Dulaney, J. T., and Hendrickson, H. (1970) J. Biol.
6. 7. 8. 9.
Sheinin, R., and Onodera, K. (1972) Biochim. Biophys. Acra 274, 49. Shopsis, C., and Sheinin, R. (1976) Biochim. Biophys. Actu 433, 101. Carlsen, S. A., and Till, J. E. (1975) Canad. J. Biochem. 53, 106. Perdue, J. F. (1974) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 31, p. 162, Academic Press, New York. Warren, L. (1974) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 31, p. 156, Academic Press, New York. Burger, M. M. (1968) Nature (London) 219, 499. Prat, M., Tarone, G., and Conoglio, P. M. (1974) Immunochemistry 12, 9. Lowry, 0. H., Rosebrough, N. H., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 246, 710. Chen, P. S., Toribara, T. Y., and Warner, H. (1956) Anal. Chem. 28, 1756.
Chem.
10. 11. 12. 13. 14.
47, 604.
106
HARSHMAN
AND CONLIN
15. Glastris, B., andPfeifIer, S. E. (1974)in Methods in Enzymology(Colowick, S. P., and Kaplan, N. O., eds.), Vol. 32, p. 124, Academic Press, New York. 16. Horvat, A., and Touster, 0. (1968) Biochim. Biophys. Acfa 148, 725. 17. Roy, A. B. (1953) B&hem. .I. 53, 12. 18. Gomori, G. (1953) J. Lab. C/in. Med. 42, 445. 19. Swanson, M. A. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, p. 541, Academic Press, New York. 20. Mahler, H. R. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 2, p. 688, Academic Press, New York. 21. Sun, A. S., and Poole, B. (1975) Anal. Biochem. 68, 260. 22. Lipmann, F., and Lee, S. G. (1977) Proc. Nat. Acad. Sci. USA 74, 163. 23. Sun, A. S., Reinach, P. S., Gelman, S., and Rubin. E., in press.