Proc. Nati. Acad. Sci. USA Vol. 74, No. 11, pp. 5069-5072, November 1977
Cell Biology
Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells (isoelectric focusing-immune electrophoresis/isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis/ S-antigen/ U-antigen)
RUPERT SCHMIDT-ULLRICH, W. SCOTT THOMPSON, PECK-SUN LIN, AND DONALD F. H. WALLACH* Tufts-New England Medical Center, Therapeutic Radiology Department, Radiobiology Division, 171 Harrison Avenue, Boston, Massachusetts 02111
Communicated by Gerhard Schmidt, September 2,1977
ABSTRACT Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the p1 4.5 and p 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pl 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively. Purified plasma membranes and mitochondria (1) from simian virus 40(SV40)-transformed hamster lymphocytes (GD248) contain immunoelectrophoretically defined antigens (2, 3) that are absent from the membranes of normal lymphocytes. Bidimensional isoelectric focusing/immune electrophoresis shows that the distinctive antigens represent protein classes with isoelectric points at pH 4.5 and 4.7 (3). We now show that these antigens occur also in the membranes of nonlymphoid SV40transformed hamster cells as well as in the membranes of SV4O-transformed mouse fibroblasts, suggesting that they are SV4O-coded. The molecular weights of the antigens, about 58,000 and 90,000-110,000, respectively, for the pI 4.5 and pI 4.7 components, fit molecular weight estimates for SV40-S (transplantation) and U antigen.
MATERIALS AND METHODS Chemicals. Triton X-100, N-2-hydroxyethylpiperazineN'-2-ethanesulfonate (Hepes), dithiothreitol, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO), dodecyl sulfate (DodSO4) and urea from Fisher Chemical Co. (Fair Lawn, NJ), agarose (lot 102D) from Litex (Glostrup, Denmark), acrylamide, N,N'-methylenebisacrylamide, N,N,N',N'-tetramethylethylenediamine, ammonium persulfate, and Coomassie brilliant blue from Bio-Rad Laboratories (Richmond, CA), ampholytes (Ampholine pH 3.5-10.0 and pH 4.0-6.0) from LKB (Upsala, Sweden), and complete Freund's adjuvant from Difco Laboratories (Detroit, MI). Eagle's minimum essential medium and RPMI 1640 were purchased from Associated Biomedic Systems, Inc. (Buffalo, NY) and fetal calf serum, from Grand Island Biological Co. (Grand Island, NY). Cells. GD248 lymphocytes were propagated subcutaneously (1-3) in outbred golden Syrian hamsters. SV40 T antigen-positive T19 cells were isolated from a hamster reticulum cell sarcoma produced by adherent phagocytic cells derived from a GD248 tumor (unpublished data). T19 cells and BALB/c The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
SV3T3 mouse fibroblasts (Flow Laboratories, Rockville, MD) were grown in RPMI 1640 and Eagle's minimum essential medium, respectively, both containing 10% heat-inactivated fetal calf serum. Membranes. GD248 plasma membranes were isolated as described (1-3). Plasma membrane-enriched membrane vesicles (fraction 5; ref. 1) were isolated from T19 and SV3T3 cells as described (1). For immune electrophoresis and isoelectric focusing, the membranes were solubilized in 1 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate, pH 8.5/1% Triton X-100 (2, 3). Antisera. Anti-GD248-membrane serum was developed in guinea pigs as described (2, 3), with boosting three times and bleeding 10 days after the last booster. To obtain anti-T19 sera, 1.5 to 6.0 X 107 cultured T19 cells were injected intravenously into rabbits. The animals were boosted with the same number of cells at weekly intervals. The serum used here was a pool of four bleedings from two rabbits, each bleeding 1 week after the booster. Rabbit antiserum against hamster immunoglobulin was purchased from Grand Island Biological Co. Normal rabbit and guinea pig sera give no immune precipitates (cf. also refs. 2 and 3). Crossed Immunoelectrophoresis. We applied this method as described (2) but with anti-hamster-immunoglobulin in the second dimension. Bidimensional Isoelectric Focusing/Immune Electrophoresis. The procedure was as described (3). Isoelectric focusing was in gel slabs (4% acrylamide, crosslinked with 2.5% bisacrylamide) containing 2% ampholytes (pH 3.5-10), 8 M urea, 1% Triton X-100, and 10% sucrose. The catholyte was 1 M NaOH and the anolyte, 1 M H3PO4. We applied about 600 ,g of T19 or SV3T3 membrane vesicle protein per lane and -400 ,ug of GD248-plasma membrane protein. After focusing, the 10 X 90 mm polyacrylamide section containing the focused proteins was sliced into two 5 X 90 mm strips. One of these was washed three times (10 min each) in 20 ml of 0.038 M Tris/0.1 M glycine, pH 8.7/1% Triton X-100 and then immunoelectrophoresed. The other strip was stained as described (3). For the second dimension, immune electrophoresis (3), we cast 80 X 80 mm immunoplates in two sections: (i) a cathodal 30 X 80 X 1.5 mm agarose strip (1%) without antibody, and (ii) a 50 X 80 X 1.5 mm area containing 0.275 ml of antiserum. Buffers and other conditions were as described (3). The washed focusing gel strip was placed on top of the serum-free agarose with the focusing axis perpendicular to the direction of immune electrophoresis and the nearest edge of the focusing strip 4 mm away from the interface between the two agarose domains. The focused proteins were then electrophoresed through the antiAbbreviations: SV40, simian virus 40; DodSO4, dodecyl sulfate. * To whom correspondence should be addressed.
5069
5070
Cell Biology: Schmidt-Ullrich et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
.AX 2 ,,
9.0
8.0
7.0
6,0
5.0
4.0
pH FIG. 1. Isoelectric focusing of solubilized (Triton X-100) T19 membrane proteins in the first dimension (horizontal), and immune electrophoresis in the second dimension (vertical) into agarose containing anti-GD248-membrane serum (46 Al/ml of agarose) in the upper compartment of the immunoplate. The pI 4.5 and 4.7 immunoprecipitates are indicated by arrows and numbered 1 and 2, respectively. The pH gradient is given in the lower compartment of the immunoplate. The results depicted are representative of three independent experiments.
body-free zone into the antibody-containing agarose at pH 8.7, 10 V per slab, for 20 hr at 6°. For demonstration of antigenic identity, we used the crossed-line immune electrophoresis approach (3, 4) in the second dimension, casting an 8 X 8 mm agarose strip between the antibody-free area and the antibody-containing area which contained the test antigen. Staining was as described (3). Bidimensional Isoelectric Focusing/DodSO4 Polyacrylamide Gel Electrophoresis. The isoelectric focusing was in cylindrical polyacrylamide gels (65 mm X 3 mm; 400-500,ug of protein per gel) of the composition given above, with both pH 3.5-10 and pH 4-6 gradients. The catholytes and anolytes were 0.03 M NaOH and 0.05 M H2SO4, respectively. For the second-dimension DodSO4/gel electrophoresis step, we used gel slabs (75 X 75 X 2.75 mm; 7.5% acrylamide, crosslinked with 2.5% bisacrylamide) on top of a "sealing gel" (10 X 75 X 2.75 mm; 15% acrylamide, crosslinked with 2.5% bisacrylamide). The gels were cast with 0.04 M Tris/0.02 M acetate/2 mM EDTA, pH 7.4, as buffer. Prior to the second-dimension step, the slabs were pre-electrophoresed with 0.04 M Tris/0.02 M acetate/2 mM EDTA/1% (wt/vol) DodSO4, pH 7.4, for 15 min at 25 mA per slab. At the same time, the focusing gels were equilibrated with DodSO4/electrophoresis buffer (3% DodSO4 and 0.12 M dithiothreitol), 10 ml of buffer per gel and buffer changed every 10 min for 50 min. These washes introduce the detergent and reducing agent required for the second-dimension separation. They also eliminate the focusing pH gradient. The focusing gel was then positioned on top of the preelectrophoresed gel slab, at the end distal from the sealing gel, with focusing axis perpendicular to the direction of electrophoresis. Electrophoresis was for 16 hr at 8 mA per slab. The slabs were washed and stained as described (3). Other Determinations. Protein was assayed as described (1-3). pH gradients were determined by using a contact-pH electrode (Ingold ES 47300-02) at 2-mm intervals.
RESULTS Membrane Protein Solubilization. More than 80% of the protein of all membranes studied here was solubilized (nonsedimentable at -300,000 X g for 60 min) by 1% Triton X-100, pH 8.5. Crossed Immunoelectrophoresis. Membrane-bound 7S IgG2 is a marker for GD248 lymphocytes (6, 7). Because T19 cells are derived from GD248 tumors, we looked for 7S IgG2 in T19 membranes. No immune precipitate was observed, al-
,
8.D
I
7.0
U-----------1---U- - - - - r-----
60
5.0
4.5
pH FIG. 2. Bidimensional isoelectric focusing/immune electrophoresis of T19 membrane proteins. Isoelectric focusing: horizontal; 0.6 mg of protein. Immune electrophoresis: vertical into agarose containing anti-T19-cell serum (90 Mul/ml of agarose). Arrows indicate the
immune precipitates.
though hamster immunoglobulin from other sources gave a strong reaction. Bidimensional Isoelectric Focusing/Immune Electrophoresis. The precipitation pattern of GD248 membrane proteins with anti-GD248-membrane serum was as before (3), showing two "multirocket" (8, 9) immune precipitates at pH 4.5 and 4.7. These precipitates represent antigens absent from normal hamster lymphocytes, as tested by crossed-line immune electrophoresis (3, 4) and by extensive absorption of antiGD248-membrane serum with membranes of normal cells. When the focused membrane proteins of T19 cells were electrophoresed into anti-GD248-membrane serum, two immune precipitates appeared (Fig. 1) at pH 4.5 and pH 4.7, and each consisted of more than one rocket. A minor component, at pH 6.0, was not detected in SV3T3 membranes (see below). The multirocket pl 4.5 and 4.7 components were also detected when T19 membranes were run against rabbit anti-T19 serum (Fig. 2). No immunoglobulin was detected with anti hamsterimmunoglobulin in immune electrophoresis. Finally, as shown in Fig. 3, when focused membrane proteins from SV3T3 cells were run against anti-GD248-membrane serum, only the complex pl 4.5 and pl 4.7 immune precipitates appeared. The data with T19 and SV3T3 cells rule out the possibility that the pl 4.5 and pI 4.7 precipitates represent clonal or species markers. Bidimensional Isoelectric Focusing/DodSO4 Gel Electrophoresis. As shown in Figs. 4-6, on second-dimension DodSO4 slab gel electrophoresis, the pl 4.5 focusing components of GD248 lymphocytes, T19 cells, and SV3T3 fibroblasts resolved into two overlapping zones with molecular weights near 58,000. However, the proportion of pI 4.5 material depended on cell type and, on simple isoelectric focusing, the pl 4.5 component was least prominent with SV3T3 cells. However, even in this case, good resolution was obtained after the second-dimension electrophoresis run. The pl 4.7 components of GD248, T19, and SV3T3 cells separated into two spots, localizing between 90,000 and 110,000 daltons (arrows in Figs. 4-6) but yielded no staining between 50,000 and 90,000 daltons. Use of expanded pH gradients (pH 4-6) showed that the weak spot at -80,000 daltons in the case of SV3T3 membranes did not coincide with either the pI 4.5 or the pI 4.7 components. Isoelectric focusing/DodSO4 gel electrophoresis of membranes of normal hamster cells revealed no pI 4.5 component (3,5) and no components at pH 4.7 between 50,000 and 110,000 daltons. Because the origins are broader in bidimensional techniques than in unidimensional electrophoresis (5 mm here), seconddimension DodSO4 electrophoresis yielded less resolution than
Cell Biology: Schmidt-Ullrich et al. .., ..w-.:,w ...
Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 5. Bidimensional isoelectric focusing/DodSO4 slab gel electrophoresis of T19 membrane proteins. Details as in Fig. 4. II
I
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pH FIG. 3. Isoelectric focusing of solubilized BALB/c SV3T3 membrane proteins (Triton X-100) in the first dimension (horizontal) and immune electrophoresis in the second dimension (vertical) with anti-GD248-membrane serum (46 Al/ml of agarose). (A) Immune precipitation (top) and focusing pattern (bottom). Arrows point to the overlapping immune precipitates at pH 4.7 and pH 4.5. (B) Schematic of the immunoplate (A), including the pH gradient. One of the pI 4.7 rockets extended into the pH 4.5 range, possibly indicating partial crossreactivity of the two focusing components.
conventional DodSO4 electrophoresis, leading to somewhat less precise molecular weight estimates. However, the relative staining intensities of the -58,000 dalton and 90,000-110,000 dalton spots followed those of pl 4.5 and pl 4.7 components, respectively. Dimension 1 A
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DISCUSSION Plasma membranes and mitochondria of SV40-transformed hamster lymphocytes (GD248) contain antigenic protein classes that focus at pH 4.5 and 4.7 and are not detectable in normal lymphocyte populations or embryonic hamster cells (3). Our new data show that both components are SV40-specific. GD248 cells are B-type lymphocytes (6, 7) that synthesize 7S IgG2 and bear this immunoglobulin on their plasma membranes (2, 6). In contrast, T19 cells are phagocytes that lack IgG2. However, membranes from both cells reveal the pI 4.5 and pI 4.7 immune precipitates with both guinea pig anti-GD248membrane serum and rabbit anti-T19 serum. The pI 4.5 and pI 4.7 antigens therefore cannot represent lymphocyte markers. Untransformed or embryonic hamster cells (2, 3, 5) or BALB/c 3T3 cells (10) gave no immune reactions with our sera. The new components at pI 4.5 and pI 4.7 are present also in the membranes of SV3T3 fibroblasts and are the only SV3T3 membrane
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FIG. 4. Bidimensional isoelectric focusing/DodSO4 gel electrophoresis of GD248 membrane proteins. First dimension: isoelectric focusing, horizontal. Second dimension: slab gel electrophoresis, vertical. The abscissas give the pH gradient and a scheme of the focusing pattern. The ordinates give scales of relative electrophoretic mobilities (RM) and molecular weights (MW), as well as a schematic of the protein distribution obtained upon unidimensional DodSO4 gel electrophoresis. The numbering of the protein bands in the first and second dimensions are as in refs. 5 and 2, respectively.
8.S
7.5
&s
5.5
4.5
pH
FIG. 6. Bidimensional isoelectric focusing/DodSO4 slab gel electrophoresis of BALB/c SV3T3-membrane proteins. Details as in Fig. 4. Expanded pH gradients (pH 4.0-6.0) showed that no proteins between 90,000 and 40,000 daltons focused at pH 4.7.
Proc. Natl. Acad. Sci. USA 74 (1977)
5072 Cell Biology: Schmidt-Ullrich et al. entities that react with anti-GD248-membrane serum. The only feature common to GD248, T19, and BALB/c SV3T3 is SV40 transformation. The pI 4.5 and pI 4.7 proteins therefore are SV40-specific. Indirect immune fluorescence, with either the anti-T19 or the anti-GD248-membrane serum, yields strong S antigen and U antigen reactions but no T antigen staining with GD248, T19, SV3T3, and W18 VA2 cells or GD248 nuclei (10). U antigen, defined as associated with the nuclear periphery (11), has recently been shown to have a molecular weight of about 94,000 in transformed cells (12). The pI 4.7 components of GD248, T19, and SV3T3 cells have molecular weights of 90,000110,000. Because our sera react with U but not T antigen, the pI 4.7 antigen is reasonably identified with the SV40 U antigen. Whether this antigen is truly a plasma membrane component or is present due to trace contamination with nuclear envelope during fractionation remains to be determined. The pI 4.5 component, a glycoprotein (5), has a molecular weight near 58,000. Recent data indicate molecular weights of 50,000-60,000 (13) and 58,000 (14) for the transplantation antigen extracted with Triton X-100 from SV40-transformed fibroblasts. Further studies (15) also show a close relation between the transplantation and surface antigens. We do not have information on transplantation antigenicity, but our reagent sera give prominent surface antigen immune fluorescence. The pI 4.5 component can thus be identified with SV40 surface antigen and, by inference, SV40 transplantation antigen. This work was supported by Grant CB-44000 from the National Cancer Institute. 1. Schmidt-Ullrich, R., Wallach, D. F. H. & Davis, F. D. G., III (1976) "Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. 1. High yield purification of plasma membrane fragments," J. Nati. Cancer Inst. 57, 1107-1116. 2. Schmidt-Ullrich, R., Wallach, D. F. H. & Davis, F. D. G., III (1976) "Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. II. Plasma membrane proteins analyzed by dodecyl sulfate polyacrylamide electrophoresis and two dimensional immune electrophoresis," J. NatI. Cancer Inst. 57, 1117-1126. 3. Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977)
4.
5.
6. 7.
8. 9.
10.
11.
12. 13.
14.
15.
"Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40," Proc. Natl. Acad. Sc. USA 74, 643-647. Bjerrum, 0. J. & Bog-Hansen, T. C. (1976) "Immunochemical gel precipitation techniques for analysis of membrane proteins," in Biochemical Analyses of Membranes, ed. Maddy, A. H. (Chapman and Hall, London), pp. 378-426. Schmidt-Ullrich, R., Verma, S. P. & Wallach, D. F. H. (1975) "Anomalous side chain amidation in plasma membrane proteins of simian virus 40-transformed lymphocytes indicated by isoelectric focusing and laser Raman spectroscopy," Biochem. Blophys. Res. Commun. 67,1062-1069. Coe, J. E. & Green, I. (1975) "B-cell origin of hamster lymphoid tumors induced by simian virus 40," J. Natl. Cancer Inst. 54, 269-270. Coe, J. E. (1976) "Immunoglobulin synthesis by an SV40-induced hamster lymphoma," Immunology 31, 495-502. Laurell, C.-B. (1966) "Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies," Anal. Biochem. 15, 45-52. Johansson, K.-E. & Hjerten, S. (1974) "Localization of the Tween 20-soluble membrane proteins of Acholeplasma laidlawii by crossed immune electrophoresis," J. Mol. Biol. 86, 341-348. Lin, P. S., Schmidt-Ullrich, R. & Wallach, D. F. H. (1977) "Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope," Proc. Natl. Acad. Sci. USA 74,2495-2499. Lewis, A. N., Jr. & Rowe, W. P. (1971) "Studies on non-defective adenovirus simian hybrid viruses. 1. A newly characterized simian virus 40 antigen induced by the Ad2+ND1 virus," J. Virol. 7, 189-197. Robb, J. S. (1977) "Identification of simian virus 40 tumor and U antigens," Proc. Natl. Acad. Sci. USA 74,447-451. Luborsky, S. W., Chang, C., Pancake, S. J. & Mora, P. T. (1976) "Detergent solubilization and molecular weight estimation of tumor specific surface antigen from SV40 transformed cells," Biochem. Biophys. Res. Commun. 71,990-996. Anderson, J. L., Martin, R. G., Chang, C., Mora, P. T. & Livingston, D. M. (1977) "Nuclear preparations of SV40-transformed cells contain tumor specific transplantation antigen activity," Virology 76,420-425. Chang, C., Pancake, S. J., Luborsky, S. W. & Mora, P. T. (1977) "Detergent solubilization and partial purification of tumor specific surface and transplantation antigens from SV40 virus transformed cells," Int. J. Cancer 19, 258-266.
Cell Biology
Simian virus 40-specific proteins in the membranes of simian virus 40-transformed hamster and mouse cells (isoelectric focusing-immune electrophoresis/isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis/ S-antigen/ U-antigen)
RUPERT SCHMIDT-ULLRICH, W. SCOTT THOMPSON, PECK-SUN LIN, AND DONALD F. H. WALLACH* Tufts-New England Medical Center, Therapeutic Radiology Department, Radiobiology Division, 171 Harrison Avenue, Boston, Massachusetts 02111
Communicated by Gerhard Schmidt, September 2,1977
ABSTRACT Membranes of simian virus 40-transformed hamster lymphocytes and phagocytes, as well as of transformed mouse fibroblasts, contain two classes of antigenic virus-specific protein. The isoelectric points of these proteins, as defined by isoelectric focusing/immune electrophoresis are at pH 4.5 and 4.7. The molecular weights of the p1 4.5 and p 4.7 components, determined by isoelectric focusing/dodecyl sulfate polyacrylamide electrophoresis, lie near 58,000 and 90,000-110,000, respectively. The pl 4.5 and pI 4.7 proteins are tentatively identified with the surface (transplantation) and U antigens, respectively. Purified plasma membranes and mitochondria (1) from simian virus 40(SV40)-transformed hamster lymphocytes (GD248) contain immunoelectrophoretically defined antigens (2, 3) that are absent from the membranes of normal lymphocytes. Bidimensional isoelectric focusing/immune electrophoresis shows that the distinctive antigens represent protein classes with isoelectric points at pH 4.5 and 4.7 (3). We now show that these antigens occur also in the membranes of nonlymphoid SV40transformed hamster cells as well as in the membranes of SV4O-transformed mouse fibroblasts, suggesting that they are SV4O-coded. The molecular weights of the antigens, about 58,000 and 90,000-110,000, respectively, for the pI 4.5 and pI 4.7 components, fit molecular weight estimates for SV40-S (transplantation) and U antigen.
MATERIALS AND METHODS Chemicals. Triton X-100, N-2-hydroxyethylpiperazineN'-2-ethanesulfonate (Hepes), dithiothreitol, and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO), dodecyl sulfate (DodSO4) and urea from Fisher Chemical Co. (Fair Lawn, NJ), agarose (lot 102D) from Litex (Glostrup, Denmark), acrylamide, N,N'-methylenebisacrylamide, N,N,N',N'-tetramethylethylenediamine, ammonium persulfate, and Coomassie brilliant blue from Bio-Rad Laboratories (Richmond, CA), ampholytes (Ampholine pH 3.5-10.0 and pH 4.0-6.0) from LKB (Upsala, Sweden), and complete Freund's adjuvant from Difco Laboratories (Detroit, MI). Eagle's minimum essential medium and RPMI 1640 were purchased from Associated Biomedic Systems, Inc. (Buffalo, NY) and fetal calf serum, from Grand Island Biological Co. (Grand Island, NY). Cells. GD248 lymphocytes were propagated subcutaneously (1-3) in outbred golden Syrian hamsters. SV40 T antigen-positive T19 cells were isolated from a hamster reticulum cell sarcoma produced by adherent phagocytic cells derived from a GD248 tumor (unpublished data). T19 cells and BALB/c The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
SV3T3 mouse fibroblasts (Flow Laboratories, Rockville, MD) were grown in RPMI 1640 and Eagle's minimum essential medium, respectively, both containing 10% heat-inactivated fetal calf serum. Membranes. GD248 plasma membranes were isolated as described (1-3). Plasma membrane-enriched membrane vesicles (fraction 5; ref. 1) were isolated from T19 and SV3T3 cells as described (1). For immune electrophoresis and isoelectric focusing, the membranes were solubilized in 1 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate, pH 8.5/1% Triton X-100 (2, 3). Antisera. Anti-GD248-membrane serum was developed in guinea pigs as described (2, 3), with boosting three times and bleeding 10 days after the last booster. To obtain anti-T19 sera, 1.5 to 6.0 X 107 cultured T19 cells were injected intravenously into rabbits. The animals were boosted with the same number of cells at weekly intervals. The serum used here was a pool of four bleedings from two rabbits, each bleeding 1 week after the booster. Rabbit antiserum against hamster immunoglobulin was purchased from Grand Island Biological Co. Normal rabbit and guinea pig sera give no immune precipitates (cf. also refs. 2 and 3). Crossed Immunoelectrophoresis. We applied this method as described (2) but with anti-hamster-immunoglobulin in the second dimension. Bidimensional Isoelectric Focusing/Immune Electrophoresis. The procedure was as described (3). Isoelectric focusing was in gel slabs (4% acrylamide, crosslinked with 2.5% bisacrylamide) containing 2% ampholytes (pH 3.5-10), 8 M urea, 1% Triton X-100, and 10% sucrose. The catholyte was 1 M NaOH and the anolyte, 1 M H3PO4. We applied about 600 ,g of T19 or SV3T3 membrane vesicle protein per lane and -400 ,ug of GD248-plasma membrane protein. After focusing, the 10 X 90 mm polyacrylamide section containing the focused proteins was sliced into two 5 X 90 mm strips. One of these was washed three times (10 min each) in 20 ml of 0.038 M Tris/0.1 M glycine, pH 8.7/1% Triton X-100 and then immunoelectrophoresed. The other strip was stained as described (3). For the second dimension, immune electrophoresis (3), we cast 80 X 80 mm immunoplates in two sections: (i) a cathodal 30 X 80 X 1.5 mm agarose strip (1%) without antibody, and (ii) a 50 X 80 X 1.5 mm area containing 0.275 ml of antiserum. Buffers and other conditions were as described (3). The washed focusing gel strip was placed on top of the serum-free agarose with the focusing axis perpendicular to the direction of immune electrophoresis and the nearest edge of the focusing strip 4 mm away from the interface between the two agarose domains. The focused proteins were then electrophoresed through the antiAbbreviations: SV40, simian virus 40; DodSO4, dodecyl sulfate. * To whom correspondence should be addressed.
5069
5070
Cell Biology: Schmidt-Ullrich et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
.AX 2 ,,
9.0
8.0
7.0
6,0
5.0
4.0
pH FIG. 1. Isoelectric focusing of solubilized (Triton X-100) T19 membrane proteins in the first dimension (horizontal), and immune electrophoresis in the second dimension (vertical) into agarose containing anti-GD248-membrane serum (46 Al/ml of agarose) in the upper compartment of the immunoplate. The pI 4.5 and 4.7 immunoprecipitates are indicated by arrows and numbered 1 and 2, respectively. The pH gradient is given in the lower compartment of the immunoplate. The results depicted are representative of three independent experiments.
body-free zone into the antibody-containing agarose at pH 8.7, 10 V per slab, for 20 hr at 6°. For demonstration of antigenic identity, we used the crossed-line immune electrophoresis approach (3, 4) in the second dimension, casting an 8 X 8 mm agarose strip between the antibody-free area and the antibody-containing area which contained the test antigen. Staining was as described (3). Bidimensional Isoelectric Focusing/DodSO4 Polyacrylamide Gel Electrophoresis. The isoelectric focusing was in cylindrical polyacrylamide gels (65 mm X 3 mm; 400-500,ug of protein per gel) of the composition given above, with both pH 3.5-10 and pH 4-6 gradients. The catholytes and anolytes were 0.03 M NaOH and 0.05 M H2SO4, respectively. For the second-dimension DodSO4/gel electrophoresis step, we used gel slabs (75 X 75 X 2.75 mm; 7.5% acrylamide, crosslinked with 2.5% bisacrylamide) on top of a "sealing gel" (10 X 75 X 2.75 mm; 15% acrylamide, crosslinked with 2.5% bisacrylamide). The gels were cast with 0.04 M Tris/0.02 M acetate/2 mM EDTA, pH 7.4, as buffer. Prior to the second-dimension step, the slabs were pre-electrophoresed with 0.04 M Tris/0.02 M acetate/2 mM EDTA/1% (wt/vol) DodSO4, pH 7.4, for 15 min at 25 mA per slab. At the same time, the focusing gels were equilibrated with DodSO4/electrophoresis buffer (3% DodSO4 and 0.12 M dithiothreitol), 10 ml of buffer per gel and buffer changed every 10 min for 50 min. These washes introduce the detergent and reducing agent required for the second-dimension separation. They also eliminate the focusing pH gradient. The focusing gel was then positioned on top of the preelectrophoresed gel slab, at the end distal from the sealing gel, with focusing axis perpendicular to the direction of electrophoresis. Electrophoresis was for 16 hr at 8 mA per slab. The slabs were washed and stained as described (3). Other Determinations. Protein was assayed as described (1-3). pH gradients were determined by using a contact-pH electrode (Ingold ES 47300-02) at 2-mm intervals.
RESULTS Membrane Protein Solubilization. More than 80% of the protein of all membranes studied here was solubilized (nonsedimentable at -300,000 X g for 60 min) by 1% Triton X-100, pH 8.5. Crossed Immunoelectrophoresis. Membrane-bound 7S IgG2 is a marker for GD248 lymphocytes (6, 7). Because T19 cells are derived from GD248 tumors, we looked for 7S IgG2 in T19 membranes. No immune precipitate was observed, al-
,
8.D
I
7.0
U-----------1---U- - - - - r-----
60
5.0
4.5
pH FIG. 2. Bidimensional isoelectric focusing/immune electrophoresis of T19 membrane proteins. Isoelectric focusing: horizontal; 0.6 mg of protein. Immune electrophoresis: vertical into agarose containing anti-T19-cell serum (90 Mul/ml of agarose). Arrows indicate the
immune precipitates.
though hamster immunoglobulin from other sources gave a strong reaction. Bidimensional Isoelectric Focusing/Immune Electrophoresis. The precipitation pattern of GD248 membrane proteins with anti-GD248-membrane serum was as before (3), showing two "multirocket" (8, 9) immune precipitates at pH 4.5 and 4.7. These precipitates represent antigens absent from normal hamster lymphocytes, as tested by crossed-line immune electrophoresis (3, 4) and by extensive absorption of antiGD248-membrane serum with membranes of normal cells. When the focused membrane proteins of T19 cells were electrophoresed into anti-GD248-membrane serum, two immune precipitates appeared (Fig. 1) at pH 4.5 and pH 4.7, and each consisted of more than one rocket. A minor component, at pH 6.0, was not detected in SV3T3 membranes (see below). The multirocket pl 4.5 and 4.7 components were also detected when T19 membranes were run against rabbit anti-T19 serum (Fig. 2). No immunoglobulin was detected with anti hamsterimmunoglobulin in immune electrophoresis. Finally, as shown in Fig. 3, when focused membrane proteins from SV3T3 cells were run against anti-GD248-membrane serum, only the complex pl 4.5 and pl 4.7 immune precipitates appeared. The data with T19 and SV3T3 cells rule out the possibility that the pl 4.5 and pI 4.7 precipitates represent clonal or species markers. Bidimensional Isoelectric Focusing/DodSO4 Gel Electrophoresis. As shown in Figs. 4-6, on second-dimension DodSO4 slab gel electrophoresis, the pl 4.5 focusing components of GD248 lymphocytes, T19 cells, and SV3T3 fibroblasts resolved into two overlapping zones with molecular weights near 58,000. However, the proportion of pI 4.5 material depended on cell type and, on simple isoelectric focusing, the pl 4.5 component was least prominent with SV3T3 cells. However, even in this case, good resolution was obtained after the second-dimension electrophoresis run. The pl 4.7 components of GD248, T19, and SV3T3 cells separated into two spots, localizing between 90,000 and 110,000 daltons (arrows in Figs. 4-6) but yielded no staining between 50,000 and 90,000 daltons. Use of expanded pH gradients (pH 4-6) showed that the weak spot at -80,000 daltons in the case of SV3T3 membranes did not coincide with either the pI 4.5 or the pI 4.7 components. Isoelectric focusing/DodSO4 gel electrophoresis of membranes of normal hamster cells revealed no pI 4.5 component (3,5) and no components at pH 4.7 between 50,000 and 110,000 daltons. Because the origins are broader in bidimensional techniques than in unidimensional electrophoresis (5 mm here), seconddimension DodSO4 electrophoresis yielded less resolution than
Cell Biology: Schmidt-Ullrich et al. .., ..w-.:,w ...
Proc. Natl. Acad. Sci. USA 74 (1977)
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pH FIG. 3. Isoelectric focusing of solubilized BALB/c SV3T3 membrane proteins (Triton X-100) in the first dimension (horizontal) and immune electrophoresis in the second dimension (vertical) with anti-GD248-membrane serum (46 Al/ml of agarose). (A) Immune precipitation (top) and focusing pattern (bottom). Arrows point to the overlapping immune precipitates at pH 4.7 and pH 4.5. (B) Schematic of the immunoplate (A), including the pH gradient. One of the pI 4.7 rockets extended into the pH 4.5 range, possibly indicating partial crossreactivity of the two focusing components.
conventional DodSO4 electrophoresis, leading to somewhat less precise molecular weight estimates. However, the relative staining intensities of the -58,000 dalton and 90,000-110,000 dalton spots followed those of pl 4.5 and pl 4.7 components, respectively. Dimension 1 A
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DISCUSSION Plasma membranes and mitochondria of SV40-transformed hamster lymphocytes (GD248) contain antigenic protein classes that focus at pH 4.5 and 4.7 and are not detectable in normal lymphocyte populations or embryonic hamster cells (3). Our new data show that both components are SV40-specific. GD248 cells are B-type lymphocytes (6, 7) that synthesize 7S IgG2 and bear this immunoglobulin on their plasma membranes (2, 6). In contrast, T19 cells are phagocytes that lack IgG2. However, membranes from both cells reveal the pI 4.5 and pI 4.7 immune precipitates with both guinea pig anti-GD248membrane serum and rabbit anti-T19 serum. The pI 4.5 and pI 4.7 antigens therefore cannot represent lymphocyte markers. Untransformed or embryonic hamster cells (2, 3, 5) or BALB/c 3T3 cells (10) gave no immune reactions with our sera. The new components at pI 4.5 and pI 4.7 are present also in the membranes of SV3T3 fibroblasts and are the only SV3T3 membrane
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FIG. 4. Bidimensional isoelectric focusing/DodSO4 gel electrophoresis of GD248 membrane proteins. First dimension: isoelectric focusing, horizontal. Second dimension: slab gel electrophoresis, vertical. The abscissas give the pH gradient and a scheme of the focusing pattern. The ordinates give scales of relative electrophoretic mobilities (RM) and molecular weights (MW), as well as a schematic of the protein distribution obtained upon unidimensional DodSO4 gel electrophoresis. The numbering of the protein bands in the first and second dimensions are as in refs. 5 and 2, respectively.
8.S
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FIG. 6. Bidimensional isoelectric focusing/DodSO4 slab gel electrophoresis of BALB/c SV3T3-membrane proteins. Details as in Fig. 4. Expanded pH gradients (pH 4.0-6.0) showed that no proteins between 90,000 and 40,000 daltons focused at pH 4.7.
Proc. Natl. Acad. Sci. USA 74 (1977)
5072 Cell Biology: Schmidt-Ullrich et al. entities that react with anti-GD248-membrane serum. The only feature common to GD248, T19, and BALB/c SV3T3 is SV40 transformation. The pI 4.5 and pI 4.7 proteins therefore are SV40-specific. Indirect immune fluorescence, with either the anti-T19 or the anti-GD248-membrane serum, yields strong S antigen and U antigen reactions but no T antigen staining with GD248, T19, SV3T3, and W18 VA2 cells or GD248 nuclei (10). U antigen, defined as associated with the nuclear periphery (11), has recently been shown to have a molecular weight of about 94,000 in transformed cells (12). The pI 4.7 components of GD248, T19, and SV3T3 cells have molecular weights of 90,000110,000. Because our sera react with U but not T antigen, the pI 4.7 antigen is reasonably identified with the SV40 U antigen. Whether this antigen is truly a plasma membrane component or is present due to trace contamination with nuclear envelope during fractionation remains to be determined. The pI 4.5 component, a glycoprotein (5), has a molecular weight near 58,000. Recent data indicate molecular weights of 50,000-60,000 (13) and 58,000 (14) for the transplantation antigen extracted with Triton X-100 from SV40-transformed fibroblasts. Further studies (15) also show a close relation between the transplantation and surface antigens. We do not have information on transplantation antigenicity, but our reagent sera give prominent surface antigen immune fluorescence. The pI 4.5 component can thus be identified with SV40 surface antigen and, by inference, SV40 transplantation antigen. This work was supported by Grant CB-44000 from the National Cancer Institute. 1. Schmidt-Ullrich, R., Wallach, D. F. H. & Davis, F. D. G., III (1976) "Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. 1. High yield purification of plasma membrane fragments," J. Nati. Cancer Inst. 57, 1107-1116. 2. Schmidt-Ullrich, R., Wallach, D. F. H. & Davis, F. D. G., III (1976) "Membranes of normal hamster lymphocytes and lymphoid cells neoplastically transformed by simian virus 40. II. Plasma membrane proteins analyzed by dodecyl sulfate polyacrylamide electrophoresis and two dimensional immune electrophoresis," J. NatI. Cancer Inst. 57, 1117-1126. 3. Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977)
4.
5.
6. 7.
8. 9.
10.
11.
12. 13.
14.
15.
"Antigenic distinctions of glycoproteins in plasma and mitochondrial membranes of lymphoid cells neoplastically transformed by simian virus 40," Proc. Natl. Acad. Sc. USA 74, 643-647. Bjerrum, 0. J. & Bog-Hansen, T. C. (1976) "Immunochemical gel precipitation techniques for analysis of membrane proteins," in Biochemical Analyses of Membranes, ed. Maddy, A. H. (Chapman and Hall, London), pp. 378-426. Schmidt-Ullrich, R., Verma, S. P. & Wallach, D. F. H. (1975) "Anomalous side chain amidation in plasma membrane proteins of simian virus 40-transformed lymphocytes indicated by isoelectric focusing and laser Raman spectroscopy," Biochem. Blophys. Res. Commun. 67,1062-1069. Coe, J. E. & Green, I. (1975) "B-cell origin of hamster lymphoid tumors induced by simian virus 40," J. Natl. Cancer Inst. 54, 269-270. Coe, J. E. (1976) "Immunoglobulin synthesis by an SV40-induced hamster lymphoma," Immunology 31, 495-502. Laurell, C.-B. (1966) "Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies," Anal. Biochem. 15, 45-52. Johansson, K.-E. & Hjerten, S. (1974) "Localization of the Tween 20-soluble membrane proteins of Acholeplasma laidlawii by crossed immune electrophoresis," J. Mol. Biol. 86, 341-348. Lin, P. S., Schmidt-Ullrich, R. & Wallach, D. F. H. (1977) "Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope," Proc. Natl. Acad. Sci. USA 74,2495-2499. Lewis, A. N., Jr. & Rowe, W. P. (1971) "Studies on non-defective adenovirus simian hybrid viruses. 1. A newly characterized simian virus 40 antigen induced by the Ad2+ND1 virus," J. Virol. 7, 189-197. Robb, J. S. (1977) "Identification of simian virus 40 tumor and U antigens," Proc. Natl. Acad. Sci. USA 74,447-451. Luborsky, S. W., Chang, C., Pancake, S. J. & Mora, P. T. (1976) "Detergent solubilization and molecular weight estimation of tumor specific surface antigen from SV40 transformed cells," Biochem. Biophys. Res. Commun. 71,990-996. Anderson, J. L., Martin, R. G., Chang, C., Mora, P. T. & Livingston, D. M. (1977) "Nuclear preparations of SV40-transformed cells contain tumor specific transplantation antigen activity," Virology 76,420-425. Chang, C., Pancake, S. J., Luborsky, S. W. & Mora, P. T. (1977) "Detergent solubilization and partial purification of tumor specific surface and transplantation antigens from SV40 virus transformed cells," Int. J. Cancer 19, 258-266.
