Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5270-5274, December 1977

Biochemistry

Moloney leukemia virus-induced cell surface antigen: Detection and characterization in sodium dodecyl sulfate gels (tumor antigens/oncovirus proteins/type C viral antigens/histocompatibility antigens/cytotoxicity)

FREDERIC A. TROY*, EVA MARIA FENYG, AND GEORGE KLEIN Department of Tumor Biology, Karolinska Institute, S-104 01 Stockholm, Sweden

Contributed by George Klein, August 26, 1977

ABSTRACT An experimental procedure for detecting and characterizing tumor-associated, virion, and histocompatibility antigens has been developed. The method takes advantage of the high resolution that proteins, solubilized by Triton X-100 and reduced, display after sodium dodecyl sulfate gel electrophoresis. The antigens can be detected as distinct molecular weight species by a highly sensitive inhibition of cytotoxic reaction. When coupled to the lactoperoxidase-catalyzed iodination of intact cells, the procedure permits the determination of externally exposed antigens. In the present study, the method has been applied to the Moloney leukemia virus-induced YAC lymphoma cells of strain A mice, which express a Moloney leukemia virus-determined cell surface antigen (MCSA) in addition to the type C viral proteins gp7l, p30, p15, p15(E), p12, and plO. MCSA was identified as an exposed surface protein distinct in size and antigenic determinants from the major envelope and core protein of Moloney leukemia virus and the histocompatibility antigens. Multiple molecular weight species possessing antigenic determinants for MCSA, gp7l, and H-2a have been detected. These results provide direct confirmation that MCSA is unrelated to the known virion structural proteins or to the H-2a antigen. This method should permit the direct identification and molecular weight characterization of any antigen whose determinants are not solely dependent on a complex quaternary structure and for which serological reagents are available. Oncovirus-infected cells are known to express a variety of membrane-associated viral antigens (1-4). The occurrence of additional virally induced antigens that differ from known virion envelope or core proteins is more controversial (5, 6). A substantial amount of indirect evidence supports the concept that the antigens are unrelated, yet no direct evidence has been presented. An accurate assessment of the chemical nature of these antigens has been handicapped by the limits of current methodology, which has not permitted a detailed study of the different antigenic activities as distinct molecular species. Based on the observation that reduced proteins in a sodium dodecyl sulfate (NaDodSO4) complex contain a high degree of order, studies were initiated to explore the possibility that both virion and virus-specified cell surface antigens could be studied as distinct molecular species after their high-resolution separation in polyacrylamide gels and subsequent detection by a sensitive inhibition of cytotoxic reaction. Accomplishment of this objective has provided an experimental approach to resolve ambiguities concerning the structural relationship between membrane antigens whose synthesis is directed by Moloney leukemia virus and known Moloney leukemia virion proteins. Working with Moloney virus-induced lymphoma cells, we have previously detected a virus-determined (7) nonviriont

antigen, designated MCSA, with sera of syngeneic mice immunized with heavily irradiated lymphoma cells. Serial in vivo passage of lymphoma cells through preimmunized mice led to the selection of sublines with reduced MCSA expression. Such sublines were no longer sensitive to the cytotoxic effect of anti-MCSA sera, but their sensitivity to goat or rabbit antisera against the virion proteins gp71, p30, p15, p15(E), and p12 remained unchanged (8), suggesting that MSCA was distinct from known virion proteins. Mice produce separate antibodies against MCSA and gp71 envelope glycoproteins. The reaction against MCSA cannot be inhibited by virus or by virion gp71.t MCSA also differs from H-2 and virion proteins by its relative refractoriness to antibody-induced redistribution and apparent molecular weight (Mr) (9, 10). The preferential recognition of MCSA by the syngeneic host suggested that MCSA may be an important rejection target. This was in line with the close parallelism between in vivo rejectability and in vitro MCSA expression in a collection of Moloney lymphomas (11). This report describes the high-resolution separation of virion proteins and of MCSA in polyacrylamide gels following Triton X-100 solubilization. Antigenic activity was monitored by cytotoxic inhibition and could be related to distinct molecular species. Surface labeling by lactoperoxidase iodination allowed the assignment of exposed membrane antigens. EXPERIMENTAL PROCEDURE Materials. All chemicals were analytical reagent grade commercial preparations. Sodium [125]iodide was purchased from the Radiochemical Centre, Amersham, and used within 1 week of activity date. All reagents for NaDodSO4/polyacrylamide gel electrophoresis were purchased from Bio-Rad Corp. High molecular weight protein markers (Mr 53,000265,000) were obtained from BDH Chemicals, Ltd. Phenylmethylsulfonyl fluoride was purchased from Pierce Chemical Co. and lactoperoxidase from Worthington Biochemical Corp. Cells. Moloney leukemia virus-induced YAC lymphoma cells of strain A mouse origin were propagated in the ascites form in syngeneic hosts (110-115 passages). Cells were harvested following growth for 7 days and washed three times in RPMI 1640 medium without serum. Cell viability, as determined by trypan blue exclusion, was 86-92%. Radiolabeling of Cells. For radiolabeling, 1 to 2 X 109 cells Abbreviations: MCSA, Moloney leukemia virus-induced cell surface antigen; H-2, histocompatibility antigen; NaDodSO4, sodium dodecyl sulfate; BSS, Hanks' balanced salt solution; Mr, molecular weight; BPB, bromphenol blue. * Present address: Department of Biological Chemistry, University of California School of Medicine, Davis, CA 95616. t E. M. Feny6, E. Yefenof, E. Klein, and G. Klein, unpublished data.

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. 5270

Biochemistry: Troy et al. were resuspended in 4 ml of serum-free RPMI 1640 and iodinated by the procedure of Gates et al. (12), using 1-2 mCi of sodium [125I]iodide. Radiolabeling was terminated after 10 min by the addition of 40 ml of serum-free RPMI 1640 containing 50 mM unlabeled sodium iodide. Labeled cells were sedimented by centrifugation and washed eight times by resuspension in 40 ml of RPMI 1640/sodium iodide buffer and centrifugation. Isolation of Radiolabeled Cell Antigens by Triton X-100 Extraction. Jodinated cells were resuspended in a total volume of 6 ml of serum-free RPM1 1640 containing 4 mM Triton X100, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and sodium azide at 0.02%. Cells were incubated at 37/for 2 hr and centrifuged at 48,000 X g for 60 min at 4°. The pellet fraction was resuspended with brief sonication in 2 ml of Hanks' balanced salt solution (BSS) containing 1 mM 2mercaptoethanol and examined directly in NaDodSO4 gel electrophoresis as described below. The Triton X-100-solubilized proteins were also examined directly in NaDodSO4 gels. NaDodSO4/Polyacrylamide Gel Electrophoresis. All gel electrophoreses were carried out in 0.1% NaDodSO4 according to a modification of the procedures of Laemmli (13). A Hofer Model SE 520 slab gel electrophoresis unit was used and run at 50. Gels were dried on a Hofer model SE 540 dryer. The Triton X-100-solubilized and reduced proteins were prepared for electrophoresis by adding the sample directly to a 2% solution of NaDodSO4 containing 5% (vol/vol) 2-mercaptoethanol, 10% glycine, and 62.5 mM Tris.HCI buffer, pH 6.8. Samples were heated at 85-900 for 10 min and electrophoresed on 30-cmlong, 15-cm-wide, 1.5-mm-thick polyacrylamide gels. Removal of NaDodSO4 and Extraction of Antigens from Polyacrylamide Gels. After electrophoresis, a single slab gel was processed in the following way in order to remove most of the NaDodSO4 prior to assay for antigenic activity, protein staining, and radioactivity measurements: the entire gel was soaked in 1.5 liters of BSS for 1-2 hr at 250 with intermittent agitation. BSS was decanted and the gel was soaked 8-10 hr at 40 in the presence of an additional 1 liter of BSS. During this procedure, most of the bromphenol blue tracking dye was removed. That no 125I was detectable in the washings showed that little, if any, of the 125I-labeled protein had been extracted. Following removal of NaDodSO4 from the gels, lanes containing Mr markers and sample were cut vertically and stained for proteins with Coomassie brilliant blue R-250. An adjacent well containing sample was then processed for antigen by laying the gel on a piece of pre-marked Whatman 3MM chromatography paper to serve as a template for slicing the gel. Threemillimeter-wide horizontal sections of the gel were then cut the length of the gel. Gel slices were added to 1-ml Eppendorf plastic test tubes and pulverized with a spatula in the presence of 0.1-0.15 ml of BSS. The tubes were incubated at 40 for at least 24 hr before aliquots were removed for assay of the various antigenic specificities. The iodination profile was determined by directly measuring the radioactivity in the tubes containing the gel slides. Analytical Methods. Protein and radioactivity measurements were carried out as previously described (14). Sera. Mouse anti-Moloney lymphoma (anti-ML) sera were produced in semisyngeneic A X C57Bl F1 by three to six injections of 6000-R-irradiated YAC lymphoma cells or in C57 leaden F1 mice by the corresponding inoculation of syngeneic YLD lymphoma cells (11). Anti-H-2a serum was produced by immunizing ASW mice with normal strain A cells as described previously (9). The goat antiserum to gp7l of Friend leukemia

Proc. Natl. Acad. Sci. USA 74 (1977)

5271

Intact YAC lymphoma cells L. P. Na 1 251

1251-Labeled exposed surface proteins 1. 4.0 mM Triton X-100 1 mM 2-Mercaptoethanof 3. 1 mM PhCH2S02F 48,000 X g, 1 hr

37, 2

Triton X-100 insoluble pellet

Triton X-100 soluble proteins ' 1. NaDodSO4

|| 1. NaDodSO4

2. 2-Mercaptoethanol

2. 2-Mercaptoethanol

NaDodSO4/pol yacrylamide gel

NaDodSO4/polyacrylamide gel BSSl

1. Antigenic assignment, based on inhibition of

cytotoxic reaction 2. Radioactivity determination

._

BSS

Na Dod SO 4

-1

=

FIG. 1. Diagram illustrating the experimental procedure devel-

oped for surface labeling, Triton X-100 solubilization, NaDodSO4 gel electrophoresis, and antigenic characterization of MCSA, Moloney leukemia virus, and H-2a proteins from YAC lymphoma cells. The details are described under Experimental Procedure. L.P., lactoperoxidase; PhCH2SO2F, phenylmethylsulfonyl fluoride; CBB, Coomassie brilliant blue; BPB, bromphenol blue.

virus was kindly provided by W. Schifer and the goat antiserum to p30 of Rauscher leukemia virus by B. Hampar. Serological Procedures: Inhibition of Cytotoxicity. The previously described (9) micro-assay was used. Antigenic material was suspended in antiserum dilutions that gave 60-90% killing. Six to ten 3- to 5-fold antigen dilutions were tested in at least two repeat experiments. Antigenic units were expressed as the reciprocal of the dilution corresponding to 20% inhibition.

RESULTS Fig. 1 is a schematic representation of the surface labeling procedure, followed by Triton X-100 solubilization, NaDodSO4 gel electrophoresis, and antigenic characterization. A material balance and quantitative determination of the solubilized components released from labeled YAC cells after treatment with Triton X-100 is summarized in Table 1. These data show that Triton X-100 solubilizes 65% of the total cellular protein, accounting for 60% of the 125I and 27% of the total MCSA antigenic activity. The remainder of the protein and radioactivity and 56% of the remaining antigenic activity are accounted for in the pellet fraction. That solubilization results in the enrichment of higher Mr proteins can be seen in Fig. 2, which compares the protein profile of the Triton X-100-soluble and insoluble fractions with the whole cell protein pattern on NaDodSO4/12% polyacrylamide gels. This is confirmed in the radioactive profile of the detergent-solubilized proteins (see below). The most notable feature of the solubilized proteins is

5272

Biochemistry: Troy et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Distribution of protein and MCSA activity after treatment of 125I-labeled YAC lymphoma cells with 4 mM Triton X-100 Total Specific MCSA radioTotal activity, activity, activity, protein, dpm/mg total units Fraction* protein X 10-6 dpm X 10-3 mg Intact cellst 4.32 115 37.5 9.72 Triton-solubilized proteins$ 2.55 74.3 34.3 2.61 Triton-insoluble

proteins 1.90 Counting efficiency was 75%. * As described in Fig. 1.

47.1

40.3

3.94

t After resuspension

in RPMI 1640 containing 4.0 mM Triton X-100, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 0.02% sodium azide and incubation at 370 for 2 hr as described under Experimental Procedure. Proteins solubilized in 4 mM Triton X-100 and isolated after centrifugation at 48,000 X g for 1 hr as described in the text.

the selective reduction in the proteins of low Mr. A more detailed analysis of the iodinated, Triton X-100-solubilized proteins is shown in Fig. 3. These data show that the majority of the proteins labeled with 125I comigrate with the proteins revealed by Coomassie blue staining. When the same gel fractions in which the radioactivity was determined were assayed for MCSA, virion gp71, p3O, and H-2a activity, the antigenic profile shown on the top was generated. This profile shows clearly that Mr x~

Mr x 0

3

2

4 5

106- -o

159-

55t-

3

5

;

25 7

177-f II

3-

BPB-

FIG. 2. NaDodSO4/12% polyacrylamide gel electrophoresis of reduced, whole cell proteins from YAC cells (lane 2) with 4 mM Triton X-100-soluble (lane 5) and insoluble (lane 4) proteins. The weight ratio of NaDodSO4 to protein in lane 2 was 20:1, in lane 4,4:1, and in lane 5, 1.8:1. The amount of protein analyzed in lanes 2,4, and 5 was 35, 73, and 56 gg, respectively. The mobilities of the proteins in lane 2 are relative to Mr standards visualized in lane 1, while the standards in lane 3 relate to the protein in lanes 4 and 5.

MCSA gp71 4401 1100' 1 ~~~~~in290i

200-

ram

p30

100-

--I

MCSA

10

18C-

20

30 40 50 Gel slice nlUrmiber I*& _

60-

60

70

~

A1

4O-t= 250 180 120 90 70 50 210 150 100 80 60 40 Molecular weight 03

30 25

FIG. 3. NaDodSOS48% polyacrylamide gel electrophoresis of 4 mM Triton X-100-solubilized and reduced proteins from l25I-labeled YAC lymphoma cells. Triton X-100-solubilized protein (141 ,g) was run on the gel. The reduced protein-NaDodSO4 complexes were prepared with a weight ratio of NaDodSO4 to protein of 1.8:1. The MCSA .(-), gp71 (- - -), p30 (-...), and H-2a (----) profile was determined on the same sample run simultaneously in an adjacent lane. NaDodSO4 was removed and the gel was sliced. Proteins were extracted from the individual slices and assayed for antigenic activity by the inhibition of cytotoxicity method. The height (length) of the lines corresponds to the relative antigenic units, which is the highest dilution showing 20% inhibition. Antigenic activity too high to plot is indicated by a number (the antigen units) after the arrow.

the major MCSA activity is associated with an exposed surface protein of an apparent Mr of approximately 92,000. Strong MCSA activity is also detected in a lower molecular weight region corresponding to an apparent Mr of 55,000-58,000. The 92,000 Mr value confirms precisely the apparent Mr determined earlier in a 12% polyacrylamide gel (results not shown). In the 12% gel, the lower molecular weight MCSA activity had an Mr of 52,000. The external exposure of the lower molecular weight MCSA antigen is less certain. The weight ratio of NaDodSO4 to protein for the experiment described in Fig. 3 was 1.8:1. For the 12% gel described, it was 6.8:1. When the Triton X-100-solubilized proteins run in Fig. 3 were treated with NaDodSO4 (weight ratio of NaDodSO4 to protein; 0.9:1) and analyzed in a preparative (3-mm-thick) 8% polyacrylamide gel, MCSA activity was also detected in a region corresponding to an apparent Mr of 180,000-192,000 (Fig. 4). This large Mr species is also an external protein but is not observed when low ratios of NaDodSO4 to protein are employed. It is therefore possible that variation in sample preparations may give rise to variation in the apparent Mr of MCSA species detected. This may relate to incomplete reduction of disulfide bonds and/or reduced detergent concentrations. Nevertheless, it is clear from these results that MCSA is an exposed surface protein distinct in size and antigenic determinants from the known major envelope and core proteins of Moloney leukemia virus and the H-2a antigens. Analysis of each gel fraction in Fig. 3 for gp7l activity showed the major antigenic determinant to be associated with a protein of apparent Mr 71,000. gp7l antigenic reactivity was also observed to be associated with the visible polypeptide chains in the Mr range 72,000-80,000. The polypeptide of Mr 80,000 show strong reactivity with anti-gp7l, results that are in support of the structural relatedness of p80 to viral gp7l. These results are confirmed in the more complete and quan-

Biochemistry: Troy et al.

Proc. Natl. Acad. Sci. USA 74 (1977)

20r MCSA (365)

gp7l

4

Issd .,5.

Table 2. Summary of the Mr values and external surface exposure of MCSA, Moloney leukemia virus, and H-2 antigens on YAC lymphoma cells

ITI

15-

iT.i,i iii

4J1

c

Antigen MCSA

111 *,i

0

gp7l

-Pco10

U0

H-2

cc5 I

H-2a

MCSA

MCSA

ii

H-2 p30 I

I'

-250 190

5273

150 90 80 70 50 i3 Molecular weight X10

35

m

IL

25

FIG. 4. High-resolution antigenic profile of MCSA, gp7l, p30, and H-26 obtained after preparative NaDodSO4/8% polyacrylamide gel electrophoresis of 4 mM Triton X-100-solubilized and reduced proteins from 1251-labeled YAC lymphoma cells. The reduced protein-NaDodSO4 complexes were prepared with a weight ratio of NaDodSO4 to protein of 0.9:1 and heated at 850 for 7.5 min. Samples containing 906 ,g of solubilized protein (same as shown in Fig. 3) were run in each of eight wells in a 3-mm-thick slab gel. Assay for antigenic activities by the inhibition of cytotoxicity method was carried out.

titative antigenic profile seen in Fig. 4. It is relevant to note that strong gp7l crossreactivity had been detected earlier in a gel region corresponding to an apparent Mr of 43,000 (10). This activity has more recently been confirmed in both YAC and an immunoresistant sub-line.t The protein exhibiting an apparent Mr of 37,500 in Fig. 3 possesses all of the p30 antigenic activity. In Fig. 4, it is observed that this same activity migrates with a mobility corresponding to a molecular weight of 28,000. No other Mr species possessing p30 activity have been detected. In contrast, H-2a activity is associated with the 52,0 4,000 Mr protein in the experiment described in Fig. 3 but with the Mr species of 38,000 and 31,000 in Fig. 4. In spite of the variability of the Mr values, it appears that in YAC cells at least two and maybe three H-2a species exist with tentative Mr assignments of 31,000, 38,000, and 52,000. Table 2 summarizes the apparent Mr values and the surface exposure of MCSA, gp7l, p30, and H-2a. DISCUSSION An experimental procedure involving NaDodSO4/polyacrylamide gel electrophoresis of Triton X-100-solubilized cell surface components has been described; the procedure can detect a potentially large number of surface antigens as separate molecular entities. The capacity to recover the proteins from the gels and test them in specific cytotoxicity assays has permitted the assignment of specific antigenic determinants to distinct Mr species. In addition, once the antigenic assignment has been made, the high resolving power of the gels permits the isolation of highly enriched, if not pure, antigens for the subsequent production of monospecific antibodies. Bolognesi et al. (15) purified the major core protein of avian tumor viruses through gel electrophoresis and, subsequently, used immunodiffusion for antigenic characterization. Recently, Burridge (16) applied 125I-labeled antibodies directly on the gels to localize cellular glycoproteins. Several aspects of the methodology described in this paper t F. A. Troy, E. M. Feny6 and G. Klein, unpublished data.

p30

Mr* X 10-3 180-192t 92 (86-97) 52 (48-58) 70-80 43 (40-48) 52 (40-54) 38 (33-40) 311 28-37

External surface exposure Todination Cytotoxicityt + +

+

? +

+

? +

?

+§

+ +

+

* The apparent NaDodSO4 Mr values summarized represent averages determined from gel electrophoretic analysis carried out on both 8% and 12% polyacrylamide gels. As described, the reduced protein-NaDodSO4 complexes were prepared with variable weight ratios of NaDodSO4 to protein. The range of all Mr values determined is in parentheses. t These data are based on the cytotoxic sensitivity of YAC to antisera against the antigens indicated. This assay does not discriminate between the Mr values of the antigens. Data taken from ref. 8. Observed only in preparative gel where weight ratio of NaDodSO4 to protein was 0.9:1. § Assignment based on fact that antibody to H-2 is lytic for YAC cells in the presence of complement (11). Tentative assignment because this activity was detected in the preparative gel described above. are noteworthy. First, the 4 mM final concentration of Triton X-100 was selected because it is above the critical micelle

concentration of this surfactant (17), a consideration important in the mechanism of solubilizing membrane-bound proteins (18), but is one-fourth the 16 mM (1%) concentration usually employed for solubilizing many antigens. This concentration solubilizes substantial amounts of total cellular protein, including MCSA activity (Table 1), and a large excess of detergent is not present. This eliminates the need of additional procedures for removal of Triton X-100. Second, this concentration of Triton X-100 permits the solubilized proteins to be run directly in NaDodSO4 gel electrophoresis and still obtain high-resolution separation of the individual polypeptide chains. Under these experimental conditions, antigenic determinants are not destroyed. Third, the NaDodSO4 is removed from the gels under conditions that do not extract the individual polypeptide chains. Fourth, direct correlation between the amount of 125I associated with each surface-exposed protein and its antigenic properties is made possible by measuring the radioactivity in the individual gel slices prior to analysis for antigenic specificities by cytotoxic inhibition. The major conclusion derived from these results is that none of the MCSA activities is associated with either the known MLV envelope or core proteins or the histocompatibility antigen H-2a. MCSA activity is associated with proteins possessing three distinct apparent Mr values; 52,000, 92,000, and 180,000-192,000. The fact that both of the two lower Mr species were detected following reduction in the presence of a very high ratio of NaDodSO4 to protein (6.8:1) renders unlikely the possibility that at least the 52,000 Mr antigen is a subunit derived from the 92,000 species. The 180,000-192,000 Mr MCSA§ was only de§ Because higher Mr proteins were excluded by molecular sieve

chromatography during purification of the MCSA species analyzed at a NaDodSO4-to-protein ratio of 6.8:1, it is not known if the sample initially contained the 180,000-192,000 Mr MCSA protein.

5274

Biochemistry: Troy et at.

Proc. Natl. Acad. Sci. USA 74 (1977)

Table 3. Possible relationship between lower Mr MCSA species and higher Mr antigen

Apparent Mr of MCSA, x i03 *

Relationship of lower Mr antigens to highest 180,000-

Mr species

Both directly related

>

No relationship

52,000

92,000

192,000

I>

2==2 sow

( )

Only lowest Mr antigen related Only intermediate Mr antigen related

EI C;_

2

(°K)4

* The tetramer, dimer, monomer representation is only a tentative aninment pending detailed analysis of the minimum and maximum Mr species possessing MCSA activity.

tected in samples prepared with a lower weight ratio of NaDodSO4 to protein (0.9:1). Several possibilities whereby the three MCSA antigenic species may be related are shown in Table 3. First, MCSA may exist as a membranous complex with a tetrameric Mr of about 180,000-192,000 consisting of four subunits of Mr 52,000.1 The subunits may be held together by disulfide bonds or by noncovalent interactions. Complete reduction and/or subunit disaggregation would give rise to the 52,000 Mr antigen, while incomplete reduction or partial aggregation might yield antigenic species of all three Mrs. In contrast, the three MCSA species may have no subunit interrelationship but rather represent three distinct Mr entities possessing common antigenic determinants. Two intermediate possibilities exist. The 180,000-192,000 Mr species may be related by subunits to only the 52,000 Mr antigen and the 92,000 Mr protein may have no subunit relatedness. Alternatively, the 92,000 Mr protein may be derived from the largest Mr species, while the 52,000 Mr antigen is unrelated by subunits to either of the other two species. The possibility that the larger Mr species may represent MCSA crosslinked through disulfide bonds to different molecular species within the membrane must also be considered. A full and precise description of the possible interrelation between the multiple MCSA activities must await further studies. The variable NaDodSO4 Mr values observed for H-2a and p30 may relate to the pre-exposure of these proteins to Triton IIt is recognized that the 180,000-192,000 Mr species may represent a minimum Mr for the native multimeric complex assembled in the membrane because it was detected in NaDodSO4 gels after reduction with 2-mercaptoethanol. No MCSA activity was detected in gels in which the sample was not reduced prior to electrophoresis.

X-100. Nothing is known, for example, about the relative affinities of these or other membrane proteins for NaDodSO4 in the presence of Triton X-100, although gp7l activity is located precisely in the expected region. It is reasonable to consider that binding of Triton X-100 to some proteins may influence the subsequent binding of NaDMdSO4 and therefore alter their mobilities. The possible role bf Triton X-100 in protecting antigenic determinants during reduction and partial denaturation in NaDodSO4 also cannot be properly evaluated at this time but may be central to the success of this procedure. The excellent technical assistance of Irma Jansson and Frida Kierszenblat is gratefully acknowledged. Special appreciation is extended to R. R. Traut for helpful suggestions in the preparation of this manuscript. This investigption was supported by National Institutes of Health Grant CA 17327 from the National Cancer Institute and by Contract N01-CP-3316 within the Virus Cancer Program of the National Cancer Institute, Grants were also received from the Swedish Cancer Society. F.A.T. is the recipient of U.S. Public Health Service Career Developmest Award CA 00095. The work reported in this paper was undertaken by F.A.T. during the tenure of an American Cancer Society-eleanor Roosevelt International Cancer Fellowship awarded by the International Union Against Cancer. E.M.F. is a recipient of the 1fdna Old Fellowship in cancer immunology awarded by the Cancer Aesearch Institute, New York. 1. Stephenson, J. R., Essex, M., Hino, S., Hardy, W. D. & Aaronson, S. A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74,1219-1223. 2. Kurth, R. & Bauer, H. (1972) Virology 47,426-433. 3. Law, L. W. & Appella, E. (1973) Nature 243, 83-87. 4. Cohen, M. H., Sibal, L. R. & Fink, M. A. (1974) Immunology 28, 37-48. 5. Friedman, M., Lilly, F. & Nathenson, S. G. (1974) J. Virol. 14, 1126-1131. 6. Hogg, N. M. (1976) Int. J. Cancer 18,439-447. 7. Fenyb, E. M. & Grundner, G. (1973) Int. J. Cancer 12, 452462. 8. Feny6, E. M.-& Klein, G. (1976) Nature 260,355-57. 9. Siegert, W., Feny6, E. M. & Klein, G. (1977) Int. J. Cancer, in press. 10. Feny6, E. M., Troy, F. A., Siegert, W. & Klein, E. (1977) in VHIIth International Symposium on Comparative Research in Leukemia, August 22-26,1977. Amsterdam, The Netherlands. 11. Klein, G., Klein, E. & Haughton, G. (1966) J. Nati. Cancer Inst.

36,607-621. 12. Gates, R. E., Phillips, D. R. & Morrison, M. (1974) Biochem. J.

147,373-376.

13. Laemmli, U. K. (1970) Nature 227,680-685. 14. Troy, F. A., Frerman, F. E. & Heath, E. C. (1971) J. Blol. Chem.

246,118-133.

15. Bolognesi, D. P., Gelderblom, H., Bauer, H., Mblling, K. & Hiiper, G. (1972) Virology, 47, 567-587. 16. Burridge, K. (1976) Proc. Natl. Acad. Sci. USA 73, 44574461. 17. Robinson, N. C. & Tanford, C. (1975) Biochemistry 14, 369378. 18. Egan, R. W., Jones, M. A. & Lehninger, A. L. (1976) J. Biol. Chem. 251, 44424447.