Diphtheria Toxin-Binding Glycoproteins on Hamster Cells:

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of adenosine 5'-diphosphate-ribose from oxi- dized nicotinamide adenine dinucleotide to elon- ... resis (SDS-PAGE) in the absence of reducing agents.
Vol. 25, No. 3

INFECTION AND IMMUNITY, Sept. 1979, p. 786-791

0019-9567/79/09-0786/06$02.00/0

Diphtheria Toxin-Binding Glycoproteins on Hamster Cells: Candidates for Diphtheria Toxin Receptors RICHARD L. PROIA, LEON EIDELS, AND DAVID A. HART* Department of Microbiology, University of Texas Health Science Center at Dallas, Dallas, Texas 75235 Received for publication 5 June 1979

Diphtheria toxin-binding glycoproteins of high molecular weight (>100,000) identified on the surface of lymph node and thymus cells from hamsters, a diphtheria toxin-sensi Jve species. These diphtheria toxin-binding glycoproteins also interacted with CRM197 protein, which possesses toxin-blocking activity, but not with diphtheria toxoid, fragment A of diphtheria toxin, or cholera toxin, all of which lack toxin-blocking activity. These observations are consistent with the hypothesis that the detected diphtheria toxin-binding glycoproteins are involved in intoxication of cells by this toxin and possibly serve as the plasma membrane receptors for diphtheria toxin.

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Diphtheria toxin (DT) is produced as a single 63,000-dalton polypeptide chain by Corynebacterium diphtheriae lysogenic for phage P3. Upon limited proteolysis, DT can be cleaved to yield two fragments, A and B, which remain associated by a disulfide bond (2, 14). Fragment A (21,000 daltons) is able to catalyze the transfer of adenosine 5'-diphosphate-ribose from oxidized nicotinamide adenine dinucleotide to elongation factor 2, an enzyme involved in eucaryotic protein synthesis (2). Since elongation factor 2 is a cytoplasmic enzyme, fragment A must penetrate the cell membrane for toxicity to be expressed. The 40,000-dalton B-fragment is essential for the initiation of the entry process by functioning as the receptor binding subunit of the toxin (11, 20). Information concerning the biochemical nature of the DT receptor is limited. Draper et al. (5) have reported evidence which suggests that the DT receptor may contain an oligosaccharide component. Recently, we have identified a highmolecular-weight DT-binding glycoprotein species from the surface of guinea pig lymph node cells and suggested it as a candidate for the DT receptor (16). If these molecules are indeed involved in DT intoxication, then they should be detectable on cells of other sensitive species. In this communication, we report the presence of DT-binding glycoproteins on the surfaces of hamster cells; these glycoproteins are biochemically similar to the molecules detected on guinea pig cells. In addition, these glycoproteins could be detected by affinity chromatography utilizing DT-Sepharose as well as the previously utilized immunoprecipitation system (16).

MATERLALS AND

METHODS Toxins, related proteins, and antisera. Purified cholera enterotoxin (choleragen) was the generous gift of Richard Finkelstein, Department of Microbiology, University of Texas Health Science Center at Dallas. The rabbit anticholera toxin was that described previously (7). Partially purified DT was purchased from Connaught Laboratories, Ontario, Canada (lot 343) and was further purified by chromatography on DE52 cellulose and ammonium sulfate fractionation according to published methods (15). The purified DT yielded a single protein band at 63,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of reducing agents. In the presence of 2-mercaptoethanol, SDS-PAGE revealed that less than 10% of the molecules had undergone limited proteolysis. Fragment A of DT and the enzymatically inactive CRM197 protein produced by C. diphtheriae lysogenic for C7 (f197)to- c"+ were generous gifts of R. K. Holmes, Department of Microbiology, Uniformed Services University of the Health Sciences. Crude diphtheria toxoid was a gift of P. Forsyth, Texas State Health Laboratories, Austin, and was further purified by ammonium sulfate fractionation and ion-exchange chromatography on DE-52 cellulose; the purified toxoid yielded a single protein band on analysis by SDS-PAGE. Antiserum to DT was produced in New Zealand white rabbits by hyperimmunization with purified toxoid. The resulting antiserum reacted with DT, fragment A, CRM197 protein, and diphtheria toxoid, as determined by Ouchterlony analysis. Preliminary experiments with this antiserum in a radioimmunoassay indicated that 20 ±l of anti-serum would completely bind 20 tg of '251-labeled DT or 20 jig of '25I-labeled diphtheria toxoid. Iodination of cells. Lymph node and thymus cells were obtained from normal female MHA hamsters (Charles River Breeding Laboratories). The cells were iodinated by the lactoperoxidase method as described 786

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and D. A. Hart, Mol. Immunol., in press). Since hamsters are sensitive to the effects of DT (1, 12), cells from this species should presumably possess membrane receptors for DT. Preliminary experiments demonstrated that subcutaneous injection of less than 1 ,ug of the purified DT was lethal for 120-g MHA hamsters within 36 h. Therefore, cells from MHA hamster lymphoid tissues appeared to be an appropriate source for the detection of membrane components which interact with DT. Experiments were performed with lymph node cells and with thymocytes. Since identical results were obtained with cells from both tissues, only the data obtained utilizing thymocytes are presented. Detection of DT-binding membrane components. Hamster thymocytes were surface labeled with "2I and solubilized in 0.5% NP-40. The labeled lysates were fractionated into a lentil lectin-adherent fraction (glycoprotein fraction) and a nonadherent fraction by affinity chromatography of the lysates on lentil lectinSepharose. The two fractions were then analyzed for the presence of DT-binding components. The first approach employed to detect labeled DT-binding molecules was an immunoprecipitation technique. Samples of radiolabeled fractions were incubated with DT to form DT-1251labeled ligand complexes, and the complexes were then immunoprecipitated with antiserum to DT. Reagent controls of DT alone or antiserum alone were also performed. The immune precipitates were dissociated, reduced with 2mercaptoethanol, and analyzed by SDS-5% polyacrylamide gel electrophoresis. Analysis of immune precipitates obtained from the nonadherent fraction of '25I-labeled cell lysates did not reveal the presence of any DT-specific peaks of radioactivity (data not shown). This result was not altered by inclusion of protease inhibitors, known to inhibit hamster thymocyte proteases (R. J. Fulton and D. A. Hart, J. Supramol. Struct. Suppl. 3, Abstr. 676, 1979), in the immunoprecipitation system. In contrast, analysis of immune precipitates obtained from the glycoprotein fraction revealed the presence of DTspecific molecules (Fig. 1A). These '251-labeled molecules were absent when either DT or the with DT or with diphtheria toxoid were analyzed by The size of SDS-PAGE as described previously (16), utilizing the antiserum was omitted (Fig. 1A). these molecules, based on five experiments, was method of Shapiro et al. (18). calculated to be 153,000 ± 4,000 daltons. AnalyRESULTS AND DISCUSSION sis of immune precipitates obtained with DT Optimal conditions have been developed for plus antiserum to DT, under nonreducing conthe radiolabeling and the subsequent detection ditions, resulted in an electrophoretic profile of cell surface glycoproteins on hamster lymph- identical to that shown in Fig. 1A. This latter oid cells (L. Eidels, R. L. Proia, J. W. Streilein, result indicates that this molecular species is previously (16). The iodinated cells were washed in an excess of phosphate-buffered saline and then lysed in 0.5% Nonidet P-40 (NP-40) (Shell Oil Co.) in tris(hydroxymethyl)aminomethane-buffered saline [TBS; 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride, 0.15 M NaCl, 0.02% NaN3, pH 7.4] at a ratio of 5 x 107 cells per ml of 0.5% NP-40 for 15 min at 4°C. Nuclei were removed by centrifugation, and the supernatant fraction was dialyzed for 3 h at 4°C against TBS. A 1-ml amount of the supernatant fraction was therefore considered to equal 5 x 10' cell equivalents. Hereafter, results obtained with cell lysates are expressed as the number of cell equivalents based on this relationship. This method normalizes the results since there is variability in the efficiency of the lactoperoxidase labeling between experiments and is also necessary since the amount of protein in the fractionated lysates is below levels detectable by conventional methods. Lentil lectin affinity chromatography. Cell lysates were fractionated on Sepharose-lentil lectin columns as described previously (16). The nonadherent fraction was collected and saved. After extensive washing of the lentil lectin-Sepharose columns with the TBS-bovine serum albumin (BSA)-NP-40 buffer, the adherent IuI-glycoprotein fraction was eluted with 0.2 M a-methylmannoside (Sigma Chemical Co.) in the same buffer. The eluted glycoprotein fraction was concentrated to the original volume of "2I-labeled cell lysate by vacuum dialysis against TBS. This entire procedure was carried out at 4°C. Immunoprecipitations Immunoprecipitation. were performed as previously described (16). Appropriate samples (1 ml) were incubated with DT (or fragment A, CRM197 protein, or toxoid) or cholera toxin for 1 h at 37°C and then allowed to incubate with the respective antitoxin for 1 h at 4°C. The resulting immune complexes were removed by centrifugation after the addition of 0.25 ml of a 10% suspension of Staphylococcus aureus. The immune precipitates were washed twice with TBS-BSA-NP-40 buffer and finally with phosphate-buffered saline. Affinity chromatography with DT and toxoid. Purified DT and diphtheria toxoid were each coupled to Sepharose 4B (1 mg/ml) by the cyanogen bromide method (8). The conjugates were first equilibrated with TBS-BSA-NP-40 buffer, and then 0.2 ml of packed DT-Sepharose or toxoid-Sepharose was incubated with samples of the adherent 1"I-glycoprotein fraction at 37°C for 1 h. The conjugates were then washed with TBS-BSA-NP-40 buffer until the radioactivity associated with the beads remained constant. SDS-PAGE. The radiolabeled proteins isolated by immunoprecipitation or by affinity chromatography

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probably not a subunit of a disulfide-linked complex. The second approach employed to detect DTbinding components was based on affinity chromatography utilizing DT or diphtheria toxoid covalently coupled to Sepharose. Diphtheria toxoid is known not to possess any receptor binding activity (20). Incubation of samples of the 125I-glycoprotein fraction with SepharoseDT or Sepharose-diphtheria toxoid, followed by analysis of the material bound to these conjugates by SDS-5% polyacrylamide gel electrophoresis revealed the presence of labeled 153,000dalton molecules bound by the Sepharose-DT which were not bound by the Sepharose-diphtheria toxoid (Fig. 1B). Therefore, in both detection systems, albeit with less efficiency in the affinity chromatography system, it appears that DT binds to 153,000dalton membrane glycoproteins from the surface of hamster lymphoid cells. Molecular weight of the DT-binding membrane component. It is well known that the molecular weights calculated for glycoproteins from SDS-PAGE analysis are normally not precise (17). Therefore, the assignment of a molecular weight for the DT-binding glycoproteins of 153,000 is valid only for 5% polyacrylamide gel analysis. Analysis of the DT-binding components on SDS-agarose-2.5% polyacrylamide gels and SDS-7.5% polyacrylamide gels yielded apparent molecular weights for the DT-binding components of 218,000 and 136,000, respectively. By using a plot of 1/apparent molecular weight versus 1/acrylamide concentration (19) and ex-

2 mA/gel for 15 h. The gels were sliced, and each fraction was analyzed for 125I and WH. The arrows indicate the molecular weights of the internal 3H markers which consisted of 3H-labeled BSA monomer (68,000), 'H-labeled cross-linked BSA dimer (136,000), and 3H-labeled cross-linked BSA trimer (204,000). (B) 0 The lentil lectin-adherent fraction from a lysate of 0 20 40 60 "2'I-labeled hamster thymus cells was incubated either with DT-Sepharose conjugate (0.2 ml, I mg of FRACTIONS toxin per ml) (A) or with diphtheria toxoid-Sepharose FIG. 1. SDS-PAGE of DT-binding glycoproteins. conjugate (0.2 ml, I mg of toxoid per ml) (A). Each (A) The lentil lectin-adherent fraction from a lysate conjugate was incubated with an amount of lentil of "25I-labeled hamster thymus cells was prepared lectin-adherent fraction corresponding to 5 x 107 cell and immunoprecipitated. Each assay contained an equivalents. The radiolabeled material adherent to amount of lentil lectin-adherent fraction correspond- the conjugates was dissociated, reduced, and applied ing to 3.7 x 10 cell equivalents. The immune precip- to SDS-5% polyacrylamide gels. SDS-PAGE analysis itates were: DT (20 pg) plus anti-DT (20 IL) (0); DT was performed as described for (A). (C) This analysis (20 p) alone (0); anti-DT (20 yIl) alone (0); and was performed as described for (A), except that the cholera toxin (20 p) plus anti-cholera toxin (20 yd) immune precipitates were: CRM197 protein (20 g) (0). The amount of antiserum employed (20 yd) com- plus anti-DT (20 yd) (V), fragment A of DT (20 a) pletely precipitated the quantity of corresponding plus anti-DT (20 Al) (V), and diphtheria toxoid (20 toxin employed (20 pg). The immune precipitates were p) plus anti-DT (20 ,ul) (x). The quantity of anti-DT dissociated, reduced, and applied to SDS-5% poly- employed (20 Al) completely precipitated the quantity acrylamide gels. The gels were electrophoresed at 7 of toxin derivatives or toxoid employed (20 p).

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trapolating to infinite acrylamide concentration, a molecular weight of 109,000 was obtained for the DT-binding glycoproteins. Specificity of the binding of the membrane component to DT-related proteins. If the membrane components detected function as receptors during DT intoxication, then they should be specific for molecules containing an active B-fragment of the toxin (11, 20). Therefore, the membrane glycoproteins should react with CRM197 protein, which has an active Bfragment and an inactive A-fragment, but should not react with the isolated A fragment or with diphtheria toxoid (2, 14). When the immunoprecipitation detection system was used, substitution of CRM197 protein for DT resulted in the precipitation of the 153,000-dalton glycoproteins (Fig. 10). However, these molecules were not precipitated when either diphtheria toxoid or fragment A was substituted for DT (Fig. 10). The antisera employed recognized both isolated fragment A and diphtheria toxoid. In addition, substitution of cholera toxin, which has a glycolipid receptor, and antiserum to cholera toxin in the immunoprecipitation system failed to precipitate the DT-binding membrane components (Fig. 1A). Therefore, the detected membrane components appear to bind specifically to DT and related proteins which have an active B-frag-

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proteins are indeed receptors for DT, then it should be possible to saturate the inmunoprecipitation system with respect to DT as well as with respect to the membrane receptors. As Fig. 2 shows, utilization of a constant amount of radiolabeled material (2 x 107 cell equivalents) and varying the amount of DT in the immunoprecipitation system led to the precipitation of increasing amounts of the membrane glycoproteins until saturation was reached. The converse experiment, in which a constant amount of DT (20 ug) was added to the reaction and the number of cell equivalents was varied (from 0.5 x 107 to 3 x 107), indicated that the quantity of the '25I-labeled glycoproteins reactive with DT was finite and proportional to the '"I-labeled cell equivalents in the system until the DT itself was saturated. To confirm the conclusion that the 1"I-glycoprotein fraction could be depleted of the DTbinding components, a sequential immunoprecipitation experiment was performed. A sample of the '25I-glycoprotein fraction (Fig. 3A) was reacted under saturating conditions with 30 ,ug of DT in the immunoprecipitation system. The immune precipitate obtained was analyzed by SDS-5% polyacrylamide gel electrophoresis (Fig. 3B), and the supernatant fraction resulting from this immunoprecipitation was again reacted with a saturating amount (30 ,ug) of DT. Analysis of this second immune precipitate revealed the presence of only a small amount of the DTbinding glycoproteins (Fig. 3B). Furthermore, SDS-5% polyacrylamide gel electrophoresis analysis of the resulting "receptor"-depleted gly-

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Saturation of the DT-membrane glycoprotein interaction. An important criterion for the identification of a receptor is saturability (3). Therefore, if the DT-binding membrane glyco-

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FIG. 2. Saturation of the DT-membrane glycoprotein interaction. (A) Samples of radiolabeled lentil lectinadherent fraction corresponding to 2 x 107 hamster thymus cells were reacted with varying amounts (4 to 30 pg) of DT. Complexes were immunoprecipitated with 30 jil of anti-DT, a concentration sufficient to completely bind the toxin. The immune precipitates were then analyzed by SDS-5% polyacrylamide gel electrophoresis as in Fig. IA. The radioactivity (counts per minute) under the peak with an apparent molecular weight of -153,000 was plotted versus the amount (micrograms) of DT. (B) Varying quantities of radiolabeled lentil lectin-adherent fraction corresponding to 0.5 x MI0 to 3 x 107 hamster thymus cell equivalents were reacted with 20pg of DTplus 20 .I of anti-DT. The immune precipitates were processed and analyzed as described for (A), except that the 21I counts per minute under the peak were plotted versus the number of cell equivalents utilized.

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-J 60 40 20 FRACTIONS FIG. 3. SDS-PAGE of the lentil lectin-acdherent fraction before and after depletion of the DT-1binding glycoproteins. (A) A sample of the lentil lectin -adherent fraction corresponding to 2 x I0d hamster thyms cell equivalents (0) was reduced and apple tan SDS-5% polyacrylamide gel, electrophorese d,d andw 0

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processed as described in the legend to Fig. IA. A similar sample was twice subjected to immune)precipitation with saturating concentrations of L)T and anti-DT as described for (A). The resulting siupernatant fraction (0) was reduced and applied to ain SDS5% polyacrylamide gel electrophoresed, an d processed as described in the legend to Fig. IAL (B) A sample of the lentil lectin-adherent fractionX corresponding to 2 x 107 cell equivalents was immiunopre-

coprotein fraction revealed an electrophoretic profile (Fig. 3A) nearly indistinguisable from that of the starting glycoprotein fraction (Fig. 3A). There are two important points to be made from these data. The first is that the DT-binding glycoproteins are minor membrane cornonents; they represent only 0.1 to 0.2% ofthe 1 6Iincorporated into cellular components. This very low percentage of 1"I in the DT-binding glycoproteins would indicate a small number of molecules per cell, if indeed all the membrane components label equally. A second point to be made is related to the observation that the DT-binding glycoproteins are a subpopulation of the molecules with an apparent molecular weight of 153,000 on SDS-5% polyacrylamide gel electrophoresis (Fig. 3). The DT-binding glycoproteins represent approximately 4 to 5% ofthe molecules in the glycoprotein fraction with this apparent molecular weight. It is of interest to note that some hormone receptors have been reported to be glycoproteins of approximately the same apparent molecular weight (135,000 to 195,000) as the DT-binding glycoproteins (4, 6, 9, 10). In addition, the purification of the receptors for these hormones (10, 13) was greatly facilitated by affinity chromatography on Sepharose-concanavalin A, a lectin with binding properties similar to those of lentil lectin. Therefore, one could speculate that the non-DT-binding glycoproteins with apparent molecular weights of -150,000 could function as receptors for other biologically active molecules. In fact, it is very unlikely that the only biological function of the DT-binding glycoproteins is to interact with DT. Interaction of DT with SDS-PAGEtreated 1251-glycoproteins. The biochemical nature of the interaction of DT with the DTbinding membrane glycoproteins is unknown. Therefore, it was of interest to determine whether DT would interact with the 153,000dalton glycoproteins after they had been denaturated by boiling in SDS-trs(hydroxymethyl)aminomethane-urea plus 1.2 M 2-mercaptoethanol and had been electrophoresed on SDS-5% polyacrylamide gels. The radioactivity from the area corresponding to the 153,000-molecular-weight range was eluted from gels of either the whole glycoprotein fraction cipitated with DT (30 pg plus anti-DT (30 itd) (A). The supernatant fraction resulting from this first immunoprecipitation was again inmunoprecipitated with DT (30 pg) plus anti-DT (30 il) (A). Both immune precipitates were dissociated, reduced, and applied to SDS-5% polyacrylanide gels, electrophoresed, and processed as described in the legend to Fig. lA.

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(Fig. 3A, fractions 16 through 22) or from the material specifically immunoprecipitated with DT plus antiserum to DT (Fig. 1A, fractions 16 through 22). Samples of the eluted material were reacted with DT plus antiserum to DT or diphtheria toxoid plus antiserum to DT. The 153,000dalton glycoproteins again specifically reacted with DT. These results imply that (i) the secondary protein structure of the glycoproteins does not play a prominent role in the interaction with DT, and (ii) the interaction with DT is probably not due to some non-iodinated molecules (lipids?) associated with the 153,000-dalton glycoproteins since such molecules should have been removed during the SDS-PAGE procedure. In conclusion, we have identified membrane glycoproteins which are candidates for the receptor for DT on eucaryotic cells. These membrane glycoproteins are present on the cell surfaces of hamster thymus and lymph node cells and have an apparent molecular weight similar to that of the DT-binding glycoproteins that we have described from lymph node cells of guinea pigs (168,000 ± 5,600) (16). In both species the solubilized membrane glycoproteins specifically interact with DT and CRM197 protein but not with diphtheria toxoid, fragment A of DT, or cholera toxin. This specificity of binding correlates well with the known specificities of the DT receptor on cells as determined by cytotoxicity methods (20) and by binding assays with radiolabeled DT (11). ACKNOWLEDGMENTS We thank R. A. Finkelstein for the gift of cholera toxin, R. K. Holmes for CRM197 protein and fragment A of DT, J. W. Uhr for support, and George C. Stewart and Jerry Jeff Fulton for critical review of the manuscript. The excellent technical assistance of Diane Apgar and the secretarial help of Daisi Marcoulides are greatly appreciated. This work was supported by Public Health Service grant AI-11851 from the National Institutes of Health and American Cancer Society grant BC-215. R.L.P. is supported by Cancer Immunology Training Grant CA 09082.

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4. Das, M., and C. F. Fox. 1978. Molecular mechanism for mitogen action: processing of receptor induced by epidermal growth factor. Proc. Natl. Acad. Sci. U.S.A. 75: 2644-2648. 5. Draper, R. K., D. Chin, and M. I. Simon. 1978. Diphtheria toxin has the properties of a lectin. Proc. Natl. Acad. Sci. U.S.A. 75:261-265. 6. Dufau, M. L, E. H. Charreau, and K. J. Catt. 1973. Characteristics of soluble gonadotropin receptor from the rat testis. J. Biol. Chem. 248:6973-6982. 7. Hart, D. A. 1975. Evidence for the non-protein nature of the receptor for the enterotoxin of Vibrio cholerae on murine lymphoid cells. Infect. Immun. 11:742-747. 8. Hayman, M. J., and M. J. Crumpton. 1972. Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohemagglutinin. Biochem. Biophys. Res. Commun. 47:923-930. 9. Hock, R. A., E. Nexo, and M. D. Hollenberg. 1979. Isolation of the human placenta receptor for epidermal growth factor-urogastrone. Nature (London) 277:403405. 10. Jacobs, S., Y. Shechter, K. Bissel, and P. Cuatrecasas. 1977. Purification and properties of insulin receptors from rat liver membranes. Biochem. Biophys. Res. Commun. 77:981-988. 11. Middlebrook, J. L, R. M. Dorland, and S. H. Leppla. 1978. Association of diphtheria toxin with Vero cells. Demonstration of a receptor. J. Biol. Chem. 253:73257330. 12. Olitzki, L, L A. Stuczynski, and N. Grossowicz. 1948. Neurotoxic symptoms produced in the syrian hamster (Cricetus auratus) by diphtherial toxin. J. Immunol.

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Acad. Sci. U.S.A. 76:685-689. 17. Segrest, J. P., R. L Jackson, E. P. Andrews, and V. T. Marchesi. 1971. Human erythrocyte membrane glycoprotein: a re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis. Biochem. Biophys. Res. Commun. 44:390-395. 18. Shapiro, A. L, E. Vinuela, and J. V. Maiel. 1967. Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem. Biophys. Res. Commun. 28:815-820. 19. Ward, C. W., and T. A. A. Dopheide. 1976. Size and chemical composition of influenza virus hemagglutinin chains. FEBS Lett. 65:365-368. 20. Zanen, J., G. Muyldermans, and N. Beugnier. 1976. Competitive antagonists of the action of diphtheria toxin in HeLa cells. FEBS Lett. 66:261-263.