Monoclonal Antibodies to Integral Membrane Transport and Receptor Proteins

Novel Reagents for Protein Purification
  • J. Craig Venter
  • Ursina Schmidt
  • Barbara Eddy
  • Giorgio Semenza
  • Claire M. Fraser


The isolation and molecular characterization of integral membrane proteins of major physiological significance, including receptors and transport proteins, are at best difficult tasks. For good results a multifaceted approach to elucidating the structure and function of membrane proteins is required. Useful approaches include affinity labeling and photoaffinity labeling (Ruoho et al, 1984), radiation inactivation/target size analysis (Venter, 1983b; Venter et al, 1983b; Lilly et al., 1983), characterization of hydrodynamic properties (Davis, 1984), and immunological approaches using conventional antibodies, monoclonal antibodies, and antiidiotypic antibodies (Venter et al, 1984; Fraser and Venter, 1980; Venter, 1983a; Venter and Fraser, 1983; Berzofsky, 1984; Berzofsky et al., 1980, 1982; Tzartos and Linstrom, 1980; Gullick et al., 1982; Yavin et al., 1981; Beisiegel et al., 1981; Greene et al., 1980; Schreiber et al., 1980;Wasserman et al., 1982). This chapter will detail the monoclonal antibody approach that we have found to be successful in isolating and characterizing the structure of membrane transport proteins and hormone and neurotransmitter receptors.


Brush Border Membrane Neurotransmitter Receptor Spend Culture Medium Brush Border Membrane Vesicle Immunoaffinity Column 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Aronson, P. A., and Sacktor, B., 1975, The Na+ gradient-dependent transport of d-glucose in renal brush border membranes, J. Biol. Chem. 250:6032–6039.PubMedGoogle Scholar
  2. Beisiegel, U., Schneider, W. J., Golstein, J. L., Anderson, R. G. W., and Brown, M. S., 1981, Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia, J. Biol. Chem. 256:11923–11931.PubMedGoogle Scholar
  3. Berzofsky, J. A., 1984, Monoclonal antibodies as probes of antigenic structure, in: Receptor Biochemistry and Methodology, Volume IV (J. C. Venter, C. M. Fraser, J. M. Lindstrom, eds.), Liss, New York, in press.Google Scholar
  4. Berzofsky, J. A., Hicks, G., Fedorko, J., and Minna, J., 1980, Properties of monoclonal antibodies specific for determinants of a protein antigen, myoglobin, J. Biol. Chem. 255:11188–11191.PubMedGoogle Scholar
  5. Berzofsky, J. A., Buckenmeyer, G. K., Hicks, G., Gurd, F. R. N., Feldmann, R. J., and Minna, J., 1982, Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin, J. Biol. Chem. 257:3189–3198.PubMedGoogle Scholar
  6. Bolger, G. T., Gengo, P. T., Luchowski, E. M., Siegel, H., Triggle, D. J., and Janis, R. A., 1982, High affinity binding of a Ca++ channel antagonist to smooth and cardiac muscle, Biochem. Biophys. Res. Commun. 104:1604–1609.PubMedCrossRefGoogle Scholar
  7. Bolger, G. T., Gengo, P. T., Kockowski, R., Luchowski, E. M., Siegel, H., Janis, R. A., Triggle, A. M., and Triggle, D. J., 1983, Characterization of binding of the Ca++ channel antagonist [3H]Nitrendipine, to guinea-pig Ileal smooth muscle, J. Pharmacol Exp. Ther. 225:291–309.PubMedGoogle Scholar
  8. Chung, S. D., Alan, N., Livingston, D., Hiller, S., and Taub, M., 1982, Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium, J. Cell Biol. 95:118–126.PubMedCrossRefGoogle Scholar
  9. Davis, A., 1984, Determination of the hydrodynamic properties of detergent solubilized proteins, in: Receptor Biochemistry and Methodology, Volume III (J. C. Venter and L. C. Harrison, eds.), Liss, New York, pp. 161–178.Google Scholar
  10. Diedrich, D. F., 1966, Competitive inhibition of intestinal glucose transport by phlorizin analogs, Arch. Biochem. Biophys. 117:248–256.PubMedCrossRefGoogle Scholar
  11. Fraser, C.M., 1984, Monoclonal antibodies to β-adrenergic receptors and receptor structure in: Receptors and Recognition (P. Cuatrecasas and M. F. Greaves, eds.), Chapman and Hall, London, in press.Google Scholar
  12. Fraser, C. M., and Lindstrom, J. M., 1984, The production and use of monoclonal antibodies in receptor characterization and purification, in: Receptor Biochemistry and Methology, Volume III (J. C. Venter, and L. C. Harrison, eds.), Liss, New York, pp. 1–30.Google Scholar
  13. Fraser, C. M., and Venter, J. C., 1980, Monoclonal antibodies to β-adrenergic receptors: Use in purification and molecular characterization of β-receptors, Proc. Natl. Acad. Sci. USA 77:7034–7038.PubMedCrossRefGoogle Scholar
  14. Fraser, C. M., and Venter, J. C., 1982, The size of the mammalian lung β2-adrenergic receptor as determined by target size analysis and immunoaffinity chromatography, Biochem. Biophys. Res. Comm. 109:21–29.PubMedCrossRefGoogle Scholar
  15. Fraser, C. M., Venter, J. C., Finkelstein, J. N., and Shapiro, D. L., 1984, Monoclonal antibodies to surface antigens of rabbit type II pneumocytes, Am. Rev. Resp. Dis., in press.Google Scholar
  16. Greene, G. L., Fitch, F. W., and Jensen, E. V., 1980, Monoclonal antibodies to estrophilin: Probes for the study of estrogen receptors, Proc. Natl. Acad. Sci. USA 77:157–161.PubMedCrossRefGoogle Scholar
  17. Gullick, W. J., Tzartos, S., and Lindstrom, J. M., 1982, Monoclonal antibodies as probes for acetylcholine receptor structure. I. Peptide mapping, Biochemistry 20:2173–2180.CrossRefGoogle Scholar
  18. Hopfer, U., Nelson, K., Perroto, J., and Isselbacher, K. J., 1973, Glucose transport in isolated brush border membrane from rat small intestine, J. Biol. Chem. 248:25–32.PubMedGoogle Scholar
  19. Janis, R. A., and Triggle, D. J., 1983, New developments in Ca++ channel antagonist, J. Med. Chem. 26:775–785.PubMedCrossRefGoogle Scholar
  20. Klausner, R. D., van Renswoude, J., Blumenthel, R., and Riunay, B., 1984, Reconstitution of membrane receptors, in: Receptor Biochemistry and Methodology, Volume III (J. C. Venter and L. C. Harrison, eds.), Liss, pp. 209-240.Google Scholar
  21. Kohler, G., and Milstein, C., 1975, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256:495–497.PubMedCrossRefGoogle Scholar
  22. Lilly, L., Fraser, C. M., Jung, C. Y., Seeman, P., and Venter, J. C., 1983, Molecular size of the canine and human brain D2 dopamine receptor as determined by radiation inactivation, Mol. Pharm. 24:10–14.Google Scholar
  23. Misfeldt, D. S., Hammamoto, S. T., and Pitelka, D. R., 1976, Transepithelial transport in cell culture, Proc. Natl. Acad. Sci. USA 73:1212–1216.PubMedCrossRefGoogle Scholar
  24. Murer, H., Hopfer, U., Kinne-Saffron, E., and Kinne, R., 1974, Glucose transport in isolated brush-border and lateral—basal plasma—membrane vesicles from intestinal epithelial cells, Biochim. Biophys. Acta 345:170–179.PubMedCrossRefGoogle Scholar
  25. Rabito, C. A., and Ausiello, D. A., 1980, Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney, J. Membrane Biol. 54:31–38.CrossRefGoogle Scholar
  26. Rindler, M. J., Taub, M., and Saier, M. H., Jr., 1979, Uptake of 22Na+ by cultured dog kidney cells (MDCK), J. Biol. Chem. 254:11935–11939.Google Scholar
  27. Ruoho, A. E., Rashidbaigi, A., and Roeder, P. E., 1984, Approaches to the identification of receptors using affinity labels, in: Receptor Biochemistry and Methodology. Vol. I (J. C. Venter and L. C. Harrison, eds.), Liss, Inc., New York, pp. 119–160.Google Scholar
  28. Schmidt, U. M., Eddy, B., Fraser, C. M., Semenza, G., and Venter, J. C., 1983a, Monoclonal antibodies and immunoaffinity purification of the Na+,d-glucose cotransporter of intestinal brush border, submitted for publication.Google Scholar
  29. Schmidt, U. M., Eddy, B., Fraser, C. M., Venter, J. C., and Semenza, G., 1983b, Purification of the Na+,d-glucose cotransporter of rabbit intestinal brush border membranes using monoclonal antibodies, FEBS Letters 161:279–283.PubMedCrossRefGoogle Scholar
  30. Schreiber, A. B., Couraud, P. O., Andre, C., Vray, B., and Strosberg, A. D., 1980, Anti-alprenolol anti-idiotypic antibodies bind to β-adrenergic receptors and modulate catecholamine-sensitive adenylate cyclase, Proc. Natl. Acad. Sci. USA 77:7385–7389.PubMedCrossRefGoogle Scholar
  31. Tannenbaum, C., Toggenburger, G., Kessler, M., Rothstein, A., and Semenza, G., 1977, High affinity phlorizin binding to brush border membranes from small intestine: Identity with (a part of) the glucose transport system. Dependence on the Na+-gradient, partial purification, J. Supramol. Struct. 6:519–533.PubMedCrossRefGoogle Scholar
  32. Taub, M., and Saier, M. H., Jr., 1981, Amiloride-resistant Madin—Darby canine kidney (MDCK) cells exhibit decreased calcium transport, J. Cell. Phys. 106:191–199.CrossRefGoogle Scholar
  33. Toggenburger, G., Kessler, M., Rothstein, A., Semenza, G., and Tannenbaum, G., 1978, Similarity in effects of Na+ gradients and membrane potentials on d-glucose transport by, and phlorizin binding to, vesicles derived from brush borders of rabbit intestinal mucosal cells, J. Membrane Biol. 40: 269–290.CrossRefGoogle Scholar
  34. Tzartos, S., and J. M. Lindstrom, 1980, Monoclonal antibodies used to probe acetylcholine receptor structure: Localization of the main immunogenic region and detection of similarities between subunits, Proc. Natl. Acad. Sci. USA 77: 755–759.PubMedCrossRefGoogle Scholar
  35. Venter, J. C., 1982, Monoclonal antibodies and autoantibodies in the isolation and characterization of neurotransmitter receptors: The future of receptor research, J. Mol. Cell. Cardiol. 14:687–693.PubMedCrossRefGoogle Scholar
  36. Venter, J. C., 1983a, Monoclonal and anti-idiotypic antibodies and the elucidation of receptor structure, Surv. Immunol. Res. 2:302–305.PubMedGoogle Scholar
  37. Venter, J. C., 1983b, Muscarinic cholinergic receptor structure I. Receptor size, membrane orientation and absence of major phylogenetic structural diversity, J. Biol. Chem. 258:4842–4848.PubMedGoogle Scholar
  38. Venter, J. C., and Fraser, C. M., 1981, The development of monoclonal antibodies to β-adrenergic receptors and their use in receptor purification and characterization, in: Monoclonal Antibodies in Endocrine Research (G. Eisenbarth and R. Fellows, eds.), Raven Press, New York, pp. 119–134.Google Scholar
  39. Venter, J. C., and Fraser, C. M., 1983, β-adrenergic receptor isolation and characterization with immobilized drugs and monoclonal antibodies, Fed. Proc. 42:273–278.PubMedGoogle Scholar
  40. Venter, J. C., Eddy, B., Hall, L. M., and Fraser, C. M., 1984, Monoclonal antibodies detect the conservation of muscarinic cholinergic receptor structure from Drosophila to human brain and possible structural homology with α-adrenergic receptors, Proc. Nat. Acad. Sci. USA 80:272–276.CrossRefGoogle Scholar
  41. Venter, J. C., Eddy, B., Schmidt, U., and Fraser, C. M., 1984, Production of monoclonal antibodies to integral membrane transport and receptor proteins and their use in structural elucidation, in: Idiotypic Manipulations in Biological Systems (H. Kohler, P. A., Cazenave, and J. Urbain, eds.), Academic Press, New York, pp. 273–301.Google Scholar
  42. Venter, J. C., Fraser, C. M., Schaber, J. S., Jung, C. Y., Bolger, G., and Triggle, D. J., 1983, Molecular properties of the slow inward calcium channel: Molecular weight determinations by radiation inactivation and covalent affinity labelling, J. Biol. Chem. 258:9344–9348.PubMedGoogle Scholar
  43. Venter, J. C., Fraser, C. M., Soiefer, A., Jeffrey, D. R., Strauss, W. L., Charlton, R. R., and Greguski, R., 1981, Autoantibodies and monoclonal antibodies to β-adrenergic receptors: Their use in receptor purification and characterization, in: Advances in Cyclic Nucleotide Research, Vol. 14 (J. Dumont, P. Greengard, and G. A. Robison, eds.), Raven Press, New York, pp. 135–143.Google Scholar
  44. Wasserman, N. H., Penn, A. S., Freimuth, P. I., Treptow, N., Wentel, S., Cleveland, W. L., and Erlanger, B. F., 1982, Anti-idiotypic route to anti-acetylcholine receptor antibodies and experimental myasthenia gravis, Proc. Natl. Acad. Sci. USA 79:4810–4814.CrossRefGoogle Scholar
  45. Yavin, E., Yavin, Z., Schneider, M., and Kohn, L. D., 1981, Monoclonal antibodies to the thyrotropin receptor: Implications for receptor structure and the action of autoantibodies in Graves disease, Proc. Natl. Acad. Sci. USA 78:3180–3184.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • J. Craig Venter
    • 1
  • Ursina Schmidt
    • 1
  • Barbara Eddy
    • 1
  • Giorgio Semenza
    • 2
  • Claire M. Fraser
    • 1
  1. 1.Department of Molecular ImmunologyRoswell Park Memorial InstituteBuffaloUSA
  2. 2.Laboratorium für BiochemieEidgenossischen Technischen Hochschule ETH-ZentrumZurichSwitzerland

Personalised recommendations