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Inhibition of ileal brush-border chloride conductance by specific antibody

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Summary

Antibody raised in mice was used in attempting to identify proteins responsible for the conductive chloride transport that can be measured in porcine ileal brush border membrane vesicles. Ileal brush-border membrane vesicle protein from pig was separated into five different molecular mass fractions by preparative SDS polyacrylamide disc gel electrophoresis. Separated protein fractions were used to immunize mice. Antibody was screened for reactivity with antigen by Western blotting, and for effects on conductive chloride transport in ileal brush border membrane vesicles. Immunization with brush-border protein from fraction I proteins (>110 kDa) produced polyclonal antisera which specifically inhibited the conductive component of chloride uptake by ileal brush border vesicle preparations. Western blotting of the antigen showed the presence of several protein species of molecular mass >100 kDa that were recognized by immune serum. Spleen cells from a mouse producing antiserum that inhibited conductive chloride transport were fused with a myeloma cell line. The resulting hybridoma colonies produced antibody that reacted with at least seven distinct protein bands by Western blot assay and inhibited chloride conductance in brush-border membrane vesicles.

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References

  1. Anderson, M.P., Gregory, R.J., Thompson, S., Souza, D.W., Paul, S., Mulligan, R.C., Smith A.E., Welsh, M.J. 1991. Demonstration that CFTR is a chloride channel by altering its selectivity. Science 253:202–205

    CAS  PubMed  Google Scholar 

  2. Field, M. 1981. Secretion of electrolytes and water by mammalian small intestine. In: Physiology of the Gastrointestinal Tract. L.R. Johnson, editor. pp. 963–982 Raven, New York

    Google Scholar 

  3. Field, M., Fromm, D., Al-Awqati, Q., Greenough, W.B. 1972. Effect of cholera enterotoxin on ion transport across isolated ileal mucosa. J. Clin. Invest. 51:796–804

    Google Scholar 

  4. Finn, A.L., Tsai, L.-M., Falk, R.J. 1989. Monoclonal antibodies to the apical chloride channel in Necturus gallbladder inhibit the chloride conductance. Proc. Natl. Acad. Sci. USA 86:7649–7652

    Google Scholar 

  5. Forsyth, G.W., Gabriel, S.E. 1988. Chloride uptake by pig jejunal brush border vesicles: Absence of NaCl cotransport and evidence for chloride conductance. J. Physiol. 402:555–564

    Google Scholar 

  6. Forsyth, G.W., Gabriel, S.E. 1989. Inhibiting conductive chloride uptake in membrane vesicles: Specificity of α-phenylcinnamate. Biochim. Biophys. Acta 977:19–25

    Google Scholar 

  7. Forsyth, G.W., Gabriel, S.E. 1989. Activation of chloride conductance in pig jejunal brush border vesicles. J. Membrane Biol. 107:137–144

    Google Scholar 

  8. Gabriel, S.E., Forsyth, G.W. 1991. Candidate proteins for conductive chloride transport in porcine ileal brush border membrane. J. Biol. Chem. 266:17764–17769

    Google Scholar 

  9. Kohler, G., Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature 256:495–497

    CAS  PubMed  Google Scholar 

  10. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  11. Landry, D.W., Akabos, M.H., Redhead, C., Edelman, A., Cragoe, E.J. Jr., Al-Awqati, Q. 1989. Purification and reconstitution of chloride channels from kidney and trachea. Science 244:1469–1472

    Google Scholar 

  12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275

    CAS  PubMed  Google Scholar 

  13. Peerce, B.E., Seifert, S., Clarke, R. 1991. Partial purification and reconstitution of an anion conductance from rabbit intestinal brush border membranes. FASEB J. 5:8078

    Google Scholar 

  14. Quinton, P.M. 1983. Chloride impermeability in cystic fibrosis. Nature 301:421–422

    Google Scholar 

  15. Ran, S., Benos, D.J. 1991. Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes. J. Biol. Chem. 266:4782–4788

    Google Scholar 

  16. Rich, D.P., Gregory, R.J., Anderson, M.P., Manaralan, P., Smith, A.E., Welsh, M.J. 1991. Effect of deleting the R domain on CFTR-generated chloride channels. Science 253:205–207

    Google Scholar 

  17. Towbin, H., Staehelin, T., Gordon, J. 1979. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354

    Google Scholar 

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Authors and Affiliations

  1. Veterinary Physiological Sciences, University of Saskatchewan, S7N 0W0, Saskatoon, Saskatchewan, Canada

    K. J. Racette, K. J. Gaspar & G. W. Forsyth

  2. Department of Medicine, Division of Pulmonary Diseases, University of North Carolina, 27599-7020, Chapel Hill, North Carolina

    S. E. Gabriel

Authors
  1. S. E. Gabriel
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  2. K. J. Racette
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  3. K. J. Gaspar
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  4. G. W. Forsyth
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Gabriel, S.E., Racette, K.J., Gaspar, K.J. et al. Inhibition of ileal brush-border chloride conductance by specific antibody. J. Membarin Biol. 129, 323–328 (1992). https://doi.org/10.1007/BF00232913

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  • DOI: https://doi.org/10.1007/BF00232913

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