Activating sheep antibodies to tyrosine hydroxylase

  • John W Haycock
  • Jack C Waymire


Antibodies to tyrosine hydroxylase (TH; EC have been extremely useful in establishing the neuronal and cellular localization of TH (Pickel et al., 1975a,b) and, recently, in demonstrating that in situ phosphorylation of TH is important in regulating the catalytic activity of TH (Waymire et al., 1979). While in the process of raising antibodies to TH, we observed an interesting property of immunoglobulins (Ig) from a sheep that was injected with TH purified from rat pheochromocytoma. Mixture of a partially purified TH and sheep Ig (0–45% ammonium sulfate) resulted in a dramatic elevation of TH activity. The following experiments were performed to establish whether this effect resulted from a specific interaction of TH and antibodies directed against TH. The results indicate that this crude Ig fraction contains anti-TH antibodies, that the majority of anti-TH activity is associated with activation of TH, and that the anti-TH antibodies are not uniformly distributed across IgG subclasses.


Tyrosine Hydroxylase Dramatic Elevation Couple Decarboxylation Peroxidase Label Antibody Method Tyrosine Hydroxylase Protein 
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. Ashe, W.K., Mage, M., Mage, R. and Notkins, A.L. (1968). Neutralization and sensitization of Herpes Simplex virus with antibody fragments from rabbits of different allotypes. J. Immunol. 101, 500–504.PubMedGoogle Scholar
  2. Bradford, M.M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248–254.CrossRefPubMedGoogle Scholar
  3. Brown, J.P., Klitzman, J.M. and Hellstrom, I. (1978), Radioimmunoassay of murine leukemia virus p30 using Staphylococcus aureus as immunoadsorbent. J. Immunol. Meth. 21, 23–33,CrossRefGoogle Scholar
  4. Choo, K.H., Myer, J., Cotton, R.G.H., Camakaris, J. and Danks, D. M. (1981). Isolation of a phenylalanine hydroxylase-stimulating monoclonal antibody by rat-myeloma--rat-spleen-cell fusion. Biochem. J. 191, 665–668.CrossRefGoogle Scholar
  5. Chuang, D.-M. and Costa, E. (1974). Biosynthesis of tyrosine hydroxylase in rat adrenal medulla after exposure to cold. Proc. Natl. Acad. Sci. USA 71, 4570–4574.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cinader, B. (1977). Meth. Immunol. Immunochem. 4, 313–375.Google Scholar
  7. Duhamel, R.C., Meezan, E. and Brendel, K. (1980). The pH-dependent binding of goat IgG1 and IgG2 to protein A-Sepharose. Molec. Immunol. 17, 29–36.CrossRefGoogle Scholar
  8. Erlichman, J., Sarkar, D., Fleischer, N. and Rubin, C.S. (1980). Identification of two subclasses of type II cAMP-dependent protein kinases. J. Biol. Chem. 255, 8179–8184.PubMedGoogle Scholar
  9. Ey, P.L., Prowse, S.J. and Jenkin, C.R. (1978). Isolation of pure IgGl, IgG2a, IgG2b immunoglobulins from mouse serum usin protein A-Sepharose. Immunochem. 15, 429–436.Google Scholar
  10. Joh, T.H., Geghman, C. and Reis, D. (1973). Immunochemical demonstration of increased accumulation of tyrosine hydroxylase protein in sympathetic ganglia and adrenal medulla elicited by reserpine. Proc. Natl. Acad. Sci. USA 70, 2767–2771.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Kelly,. P.T. and Luttges, M.W. (1975). Electrophoretic separation of nervous system proteins on exponential gradient polyacrylamide gels. J. Neurochem. 24, 1077–1079.Google Scholar
  12. Kessler, S.W. (1975). Rapid isolation of antigens from cells with a Staphylococcal protein A-antibody adsorbent. J. Immunol. 115, 1617–1624.PubMedGoogle Scholar
  13. Kessler, S.W. (1976). Cell membrane antigen isolation with the Staphylococcal protein A-antibody adsorbent. J. Immunol. 117, 1482–1490.PubMedGoogle Scholar
  14. Lloyd, T. and Kaufman, S. (1973). Production of antibodies to bovine adrenal tyrosine hydroxylase. Molec. Pharmacol. 9, 438–444.Google Scholar
  15. Markey, K.A., Kondo, S., Shenkman, L. and Goldstein, M. (1980). Purification and characterization of tyrosine hydroxylase from a clonal pheochromocytoma cell line. Molec. Pharmacol. 17, 79–85.Google Scholar
  16. Natali, P.G., Pellegrino, M.A., Walker, L., Ferrone, S. and Reisfeld, R.A. (1979). Antibody-coated protein A-bearing Staphylococcus aureus: A versitile and stable immune reagent. J. Immunol. Meth. 25, 255–264.CrossRefGoogle Scholar
  17. Okada, Y., Ikenaka, T., Yagura, T. and Yamamura, Y. (1963). Immunological heterogeneity of rabbit antibody fragments against Taka-amylase A. J. Biochem. (Tokyo) 54, 101–102.Google Scholar
  18. O’Keefe, E. and Bennett, V. (1980). Use of immunoglobulin-loaded protein A-bearing Staphylococci as a primary solid phase immunoadsorbent in radioimmunoassay. J. Biol. Chem. 255, 561–568.PubMedGoogle Scholar
  19. Park, D.H. and Goldstein, M. (1975). Purification of tyrosine hydroxylase from pheochromocytoma tumors. Life Sci. 18, 55–60.CrossRefGoogle Scholar
  20. Pickel, V.M., Joh, T.H., Field, P.M., Becker, C.G. and Reis, D.J. (1975a). Cellular localization of tyrosine hydroxylase by immunocytochemistry. J. Histochem. Cytochem. 23, 1–12.CrossRefPubMedGoogle Scholar
  21. Pickel, V.M., Joh, T.H. and Reis, D.J. (1975b). Ultrastructural localization of tyrosine hydroxylase in noradrenergic neurons of brain. Proc. Natl. Acad. Sci. USA 72, 659–663.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Pollock, M.R. (1964). Stimulating and inhibiting antibodies for bacterial penicillinase. Immunol. 7, 707–723.Google Scholar
  23. Richmond, M.H. (1963). Purification andproperties of the exopenicillinase from Staphylococcus aureus. Biochem. J. 88, 452–459.Google Scholar
  24. Ross, M.E., Joh, T.H. and Reis, D.J. (1981) Monoclonal antibodies to tyrosine hydroxylase: Production and characterization. Brain Res. 207, in press.Google Scholar
  25. Spector, T. (1978). Refinement of the Coomassie blue method of protein quantitation. Analyt. Biochem. 86, 142–146.CrossRefPubMedGoogle Scholar
  26. Spiegelberg, H.L. (1964). Biological activities of immunoglobulins of different classes and subclasses. Adv. Immunol. 19, 259–294.CrossRefGoogle Scholar
  27. Stephens, J.K., Masserano, J.M., Vulliet, P.R., Weiner, N. and Nakame, P.K. (1981). Immunocytochemical localization of tyrosine hydroxylase in rat adrenal medulla by the peroxidase labelled antibody method. Brain Res. 207, in press.Google Scholar
  28. Suzuki, T., Pelichova, H. and Cinader, B. (1969). Enzyme activation by antibody. J. Immunol. 103, 1366–1376.PubMedGoogle Scholar
  29. Vulliet, P.R., Langan, T.A. and Weiner, N. (1980). Tyrosine hydroxylase: A substrate of cyclic AMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 77, 92–96.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Waymire, J.C., Bjur, R. and Weiner, N. (1971). Assay of tyrosine hydroxylase by coupled decarboxylation of DOPA formed from l–14C-L-tyrosine. Analyt. Biochem. 43, 588–600.CrossRefPubMedGoogle Scholar
  31. Waymire, J.C., Haycock, J.W., Meligeni, J.A. and Browning, M.D. (1979). Activation and phosphorylation of tyrosine hydroxylase by cAMP. In Catecholamines: Basic and Clinical Frontiers, (eds. E. Usdin, I.J. Kopin and J. Barchas), pp. 40–42, Pergamon Press, New York.CrossRefGoogle Scholar

Copyright information

© The Contributors 1981

Authors and Affiliations

  • John W Haycock
    • 1
  • Jack C Waymire
    • 1
  1. 1.Department of Neurobiology & AnatomyUniversity of Texas Medical School POB 20708HoustonUSA

Personalised recommendations