Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Jan 1;100(1):276–281. doi: 10.1083/jcb.100.1.276

Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods

PMCID: PMC2113468  PMID: 3965474

Abstract

Tubulin-tyrosine ligase (TTL), the enzyme responsible for the reversible addition of a tyrosine residue at the carboxyl end of alpha- tubulin, has been purified from porcine brain using a purification scheme based on standard biochemical procedures. The enzyme preparation was nearly homogeneous (purity greater than 95%), was free of tubulin, and could be stored in the presence of glycerol for several months without loss in activity. To develop a more convenient purification of TTL, we have isolated mouse hybridoma cells secreting antibodies to TTL. These monoclonal antibodies recognize TTL not only in brain tissue but also in the liver of various mammals. Monoclonal antibodies isolated from ascites fluid allowed a rapid purification of TTL from a crude brain extract. TTL stayed bound to the immunoaffinity column in 1.5 M NaCl and was eluted with 3 M MgCl2. Highly active TTL was recovered nearly quantitatively at greater than 95% purity and could be stabilized in the presence of glycerol. Glycerol gradient centrifugation, SDS gel electrophoresis and immunoblots identified TTL as a monomeric protein with an apparent polypeptide molecular weight of about 40,000. A one to one complex of TTL with alpha beta-tubulin was observed by gradient centrifugation.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arce C. A., Rodriguez J. A., Barra H. S., Caputo R. Incorporation of L-tyrosine, L-phenylalanine and L-3,4-dihydroxyphenylalanine as single units into rat brain tubulin. Eur J Biochem. 1975 Nov 1;59(1):145–149. doi: 10.1111/j.1432-1033.1975.tb02435.x. [DOI] [PubMed] [Google Scholar]
  2. Argarana C. E., Barra H. S., Caputto R. Tubulinyl-tyrosine carboxypeptidase from chicken brain: properties and partial purification. J Neurochem. 1980 Jan;34(1):114–118. doi: 10.1111/j.1471-4159.1980.tb04628.x. [DOI] [PubMed] [Google Scholar]
  3. Debus E., Weber K., Osborn M. Monoclonal antibodies to desmin, the muscle-specific intermediate filament protein. EMBO J. 1983;2(12):2305–2312. doi: 10.1002/j.1460-2075.1983.tb01738.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Flavin M., Kobayashi T., Martensen T. M. Tubulin-tyrosine ligase from brain. Methods Cell Biol. 1982;24:257–263. doi: 10.1016/s0091-679x(08)60659-7. [DOI] [PubMed] [Google Scholar]
  5. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  6. Herzog W., Weber K. In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol. Proc Natl Acad Sci U S A. 1977 May;74(5):1860–1864. doi: 10.1073/pnas.74.5.1860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kumar N., Flavin M. Modulation of some parameters of assembly of microtubules in vitro by tyrosinolation of tubulin. Eur J Biochem. 1982 Nov;128(1):215–222. doi: 10.1111/j.1432-1033.1982.tb06954.x. [DOI] [PubMed] [Google Scholar]
  9. Kumar N., Flavin M. Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin. J Biol Chem. 1981 Jul 25;256(14):7678–7686. [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lamed R., Oplatka A. Applications of immobilized adenosine triphosphate in the study of myosin. Biochemistry. 1974 Jul 16;13(15):3137–3142. doi: 10.1021/bi00712a021. [DOI] [PubMed] [Google Scholar]
  13. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  14. Martensen T. M. Preparation of brain tyrosinotubulin carboxypeptidase. Methods Cell Biol. 1982;24:265–269. doi: 10.1016/s0091-679x(08)60660-3. [DOI] [PubMed] [Google Scholar]
  15. Matsumoto G., Kobayashi T., Sakai H. Restoration of the excitability of squid giant axon by tubulin-tyrosine ligase and microtubule proteins. J Biochem. 1979 Oct;86(4):1155–1158. doi: 10.1093/oxfordjournals.jbchem.a132611. [DOI] [PubMed] [Google Scholar]
  16. Murofushi H. Purification and characterization of tubulin-tyrosine ligase from porcine brain. J Biochem. 1980 Mar;87(3):979–984. doi: 10.1093/oxfordjournals.jbchem.a132828. [DOI] [PubMed] [Google Scholar]
  17. Nath J., Flavin M. An apparent paradox in the occurrence, and the in vivo turnover, of C-terminal tyrosine in membrane-bound tubulin of brain. J Neurochem. 1980 Sep;35(3):693–706. doi: 10.1111/j.1471-4159.1980.tb03708.x. [DOI] [PubMed] [Google Scholar]
  18. Preston S. F., Deanin G. G., Hanson R. K., Gordon M. W. The phylogenetic distribution of tubulin:tyrosine ligase. J Mol Evol. 1979 Oct;13(3):233–244. doi: 10.1007/BF01739482. [DOI] [PubMed] [Google Scholar]
  19. Raybin D., Flavin M. An enzyme tyrosylating alpha-tubulin and its role in microtubule assembly. Biochem Biophys Res Commun. 1975 Aug 4;65(3):1088–1095. doi: 10.1016/s0006-291x(75)80497-9. [DOI] [PubMed] [Google Scholar]
  20. Raybin D., Flavin M. Enzyme which specifically adds tyrosine to the alpha chain of tubulin. Biochemistry. 1977 May 17;16(10):2189–2194. doi: 10.1021/bi00629a023. [DOI] [PubMed] [Google Scholar]
  21. Rodriguez J. A., Barra H. S., Arce C. A., Hallak M. E., Caputto R. The reciprocal exclusion by L-dopa (L-3,4-dihydroxyphenylalanine) and L-tyrosine of their incorporation as single units into a soluble rat brain protein. Biochem J. 1975 Jul;149(1):115–121. doi: 10.1042/bj1490115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thompson W. C., Deanin G. G., Gordon M. W. Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1318–1322. doi: 10.1073/pnas.76.3.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Thompson W. C. The cyclic tyrosination/detyrosination of alpha tubulin. Methods Cell Biol. 1982;24:235–255. doi: 10.1016/s0091-679x(08)60658-5. [DOI] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Valenzuela P., Quiroga M., Zaldivar J., Rutter W. J., Kirschner M. W., Cleveland D. W. Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs. Nature. 1981 Feb 19;289(5799):650–655. doi: 10.1038/289650a0. [DOI] [PubMed] [Google Scholar]
  27. Wehland J., Schröder H. C., Weber K. Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2. EMBO J. 1984 Jun;3(6):1295–1300. doi: 10.1002/j.1460-2075.1984.tb01965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wehland J., Willingham M. C., Sandoval I. V. A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo. J Cell Biol. 1983 Nov;97(5 Pt 1):1467–1475. doi: 10.1083/jcb.97.5.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES