Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1976 Jun 1;69(3):599–607. doi: 10.1083/jcb.69.3.599

Properties of tubulin in unfertilized sea urchin eggs. Quantitation and characterization by the colchicine-binding reaction

PMCID: PMC2109704  PMID: 944700

Abstract

The colchicine-binding assay was used to quantitate the tubulin concentration in unfertilized Strongylocentrotus purpuratus eggs and to characterize pharmacological properties of this tubulin. Specificity of colchicine binding to tubulin was demonstrated by apparent first-order decay colchicine-binding activity with stabilization by vinblastine sulfate, time and temperature dependence of the reaction, competitive inhibition by podophyllotoxin, and lack of effect of lumicolchicine. The results demonstrate that the minimum tubulin concentration in the unfertilized egg is 2.71 mg per milliliter or 5.0% of the total soluble cell protein. Binding constants and decay rates were determined at six different temperatures between 8 degrees C and 37 degrees C, and the thermodynamic parameters of the reaction were calculated. delta H0=6.6 kcal/mol, delta S0=46.5 eu, and, at 13 degrees C, delta G=-6.7 kcal/mol. The association constants obtained were similar to those of isolated sea urchin egg vinblastine paracrystals (Bryan, J. 1972. Biochemistry. 11:2611-2616) but approximately 10 times lower than that obtained for purified chick embryo brain tubulin at 37 degrees C (Wilson, L.J.R. Bamburg, S.B. Mizel, L. Grisham, and K. Creswell. 1974. Fed Proc. 33:158-166). Therefore, the lower binding constants for colchicine in tubulin-vinblastine paracrystals are not due to the paracrystalline organization of the tubulin, but are properties of the sea urchin egg tubulin itself.

Full Text

The Full Text of this article is available as a PDF (580.5 KB).

Selected References

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

  1. Anderson L. E., McClure W. O. An improved scintillation cocktail of high-solubilizing power. Anal Biochem. 1973 Jan;51(1):173–179. doi: 10.1016/0003-2697(73)90465-x. [DOI] [PubMed] [Google Scholar]
  2. Bamburg J. R., Shooter E. M., Wilson L. Developmental changes in microtubule protein of chick brain. Biochemistry. 1973 Apr 10;12(8):1476–1482. doi: 10.1021/bi00732a002. [DOI] [PubMed] [Google Scholar]
  3. Behnke O., Forer A. Evidence for four classes of microtubules in individual cells. J Cell Sci. 1967 Jun;2(2):169–192. doi: 10.1242/jcs.2.2.169. [DOI] [PubMed] [Google Scholar]
  4. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. J Cell Biol. 1967 Aug;34(2):525–533. doi: 10.1083/jcb.34.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. J Cell Biol. 1967 Aug;34(2):535–548. doi: 10.1083/jcb.34.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bryan J. Definition of three classes of binding sites in isolated microtubule crystals. Biochemistry. 1972 Jul 4;11(14):2611–2616. doi: 10.1021/bi00764a010. [DOI] [PubMed] [Google Scholar]
  7. Bryan J. Vinblastine and microtubules. I. Induction and isolation of crystals from sea urchin oocytes. Exp Cell Res. 1971 May;66(1):129–136. doi: 10.1016/s0014-4827(71)80020-4. [DOI] [PubMed] [Google Scholar]
  8. Burnside B., Kozak C., Kafatos F. C. Tubulin determination by an isotope dilution-vinblastine precipitation method. The tubulin content of Spisula eggs and embryos. J Cell Biol. 1973 Dec;59(3):755–762. doi: 10.1083/jcb.59.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garland D., Teller D. C. A reexamination of the reaction between colchicine and tubulin. Ann N Y Acad Sci. 1975 Jun 30;253:232–238. doi: 10.1111/j.1749-6632.1975.tb19202.x. [DOI] [PubMed] [Google Scholar]
  10. Haber J. E., Peloquin J. G., Halvorson H. O., Borisy G. G. Colcemid inhibition of cell growth and the characterization of a colcemid-binding activity in Saccharomyces cerevisiae. J Cell Biol. 1972 Nov;55(2):355–367. doi: 10.1083/jcb.55.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hynes R. O., Raff R. A., Gross P. R. Properties of the three cell types in sixteen-cell sea urchin embryos: aggregation and microtuble protein synthesis. Dev Biol. 1972 Feb;27(2):150–164. doi: 10.1016/0012-1606(72)90094-2. [DOI] [PubMed] [Google Scholar]
  12. Inoué S., Sato H. Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement. J Gen Physiol. 1967 Jul;50(6 Suppl):259–292. [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Meza I., Huang B., Bryan J. Chemical heterogeneity of protofilaments forming the outer doublets from sea urchin flagella. Exp Cell Res. 1972 Oct;74(2):535–540. doi: 10.1016/0014-4827(72)90413-2. [DOI] [PubMed] [Google Scholar]
  15. Mizel S. B., Wilson L. Nucleoside transport in mammalian cells. Inhibition by colchicine. Biochemistry. 1972 Jul 4;11(14):2573–2578. doi: 10.1021/bi00764a003. [DOI] [PubMed] [Google Scholar]
  16. Olmsted J. B., Borisy G. G. Microtubules. Annu Rev Biochem. 1973;42:507–540. doi: 10.1146/annurev.bi.42.070173.002451. [DOI] [PubMed] [Google Scholar]
  17. Owellen R. J., Owens A. H., Jr, Donigian D. W. The binding of vincristine, vinblastine and colchicine to tubulin. Biochem Biophys Res Commun. 1972 May 26;47(4):685–691. doi: 10.1016/0006-291x(72)90546-3. [DOI] [PubMed] [Google Scholar]
  18. Raff R. A., Colot H. V., Selvig S. E., Gross P. R. Oogenetic origin of messenger RNA for embryonic synthesis of microtubule proteins. Nature. 1972 Jan 28;235(5335):211–214. doi: 10.1038/235211a0. [DOI] [PubMed] [Google Scholar]
  19. Raff R. A., Greenhouse G., Gross K. W., Gross P. R. Synthesis and storage of microtubule proteins by sea urchin embryos. J Cell Biol. 1971 Aug;50(2):516–527. doi: 10.1083/jcb.50.2.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Raff R. A., Kaumeyer J. F. Soluble microtubule proteins of the sea urchin embryo: partial characterization of the proteins and behavior of the pool in early development. Dev Biol. 1973 Jun;32(2):309–320. doi: 10.1016/0012-1606(73)90243-1. [DOI] [PubMed] [Google Scholar]
  21. Shelanski M. L., Taylor E. W. Isolation of a protein subunit from microtubules. J Cell Biol. 1967 Aug;34(2):549–554. doi: 10.1083/jcb.34.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sherline P., Leung J. T., Kipnis D. M. Binding of colchicine to purified microtubule protein. J Biol Chem. 1975 Jul 25;250(14):5481–5486. [PubMed] [Google Scholar]
  23. Stephens R. E. Thermal fractionation of outer fiber doublet microtubules into A- and B-subfiber components. A- and B-tubulin. J Mol Biol. 1970 Feb 14;47(3):353–363. doi: 10.1016/0022-2836(70)90307-4. [DOI] [PubMed] [Google Scholar]
  24. Tilney L. G., Gibbins J. R. Differential effects of antimitotic agents on the stability and behavior of cytoplasmic and ciliary microtubules. Protoplasma. 1968;65(1):167–179. doi: 10.1007/BF01666377. [DOI] [PubMed] [Google Scholar]
  25. Ventilla M., Cantor C. R., Shelanski M. A circular dichroism study of microtubule protein. Biochemistry. 1972 Apr 25;11(9):1554–1561. [PubMed] [Google Scholar]
  26. Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]
  27. Wilson L., Bamburg J. R., Mizel S. B., Grisham L. M., Creswell K. M. Interaction of drugs with microtubule proteins. Fed Proc. 1974 Feb;33(2):158–166. [PubMed] [Google Scholar]
  28. Wilson L., Bryan J., Ruby A., Mazia D. Precipitation of proteins by vinblastine and calcium ions. Proc Natl Acad Sci U S A. 1970 Jul;66(3):807–814. doi: 10.1073/pnas.66.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wilson L., Friedkin M. The biochemical events of mitosis. II. The in vivo and in vitro binding of colchicine in grasshopper embryos and its possible relation to inhibition of mitosis. Biochemistry. 1967 Oct;6(10):3126–3135. doi: 10.1021/bi00862a021. [DOI] [PubMed] [Google Scholar]
  30. Wilson L., Meza I. The mechanism of action of colchicine. Colchicine binding properties of sea urchin sperm tail outer doublet tubulin. J Cell Biol. 1973 Sep;58(3):709–719. doi: 10.1083/jcb.58.3.709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wilson L. Microtubules as drug receptors: pharmacological properties of microtubule protein. Ann N Y Acad Sci. 1975 Jun 30;253:213–231. doi: 10.1111/j.1749-6632.1975.tb19201.x. [DOI] [PubMed] [Google Scholar]
  32. Wilson L. Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyllotoxin. Biochemistry. 1970 Dec 8;9(25):4999–5007. doi: 10.1021/bi00827a026. [DOI] [PubMed] [Google Scholar]
  33. Wolff J., Bhattacharyya B. Microtubules and thyroid hormone mobilization. Ann N Y Acad Sci. 1975 Jun 30;253:763–770. doi: 10.1111/j.1749-6632.1975.tb19244.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES