Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1975 Sep 1;66(3):609–620. doi: 10.1083/jcb.66.3.609

Colchicine-binding protein of the liver. Its characterization and relation to microtubules

PMCID: PMC2109447  PMID: 808552

Abstract

Colchicine-binding activity of mouse liver high-speed supernate has been investigated. It has been found to be time and temperature dependent. Two binding activities with different affinities for colchicine seem to be present in this high-speed supernate, of which only the high-affinity binding site (half maximal binding at 5 x 10(-6) M colchicine) can be attributed to microtubular protein by comparison with purified tubulin. Vinblastine interacted with this binding activity by precipitating it when used at high concentrations (2 x 10(- 3) M), and by stabilizing it at low concentrations (10(-5) M). Lumicolchicine was found not to compete with colchicine. The colchicine-binding activity was purified from liver and compared with that of microtubular protein from brain. The specific binding activity of the resulting preparation, its electrophoretic behavior, and the electron microscope appearance of the paracrystals obtained upon its precipitation with vinblastine permitted its identification as microtubular protein (tubulin). Electrophoretic analysis of the proteins from liver supernate that were precipitated by vinblastine indicated that this drug was not specific for liver tubulin. Preincubation of liver supernate with 5 mM EGTA resulted in a time- dependent decrease of colchicine-binding activity, which was partly reversed by the addition of Ca++. However, an in vitro formation of microtubules upon lowering the Ca++ concentration could not be detected. Finally, a method was developed enabling that portion of microtubular protein which was present as free tubulin to be measured and to be compared with the total amount of this protein in the tissue. This procedure permitted demonstration of the fact that, under normal conditions, only about 40% of the tubulin of the liver was assemled as microtubules. It is suggested that, in the liver, rapid polymerization and depolymerization of microtubules occur and may be an important facet of the functional role of the microtubular system.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

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

  1. Allison A. C. The role of microfilaments and microtubules in cell movement, endocytosis and exocytosis. Ciba Found Symp. 1973;14:109–148. doi: 10.1002/9780470719978.ch6. [DOI] [PubMed] [Google Scholar]
  2. Bamburg J. R., Shooter E. M., Wilson L. Assay of microtuble protein in embryonic chick dorsal root ganglia. Neurobiology. 1973;3(3):162–173. [PubMed] [Google Scholar]
  3. Bensch K. G., Marantz R., Wisniewski H., Shelanski M. Induction in vitro of microtubular crystals by vinca alkaloids. Science. 1969 Aug 1;165(3892):495–496. doi: 10.1126/science.165.3892.495. [DOI] [PubMed] [Google Scholar]
  4. Berry R. W., Shelanski M. L. Interactions of tubulin with vinblastine and guanosine triphosphate. J Mol Biol. 1972 Oct 28;71(1):71–80. doi: 10.1016/0022-2836(72)90401-9. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  7. Kirschner M. W., Williams R. C., Weingarten M., Gerhart J. C. Microtubules from mammalian brain: some properties of their depolymerization products and a proposed mechanism of assembly and disassembly. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1159–1163. doi: 10.1073/pnas.71.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Le Marchand Y., Patzelt C., Assimacopoulos-Jeannet F., Loten E. G., Jeanrenaud B. Evidence for a role of the microtubular system in the secretion of newly synthesized albumin and other proteins by the liver. J Clin Invest. 1974 Jun;53(6):1512–1517. doi: 10.1172/JCI107701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Le Marchand Y., Singh A., Assimacopoulos-Jeannet F., Orci L., Rouiller C., Jeanrenaud B. A role for the microtubular system in the release of very low density lipoproteins by perfused mouse livers. J Biol Chem. 1973 Oct 10;248(19):6862–6870. [PubMed] [Google Scholar]
  11. Lee J. C., Frigon R. P., Timasheff S. N. The chemical characterization of calf brain microtubule protein subunits. J Biol Chem. 1973 Oct 25;248(20):7253–7262. [PubMed] [Google Scholar]
  12. Marantz R., Shelanski M. L. Structure of microtubular crystals induced by vinblastine in vitro. J Cell Biol. 1970 Jan;44(1):234–238. doi: 10.1083/jcb.44.1.234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Murphy D. B., Tilney L. G. The role of microtubules in the movement of pigment granules in teleost melanophores. J Cell Biol. 1974 Jun;61(3):757–779. doi: 10.1083/jcb.61.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Olmsted J. B., Borisy G. G. Characterization of microtubule assembly in porcine brain extracts by viscometry. Biochemistry. 1973 Oct 9;12(21):4282–4289. doi: 10.1021/bi00745a037. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Orci L., Le Marchand Y., Singh A., Assimacopoulos-Jeannet F., Rouiller C., Jeanrenaud B. Letter: Role of microtubules in lipoprotein secretion by the liver. Nature. 1973 Jul 6;244(5410):30–32. doi: 10.1038/244030a0. [DOI] [PubMed] [Google Scholar]
  17. Porter K. R. Microtubules in intracellular locomotion. Ciba Found Symp. 1973;14:149–169. doi: 10.1002/9780470719978.ch7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Stadler J., Franke W. W. Characterization of the colchicine binding of membrane fractions from rat and mouse liver. J Cell Biol. 1974 Jan;60(1):297–303. doi: 10.1083/jcb.60.1.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stein O., Sanger L., Stein Y. Colchicine-induced inhibition of lipoprotein and protein secretion into the serum and lack of interference with secretion of biliary phospholipids and cholesterol by rat liver in vivo. J Cell Biol. 1974 Jul;62(1):90–103. doi: 10.1083/jcb.62.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stein O., Stein Y. Colchicine-induced inhibition of very low density lipoprotein release by rat liver in vivo. Biochim Biophys Acta. 1973 Apr 13;306(1):142–147. doi: 10.1016/0005-2760(73)90219-1. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Weisenberg R. C. Changes in the organization of tubulin during meiosis in the eggs of the surf clam, Spisula solidissima. J Cell Biol. 1972 Aug;54(2):266–278. doi: 10.1083/jcb.54.2.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Weisenberg R. C. Microtubule formation in vitro in solutions containing low calcium concentrations. Science. 1972 Sep 22;177(4054):1104–1105. doi: 10.1126/science.177.4054.1104. [DOI] [PubMed] [Google Scholar]
  25. Weisenberg R. C., Timasheff S. N. Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry. 1970 Oct 13;9(21):4110–4116. doi: 10.1021/bi00823a012. [DOI] [PubMed] [Google Scholar]
  26. Wilson L., Friedkin M. The biochemical events of mitosis. I. Synthesis and properties of colchicine labeled with tritium in its acetyl moiety. Biochemistry. 1966 Jul;5(7):2463–2468. doi: 10.1021/bi00871a042. [DOI] [PubMed] [Google Scholar]
  27. 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]

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

RESOURCES