Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Apr 1;323(Pt 1):189–196. doi: 10.1042/bj3230189

Response of microtubules to the addition of colchicine and tubulin-colchicine: evaluation of models for the interaction of drugs with microtubules.

A Vandecandelaere 1, S R Martin 1, Y Engelborghs 1
PMCID: PMC1218294  PMID: 9173881

Abstract

The effects of free drug and tubulin-drug complexes on steady-state GTP/GDP-associated microtubules and on equilibrium guanosine 5'[beta,gamma-imido]triphosphate-associated microtubules are compared. The addition of colchicine or the tubulin-colchicine complex (TuCol) to steady-state microtubules induces microtubule disassembly. Only limited disassembly of equilibrium microtubules is observed under similar conditions. Addition of colchicine or the bifunctional colchicine analogue 2-methoxy-5-(2'3',4'-trimethoxyphenyl)tropone to preassembled steady-state or equilibrium microtubules does induce disassembly, but establishment of the new steady state or equilibrium is very slow. These observations are related to the fact that TuCol readily adds to the microtubule end, but is only incorporated into the lattice with difficulty. As a result, microtubule growth is effectively inhibited and the critical concentration is significantly increased. Nevertheless, drug-induced disassembly can be extremely slow, because the frequency of addition reactions increases as the concentration of soluble dimers increases. The efficiency of incorporation of TuCol decreases as it concentration increases. The work further confirms the existence of colchicine-binding sites with low affinity (association constant KMT approximately 3 x 10(2) M-1) along the microtubule lattice. This value suggests that part of the colchicine-binding site on tubulin remains available in the polymer. The interaction of colchicine with these sites has no appreciable effect on microtubule dynamics. These observations are reproduced and rationalized by the model described elsewhere [Vandecandelaere, Martin, Bayley and Schilstra (1994) Biochemistry 33, 2792-2801], and the possibility that there are co-operative effects in the inhibition is considered.

Full Text

The Full Text of this article is available as a PDF (499.1 KB).

Selected References

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

  1. Andreu J. M., Timasheff S. N. Interaction of tubulin with single ring analogues of colchicine. Biochemistry. 1982 Feb 2;21(3):534–543. doi: 10.1021/bi00532a019. [DOI] [PubMed] [Google Scholar]
  2. Andreu J. M., Timasheff S. N. Tubulin bound to colchicine forms polymers different from microtubules. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6753–6756. doi: 10.1073/pnas.79.22.6753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andreu J. M., Wagenknecht T., Timasheff S. N. Polymerization of the tubulin-colchicine complex: relation to microtubule assembly. Biochemistry. 1983 Mar 29;22(7):1556–1566. doi: 10.1021/bi00276a006. [DOI] [PubMed] [Google Scholar]
  4. Bergen L. G., Borisy G. G. Tubulin-colchicine complex inhibits microtubule elongation at both plus and minus ends. J Biol Chem. 1983 Apr 10;258(7):4190–4194. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Deery W. J., Weisenberg R. C. Kinetic and steady-state analysis of microtubules in the presence of colchicine. Biochemistry. 1981 Apr 14;20(8):2316–2324. doi: 10.1021/bi00511a038. [DOI] [PubMed] [Google Scholar]
  7. Engelborghs Y., De Maeyer L. C., Overbergh N. A kinetic analysis of the assembly of microtubules in vitro. FEBS Lett. 1977 Aug 1;80(1):81–85. doi: 10.1016/0014-5793(77)80411-0. [DOI] [PubMed] [Google Scholar]
  8. Engelborghs Y., Dumortier C., D'Hoore A., Vandecandelaere A., Fitzgerald T. J. Evidence for an alternative pathway for colchicine binding to tubulin, based on the binding kinetics of the constituent rings. J Biol Chem. 1993 Jan 5;268(1):107–112. [PubMed] [Google Scholar]
  9. Farrell K. W., Wilson L. Tubulin-colchicine complexes differentially poison opposite microtubule ends. Biochemistry. 1984 Jul 31;23(16):3741–3748. doi: 10.1021/bi00311a027. [DOI] [PubMed] [Google Scholar]
  10. Fernando Díaz J., Andreu J. M. Kinetics of dissociation of the tubulin-colchicine complex. Complete reaction scheme and comparison to thermodynamic measurements. J Biol Chem. 1991 Feb 15;266(5):2890–2896. [PubMed] [Google Scholar]
  11. Garland D. L. Kinetics and mechanism of colchicine binding to tubulin: evidence for ligand-induced conformational change. Biochemistry. 1978 Oct 3;17(20):4266–4272. doi: 10.1021/bi00613a024. [DOI] [PubMed] [Google Scholar]
  12. Hinz H. J., Timasheff S. N. Enthalpy changes in microtubule assembly from pure tubulin. Biochemistry. 1986 Dec 16;25(25):8285–8291. doi: 10.1021/bi00373a024. [DOI] [PubMed] [Google Scholar]
  13. Hiratsuka T., Kato T. A fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid, as a probe for the colcemid-binding sites of tubulin and microtubules. J Biol Chem. 1987 May 5;262(13):6318–6322. [PubMed] [Google Scholar]
  14. Karr T. L., Podrasky A. E., Purich D. L. Participation of guanine nucleotides in nucleation and elongation steps of microtubule assembly. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5475–5479. doi: 10.1073/pnas.76.11.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keates R. A., Mason G. B. Inhibition of microtubule polymerization by the tubulin-colchicine complex: inhibition of spontaneous assembly. Can J Biochem. 1981 May;59(5):361–370. doi: 10.1139/o81-050. [DOI] [PubMed] [Google Scholar]
  16. Lambeir A., Engelborghs Y. A fluorescence stopped flow study of colchicine binding to tubulin. J Biol Chem. 1981 Apr 10;256(7):3279–3282. [PubMed] [Google Scholar]
  17. Lambeir A., Engelborghs Y. A quantitative description of microtubule formation in the presence of tubulin-colchicine. Eur J Biochem. 1983 May 2;132(2):369–373. doi: 10.1111/j.1432-1033.1983.tb07372.x. [DOI] [PubMed] [Google Scholar]
  18. Lambier A., Engelborghs Y. A quantitative analysis of tubulin-colchicine binding to microtubules. Eur J Biochem. 1980 Aug;109(2):619–624. doi: 10.1111/j.1432-1033.1980.tb04835.x. [DOI] [PubMed] [Google Scholar]
  19. Margolis R. L., Rauch C. T., Wilson L. Mechanism of colchicine-dimer addition to microtubule ends: implications for the microtubule polymerization mechanism. Biochemistry. 1980 Nov 25;19(24):5550–5557. doi: 10.1021/bi00565a014. [DOI] [PubMed] [Google Scholar]
  20. Margolis R. L., Wilson L. Addition of colchicine--tubulin complex to microtubule ends: the mechanism of substoichiometric colchicine poisoning. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3466–3470. doi: 10.1073/pnas.74.8.3466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Martin S. R., Schilstra M. J., Bayley P. M. Dynamic instability of microtubules: Monte Carlo simulation and application to different types of microtubule lattice. Biophys J. 1993 Aug;65(2):578–596. doi: 10.1016/S0006-3495(93)81091-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Palanivelu P., Ludueña R. F. Interactions of the tau-tubulin-vinblastine complex with colchicine, podophyllotoxin, and N,N'-ethylenebis(iodoacetamide). J Biol Chem. 1982 Jun 10;257(11):6311–6315. [PubMed] [Google Scholar]
  24. Panda D., Daijo J. E., Jordan M. A., Wilson L. Kinetic stabilization of microtubule dynamics at steady state in vitro by substoichiometric concentrations of tubulin-colchicine complex. Biochemistry. 1995 Aug 8;34(31):9921–9929. doi: 10.1021/bi00031a014. [DOI] [PubMed] [Google Scholar]
  25. Pantaloni D., Carlier M. F. Involvement of guanosine triphosphate (GTP) hydrolysis in the mechanism of tubulin polymerization: regulation of microtubule dynamics at steady state by a GTP cap. Ann N Y Acad Sci. 1986;466:496–509. doi: 10.1111/j.1749-6632.1986.tb38427.x. [DOI] [PubMed] [Google Scholar]
  26. Perez-Ramirez B., Andreu J. M., Gorbunoff M. J., Timasheff S. N. Stoichiometric and substoichiometric inhibition of tubulin self-assembly by colchicine analogues. Biochemistry. 1996 Mar 12;35(10):3277–3285. doi: 10.1021/bi950523x. [DOI] [PubMed] [Google Scholar]
  27. Purich D. L., MacNeal R. K. Properties of tubulin treated with alkaline phosphatase to remove guanine nucleotides from the exchangeable binding site. FEBS Lett. 1978 Dec 1;96(1):83–86. doi: 10.1016/0014-5793(78)81067-9. [DOI] [PubMed] [Google Scholar]
  28. Saltarelli D., Pantaloni D. Copolymerization of tubulin-colchicine complex and unliganded tubulin in a nonmicrotubular polymer. Biochemistry. 1983 Sep 13;22(19):4607–4614. doi: 10.1021/bi00288a040. [DOI] [PubMed] [Google Scholar]
  29. Saltarelli D., Pantaloni D. Polymerization of the tubulin-colchicine complex and guanosine 5'-triphosphate hydrolysis. Biochemistry. 1982 Jun 8;21(12):2996–3006. doi: 10.1021/bi00541a030. [DOI] [PubMed] [Google Scholar]
  30. Shearwin K. E., Timasheff S. N. Effect of colchicine analogues on the dissociation of alpha beta tubulin into subunits: the locus of colchicine binding. Biochemistry. 1994 Feb 1;33(4):894–901. doi: 10.1021/bi00170a007. [DOI] [PubMed] [Google Scholar]
  31. Skoufias D. A., Wilson L. Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes. Biochemistry. 1992 Jan 28;31(3):738–746. doi: 10.1021/bi00118a015. [DOI] [PubMed] [Google Scholar]
  32. Sternlicht H., Ringel I. Colchicine inhibition of microtubule assembly via copolymer formation. J Biol Chem. 1979 Oct 25;254(20):10540–10550. [PubMed] [Google Scholar]
  33. Sternlicht H., Ringel I., Szasz J. Theory for modeling the copolymerization of tubulin and tubulin-colchicine complex. Biophys J. 1983 Jun;42(3):255–267. doi: 10.1016/S0006-3495(83)84393-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. TAYLOR E. W. THE MECHANISM OF COLCHICINE INHIBITION OF MITOSIS. I. KINETICS OF INHIBITION AND THE BINDING OF H3-COLCHICINE. J Cell Biol. 1965 Apr;25:SUPPL–SUPPL:160. doi: 10.1083/jcb.25.1.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vandecandelaere A., Martin S. R., Schilstra M. J., Bayley P. M. Effects of the tubulin-colchicine complex on microtubule dynamic instability. Biochemistry. 1994 Mar 15;33(10):2792–2801. doi: 10.1021/bi00176a007. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Zimmermann H. P., Doenges K. H. Decoration of microtubules by a colchicine derivative. Cell Biol Int Rep. 1981 Jul;5(7):699–703. doi: 10.1016/0309-1651(81)90190-9. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES