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. 1977 May;74(5):1860–1864. doi: 10.1073/pnas.74.5.1860

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

W Herzog, K Weber
PMCID: PMC431031  PMID: 266708

Abstract

Microtubule protein from porcine cerebrum was fractionate into pure tubulin and microtubule-associated proteins by chromatography on phosphocellulose. In agreement with previous studies, pure tubulin does not form microtubules to a significant extent at 37 degrees in normal assembly buffers, which are characterized by a low concentration of Mg2+ ions. If, however, the Mg2+ concentration is raised to approximately 10 mM, rapid and extensive self-assembly of pure tubulin into microtubules is observed, provided the tubulin concentration is above 2.5 mg/ml. At a protein concentration of 3 mg/ml, the lag period is 1.5 min and the assembly process is virtually complete after 6 min at 37 degrees. These microtubules are like normal microtubules--sensitive to calcium ions, colchicine, and low temperature.

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Selected References

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

  1. Bloodgood R. A., Rosenbaum J. L. Initiation of brain tubulin assembly by a high molecular weight flagellar protein factor. J Cell Biol. 1976 Oct;71(1):322–331. doi: 10.1083/jcb.71.1.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bryan J. B., Nagle B. W., Doenges K. H. Inhibition of tubulin assembly by RNA and other polyanions: evidence for a required protein. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3570–3574. doi: 10.1073/pnas.72.9.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dentler W. L., Granett S., Rosenbaum J. L. Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. J Cell Biol. 1975 Apr;65(1):237–241. doi: 10.1083/jcb.65.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Erickson H. P. Assembly of microtubules from preformed, ring-shaped protofilaments and 6-S tubulin. J Supramol Struct. 1974;2(2-4):393–411. doi: 10.1002/jss.400020228. [DOI] [PubMed] [Google Scholar]
  5. Erickson H. P., Voter W. A. Polycation-induced assembly of purified tubulin. Proc Natl Acad Sci U S A. 1976 Aug;73(8):2813–2817. doi: 10.1073/pnas.73.8.2813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frigon R. P., Timasheff S. N. Magnesium-induced self-association of calf brain tubulin. II. Thermodynamics. Biochemistry. 1975 Oct 21;14(21):4567–4573. doi: 10.1021/bi00692a002. [DOI] [PubMed] [Google Scholar]
  7. Gaskin F., Cantor C. R., Shelanski M. L. Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules. J Mol Biol. 1974 Nov 15;89(4):737–755. doi: 10.1016/0022-2836(74)90048-5. [DOI] [PubMed] [Google Scholar]
  8. Gaskin F., Kramer S. B., Cantor C. R., Adelstein R., Shelanski M. L. A dynein-like protein associated with neurotubules. FEBS Lett. 1974 Apr 1;40(2):281–286. doi: 10.1016/0014-5793(74)80244-9. [DOI] [PubMed] [Google Scholar]
  9. Gillespie E. The mechanism of breakdown of tubulin in vitro. FEBS Lett. 1975 Oct 15;58(1):119–121. doi: 10.1016/0014-5793(75)80238-9. [DOI] [PubMed] [Google Scholar]
  10. Kirschner M. W., Williams R. C. The mechanism of microtubule assembly in vitro. J Supramol Struct. 1974;2(2-4):412–428. doi: 10.1002/jss.400020229. [DOI] [PubMed] [Google Scholar]
  11. Lee J. C., Timasheff S. N. The reconstitution of microtubules from purified calf brain tubulin. Biochemistry. 1975 Nov 18;14(23):5183–5187. doi: 10.1021/bi00694a025. [DOI] [PubMed] [Google Scholar]
  12. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Olmsted J. B., Marcum J. M., Johnson K. A., Allen C., Borisy G. G. Microtuble assembly: some possible regulatory mechanisms. J Supramol Struct. 1974;2(2-4):429–450. doi: 10.1002/jss.400020230. [DOI] [PubMed] [Google Scholar]
  15. Osborn M., Weber K. Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane. Proc Natl Acad Sci U S A. 1976 Mar;73(3):867–871. doi: 10.1073/pnas.73.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Osborn M., Weber K. Tubulin-specific antibody and the expression of microtubules in 3T3 cells after attachment to a substratum. Further evidence for the polar growth of cytoplasmic microtubules in vivo. Exp Cell Res. 1976 Dec;103(2):331–340. doi: 10.1016/0014-4827(76)90270-6. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Sloboda R. D., Dentler W. L., Rosenbaum J. L. Microtubule-associated proteins and the stimulation of tubulin assembly in vitro. Biochemistry. 1976 Oct 5;15(20):4497–4505. doi: 10.1021/bi00665a026. [DOI] [PubMed] [Google Scholar]
  19. Snyder J. A., McIntosh J. R. Biochemistry and physiology of microtubules. Annu Rev Biochem. 1976;45:699–720. doi: 10.1146/annurev.bi.45.070176.003411. [DOI] [PubMed] [Google Scholar]
  20. Weber K., Wehland J., Herzog W. Griseofulvin interacts with microtubules both in vivo and in vitro. J Mol Biol. 1976 Apr 25;102(4):817–829. doi: 10.1016/0022-2836(76)90293-x. [DOI] [PubMed] [Google Scholar]
  21. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Weisenberg R. C., Deery W. J. Role of nucleotide hydrolysis in microtubule assembly. Nature. 1976 Oct 28;263(5580):792–793. doi: 10.1038/263792a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Witman G. B., Cleveland D. W., Weingarten M. D., Kirschner M. W. Tubulin requires tau for growth onto microtubule initiating sites. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4070–4074. doi: 10.1073/pnas.73.11.4070. [DOI] [PMC free article] [PubMed] [Google Scholar]

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