Assembly of purified GDP-tubulin into microtubules induced by taxol and taxotere: reversibility, ligand stoichiometry, and competition

Biochemistry. 1993 Mar 23;32(11):2747-55. doi: 10.1021/bi00062a003.


Purified tubulin fully liganded to GDP at the exchangeable nucleotide binding site has been prepared by a new direct nucleotide exchange procedure. This normally inactive GDP-tubulin is driven to assemble into microtubules by the binding of the antitumor drug taxol or its more soluble side-chain analogue Taxotere in Mg(2+)-containing buffer, and it disassembles by cooling the solution. Therefore this ligand-induced equilibrium microtubule assembly system dispenses with the requirement of a gamma-phosphate-metal cation ligand bound at the nucleotide site for tubulin to be active. GDP-tubulin can also form characteristic pseudo-ordered aggregates of double rings. These aggregates dissociate upon warming or by addition of GTP. Back-substitution of the nucleotide gamma-phosphate permits glycerol-induced assembly without taxol and reduces the critical protein concentration required for drug-induced microtubule assembly by a factor of 2.6 +/- 0.1. The ligand-induced assembly is maximal at taxol or Taxotere concentrations equimolar with tubulin, and both drugs bind to assembled tubulin with a stoichiometry of 0.99 +/- 0.04 ligand per alpha beta dimer. Taxotere apparently competes with taxol for the same binding site, with 1.9 +/- 0.1 times larger effective affinity. Similarly, the Taxotere-induced assembly of GDP-tubulin or GTP-tubulin proceeds with a critical protein concentration 2.1 +/- 0.1 times smaller than with taxol.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents, Phytogenic / pharmacology*
  • Binding, Competitive
  • Brain / metabolism*
  • Cattle
  • Docetaxel
  • Guanosine Diphosphate / metabolism*
  • Guanosine Triphosphate / metabolism*
  • Kinetics
  • Ligands
  • Mathematics
  • Microscopy, Electron
  • Microtubules / drug effects
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Paclitaxel / analogs & derivatives*
  • Paclitaxel / metabolism
  • Paclitaxel / pharmacology*
  • Taxoids*
  • Tubulin / isolation & purification
  • Tubulin / metabolism*


  • Antineoplastic Agents, Phytogenic
  • Ligands
  • Taxoids
  • Tubulin
  • Guanosine Diphosphate
  • Docetaxel
  • Guanosine Triphosphate
  • Paclitaxel