Assembly of purified GDP-tubulin into microtubules induced by taxol and taxotere: reversibility, ligand stoichiometry, and competition.
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.
Díaz
J F
JF
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Andreu
J M
JM
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D000077143:Docetaxel; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D017239:Paclitaxel / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D043823:Taxoids; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D000972:Antineoplastic Agents, Phytogenic; D008024:Ligands; D043823:Taxoids; D014404:Tubulin; D006153:Guanosine Diphosphate; D000077143:Docetaxel; D006160:Guanosine Triphosphate; D017239:Paclitaxel