Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

Abstract

Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.

A Specific Domain of Giα Required for the Transactivation of Giα by Tubulin Is Implicated in the Organization of Cellular Microtubules

“…Tubulin is a GTP-binding protein, two GTP molecules are bound noncovalently in exchangeable (E-site on ␤-tubulin) and nonexchangeable (N-site on ␣-tubulin) sites. Both G␣ subunits and tubulin have intrinsic GTPase activity but that of tubulin is activated during the process of polymerization (10) or when complexed with G␣ (11).…”

Molecular Recognition of Taxol by Microtubules

“…Unassembled tubulin shows insignificant affinity for Taxol (12)(13)(14), indicating that the binding site is mostly formed by the polymerization process. The interaction of microtubules with Taxol has been widely studied (10,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). However, since Taxol binding and microtubule assembly are linked reactions and the binding affinity seems to be high (12,14), direct measurements of the binding thermodynamics are extremely difficult.…”

Paclitaxel as an anticancer agent: isolation, activity, synthesis and stability

“…Paclitaxel is a complex diterpene with antitumor activity against ovarian, breast, lung, and prostate cancer [14,48], and acts as a promoter of tubulin polymerization and stabilizes microtubules to depolymerization by different agents, both in vitro and in vivo [49,50]. Paclitaxel alters the normal equilibrium between tubulin dimmers and microtubules, and, therefore, disrupts cell division [51].…”

“…The proposed mechanism of action, metabolism, relationship between structure and activity, such as pharmacokinetics of paclitaxel, have been explained in the literature [14,49,[52][53][54][55][56][57][58]. Analogs of paclitaxel having good biological activity have been synthesized [14,48,51,52,57,59,60].…”