HOW TAXOL MODULATES MICROTUBULE DISASSEMBLY

Measurement of the affinity of microtubules for the anti-cancer drug t axol is problematic, because microtubules are not stable at the very l ow concentrations required to detect taxol dissociation. We have circu mvented this problem by using the GTP analogue GMPCPP (guanylyl alpha, beta-methylenediphosphonate), which renders microtubules sufficiently stable to allow binding studies with nonsaturating concentrations of t axol. A K-d value equal to about 10 nM was estimated from the effect o f taxol concentration on the dilution-induced disassembly rate and on the binding of [H-3]taxol. With GTP-microtubules the K-d value for tax ol binding by tubulin-GDP subunits in the core of the microtubule appe ars to be comparable with that of GMPCPP-microtubules. However, the st abilizing effect of the drug bound to tubulin subunits that arrive at ends of disassembling microtubules is attenuated by a two-step reactio n sequence in which taxol dissociates (k = 30 s(-1)), followed by rapi d (k = 1000 s(-1)) loss of the taxol-free tubulin subunit. This sequen tial reaction can be disrupted by high (micromolar) concentrations of taxol, which react rapidly with tubulin subunits at the ends of microt ubules (k = 2 x 10(9) M(-1) s(-1)). The inhibitory effect of taxol on microtubule disassembly at concentrations a thousand-fold greater than the K-d value suggests the desirability of using high taxol concentra tions in chemotherapy with this compound.