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.