Inhibition of bovine brain microtubule assembly in vitro by stypoldione

Stypolidione, an orthoquinone derived from the brown seaweed Stypopodium zonale, inhibited the polymerization of three-cycle-purified bovine brain microtubule protein in vitro in a concentration-dependent manner. Fifty per cent inhibition of the extent of polymerization beginning under initiating conditions occurred at a stypoldione concentration of approximately 25 microM, and 50% inhibition of tubulin addition to the assembly ends of microtubules at steady state occurred at a concentration of approximately 8 microM. Only slight structural abnormalities could be detected by negative stain electron microscopy in some of the microtubules that did assemble in the presence of the drug, and no aberrant structural forms of microtubule protein were detected. Stypoldione inhibited the binding of [3H]colchicine to tubulin, with 50% inhibition of colchicine binding activity occurring at a stypoldione concentration of 12-15 microM. Inhibition of colchicine binding activity appeared noncompetitive and was at least partially reversible, suggesting that stypoldione and colchicine bind at separate sites. By assuming that the inhibition constant for the ability of stypoldione to prevent the binding of colchicine to tubulin was equivalent to the dissociation constant for the binding of stypoldione to tubulin, we calculated that approximately 62% of the tubulin present free in solution under initiating conditions and 35-37% of the soluble tubulin under steady-state conditions was complexed with stypoldione when polymerization was inhibited by 50%. These data are consistent with a mechanism in which stypoldione interacts with soluble tubulin and inactivates the tubulin so that it is unable to add to microtubule ends, although a colchicine-like mechanism involving an action of stypoldione at microtubule ends has not been eliminated.