Biosynthesis of Radiolabeled Curacin A and Its Rapid and Apparently Irreversible Binding to the Colchicine Site of Tubulin

Curacin A is a potent competitive inhibitor of colchicine binding to tubulin, and it inhibits the growth of tumor cells. We prepared [14C]curacin A biosynthetically to investigate its interaction with tubulin. Binding was rapid, even at 0°C, with a minimum kf of 4.4 × 103 M−1 s−1. We were unable to demonstrate any dissociation of the [14C]curacin A from tubulin. Consistent with these observations, the Ka value was so high that an accurate determination by Scatchard analysis was not possible. The [14C]curacin A was released from tubulin following urea treatment, indicating that covalent bond formation does not occur. We concluded that curacin A binds more tightly to tubulin than does colchicine. Besides high-affinity binding to the colchicine site, we observed significant superstoichiometric amounts of the [14C]curacin A bound to tubulin, and Scatchard analysis confirmed the presence of two binding sites of relatively low affinity with a Ka of 3.2 × 10−5 M−1.