Maytansine binding to the vinblastine sites of tubulin

Tubulin possesses two distinct binding sites for vinblastine; one of high affinity (Ka = 6.2 X 106M” ) occupancy of which prevents polymerization of tubulin and a second, lower affinity site (K, = 8 X 104M”) occupancy of which correlates with the aggregating effects of the alkaloid on tubulin [l]. The recent report [2] that maytansine, a new antitumor agent of plant origin [3,4] , was a potent competitive inhibitor of 3H vincristine binding in rat brain homogenates, led us to investigate which of the two vinblastine-binding sites would be involved in this effect. The formulae of these two compounds are shown below (fig.1). For polymerization experiments, tubulin was purified from rat brain extracts by three cycles of polymerization and depolymerization, according to the method of Shelanski et al. [5]. The kinetics of tubule assembly have been studied by turbidimetric measurements at 400 nm as described by Gaskin et al. [6] in a temperature-controlled chamber of a Cary spectrophotometer (model 14) at 37’C. The polymerization buffer contained 0.1 M 2-(N-morpholinoethane-sulfonic acid) (Mes) buffer, pH 6.4, 1 mM ethylene glycol-bis@amino-ethyl ether)-N,N’tetraacetic acid (EGTA), 1 mM GTP and 0.5 n&i MgClz. Colchicine binding was determined by a modification of the DEAE-filter paper method [7].