A metabolite of estradiol, 2-methoxyestradiol (2ME), inhibits angiogenesis In the chicken embryo chorioallantoic membrane assay. Since 2ME causes mitotic perturbalions, we ea ined Its interactions with tubulin. In our standard 1.0 M glutamate system (plus 1.0 mM MgCl2 at 3rC), superstoichiometric concentrations (relative to tubulin) of 2ME inhibited the nucleation and propagation phases of tubulin assembly but did not affect the reaction extent. Although polymer formed In the presence of 2ME was more cold-stable than control polymer, morphology was little changed. Under suboptimal reaction conditions (0.8 M glutamate/no MgCl2 at 26WC), substoichiometric 2ME totally inhibited polymerization. No other estrogenic compound was as effective as 2ME as an inhibitor of polymerization or of the binding of colchicine to tubulin. Inhibition ofcolchicine binding was competitive (KM,22 aM). Thus, a m an metabolite of estradiol binds to the colchicine site of tubulin and, depending on reaction conditions, either inhibits assembly or seems to be incorporated into a polymer with altered stability properties.There is growing evidence that estrogenic compounds affect cell division and act directly on microtubules by interacting with tubulin. The most extensively studied of these compounds is the synthetic analog diethylstilbestrol (DES; Fig. 1, compound 1) and related agents (1-3). These drugs cause disturbances in mitosis, inhibit microtubule assembly at relatively high concentrations, and inhibit the binding of colchicine to tubulin. Estradiol (compound 2), the major estrogenic hormone of human beings, also causes disturbances ofmitosis in cultured cells (1,4,5). These perturbations include aneuploidy, multinucleation, and mitotic arrest, and estradiol has been reported to inhibit the polymerization of rat brain tubulin (6). Seegers et al. (5) found that 2-methoxyestradiol (2ME; compound 3), a metabolite of both estradiol (7) and the oral contraceptive agent 17-ethynylestradiol (compound 4), was more potent than estradiol in producing mitotic perturbations and proposed that it was 2ME rather than estradiol that caused the observed disturbances. Although 2-methoxyestrogens are extremely weak in binding to cytosol estrogen receptors (8), 2ME is found in blood and urine after sequential hepatic hydroxylation and methylation ofestradiol (7).We recently found that 2ME inhibited angiogenesis in the chicken embryo chorioallantoic membrane assay of Crum et al. (9). Disks of2ME (100 Mg) produced large avascular zones 48 hr after implantation on a 6-day embryo, similar to in vitro results of others (10). In attempting to define the mechanistic basis, we observed that 2ME inhibited microtubule assembly. This finding led us to study the interactions of 2ME with purified tubulin.MATERIALS AND METHODS Materials. Purified bovine brain tubulin and H2-CSA4 were prepared as described (11,12). Monosodium glutamate (2.0 M) was adjusted to pH 6.6 with HCl. 2ME, 2-fluoroestradiol, 2-methoxy-17-ethynylestradiol, 2-methoxyestradiol 3-0-meth...