Identification of the cysteine residue of .beta.-tubulin alkylated by the antimitotic agent 2,4-dichlorobenzyl thiocyanate, facilitated by separatio of the protein subunits of tubulin by hydrophobic column chromatography

Abstract

The mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate (DCBT) has been examined in detail. Shown in previous studies to inhibit tubulin polymerization [Abraham. I., Dion, R. L., Duanmu, C., Gottesman, M. M., and Hamel, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6839-6843] and to form a covalent bond preferentially with .beta.-tubulin [Bai, R., Duanmu, C., and Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20], DCBT has now been documented to interact at low concentrations with a high degree of specificity at cysteine residue 239 of .beta.-tubulin. These low DCBT concentrations also result in the partial inhibition of tubulin polymerization. Such findings strongly indicate athat cysteine-239 of .beta.-tubulin is essential for microtubule assembly. Although .alpha.-tubulin is alkylated almost as well as .beta.-tubulin when the drug:tubulin ratio = 5:1 (Bai et al., 1989), .beta.-tubulin is alkylated about 25 times as extensively as .alpha.-tubulin, almost excusively at Cys-239, when the drug:tubulin ratio = 1:5. In addition, we find that low concentrations of DCBT do not affect the binding of colchicine to tubulin but that colchicine and related compounds do reduce the alkylation of tubulin by DCBT. This suggests that Cys-239 of .beta.-tubulin is not involved in the binding of colchicine to tubulin but that this amino acid residue is at least partially masked by the drug when it is bound to the protein. We also describe a column chromatography procedure (hydrophobic chromatography on decylagarose) useful for the preparative resolution of unalkylated, although denatured, .alpha.- and .beta.-tubulin.