Interaction of Tubulin with Drugs and Alkylating Agents. 1. Alkylation of Tubulin by Iodo[14C]acetamide and N,N-Ethylenebis(iodoacetamide)

The sulfhydryl groups of tubulin are reported to play a role in regulating microtubule assembly and colchicine binding to tubulin. The alkylating agents iodo[¹⁴C]acetamide and its bifunctional analogue N,N'-ethylenebis(iodoacetamide) are used as probes for the sulfhydryl groups of tubulin. In the presence of 8 M urea, α- and β-tubulin have 10-11 and 8 alkylatable sulfhydryls, respectively, and one of the high molecular weight proteins (HMW 2) has 5 sulfhydryls/M_r 271000. In the absence of urea, the rates of alkylation of aand β-tubulin are significantly lower but that of HMW 2 is unaffected. The sulfhydryls of tubulin are masked in intact microtubules. N,N'-Ethylenebis(iodoacetamide) reacts with free tubulin to generate a band, designated β*, which migrates ahead of β on polyacrylamide gels. β* appears to represent a form of β-tubulin containing at least one intrachain cross-link between sulfhydryl groups. Formation of β* is inhibited in intact microtubules and is abolished if tubulin is denatured by 8 M urea, 1% sodium dodecyl sulfate, or boiling. N,N'-Ethylenebis(iodoacetamide) may thus be used as a probe for the native conformation of free tubulin.