Interaction of tubulin with drugs and alkylating agents. 1. Alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide).

Abstract

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, alpha- and beta-tubulin have 10-11 and 8 alkylatable sulfhydryls, respectively, and one of the high molecular weight proteins (HMW 2) has 5 sulfhydryls/Mr 271 000. In the absence of urea, the rates of alkylation of alpha- and beta-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 beta, which migrates ahead of beta on polyacrylamide gels. beta appears to represent a form of beta-tubulin containing at least one intrachain cross-link between sulfhydryl groups. Formation of beta* 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.