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

Abstract

The SH groups of [bovine brain] 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(iodacetamide) were used as probes for the SH groups of tubulin. In the presence of 8 M urea, .alpha.- and .beta.-tubulin have 10-11 and 8 alkylatable SH, respectively, and one of the high MW proteins (HMW 2) has 5 SH/MW 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 SH of tubulin were 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 1 intrachain cross-link between SH 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.