Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding

Abstract

Sulfhydryls and disulfides of [human] platelet tubulin were quantified, their accessibility and reactivity measured, and their role in polymerization and colchicine binding evaluated. Platelet tubulin isolated by 2 cycles of temperature-dependent polymerization-depolymerization contained 12 free sulfhydryl groups per tubulin monomer all of which reacted rapidly with p-chloromercuribenzoate. One sulfhydryl was inaccessible to dithiobis(nitrobenzoic acid). Under anaerobic conditions of tubulin extraction, 1 intrachain disulfide bridge was found per tubulin monomer. Polymerization of tubulin reduced the number of sulfhydryls by 1 which were able to react with p-chloromercuribenzoate or dithiobis(nicotinic acid) but did not affect the disulfide bridge. Polymerizability of platelet tubulin was very sensitive to blocking of free sulfhydryl groups. Complete inhibition of microtubule assembly was obtained when the number of free sulfhydryls per tubulin was reduced by 3 but could be reversed by the addition of dithiothreitol. Colchicine binding was only minimally influenced by blocking of sulfhydryls.