Specific affinity labelling of tubulin with bromocolchicine

Henri Schmitt*, Daphne Atlas

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

Abstract

Bromocolchicine, synthesized by substituting tho N-acetyl moiety of colchicine with a reactive bromoacetyl group, was found to be an affinity label for tubulin. Binding of [3H]colchicine to tubulin was competitively and irreversibly inhibited by bromocolchicine with a Ki value of 2.3 × 10-5m. The affinity label could not be displaced by precipitating the protein with trichloroacetic acid and is thus covalently bound. Autoradiographs of brain high-speed supernatant proteins after their electrophoretic separation on sodium dodecyl sulphate/polyacrylamide gels showed that [3H]bromocolchicine reacted with four proteins, of which tubulin was one. Labelling of two of these proteins could be prevented by pretreatment of the brain extracts with α-bromoacetic acid, after which 70% of the covalently bound label was specifically located in the tubulin band. Up to 1.6 mol of affinity label could be bound per mol of tubulin, while under our experimental conditions 1 mol of protein bound irreversibly only 0.2 mol of [3H]colchicine. Autoradiography of sodium dodecyl sulphate/urea-polyacrylamide gels, which separate the subunits of tubulin, showed about 30% [3H] bromocolchicine bound to the α-subunit of tubulin and 70% to tho β-subunit. The irreversible binding site of colchicine was localized to the α-subunit, as labelling of only this subunit was inhibited by colchicine at high affinity label concentrations. At lower concentrations, colchicine inhibited the labelling of both subunits. Bromoacetic acid did not inhibit the reaction of the affinity label with the tubulin subunits, but increased the inhibition of [3H]bromocolchicine binding at lower concentrations of the affinity label in brain extracts preincubated with cold colchicine. This is interpreted to show a conformational change which takes place in the two subunits of tubulin upon binding of colchicine and results in the exposure of some of the binding sites of [3H]bromocolchicine to bromoacetic acid.

Original languageEnglish
Pages (from-to)743-758
Number of pages16
JournalJournal of Molecular Biology
Volume102
Issue number4
DOIs
StatePublished - 25 Apr 1976
Externally publishedYes

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