TY - JOUR
T1 - Specific affinity labelling of tubulin with bromocolchicine
AU - Schmitt, Henri
AU - Atlas, Daphne
PY - 1976/4/25
Y1 - 1976/4/25
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0017261734&partnerID=8YFLogxK
U2 - 10.1016/0022-2836(76)90289-8
DO - 10.1016/0022-2836(76)90289-8
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C2 - 940153
AN - SCOPUS:0017261734
SN - 0022-2836
VL - 102
SP - 743
EP - 758
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -