Binding to tubulin of an allocolchicine spin probe: interaction with the essential SH groups and other active sites.
EPR titration of tubulin with an allocolchicine spin probe showed more than one binding site: one high-affinity binding site (Kd = 8 microM), consistent with the Ki found for competition with colchicine, and one or more low-affinity site(s) (Kd higher than 50 microM). No disturbance of the EPR signal of the tubulin-bound allocolchicine spin probe could be observed at room temperature in the presence of other paramagnetic probes: Mn(II) for the binding site of Mg(II), Co(II) for the Zn(II) binding site and Cr(III)GTP for the binding site of the exchangeable GTP. Labelling of tubulin with both the allocolchicine and a SH-group spin probe also showed lack of interaction. The colchicine-binding site is thus sterically isolated from the binding sites for GTP, Mg(II), Zn(II) and the two essential SH-groups. In the tubulin-colchicin complex, all SH-groups could still be labelled with an excess of the SH-reagent, N-ethylmaleimide. Furthermore, colchicine binding was only minimally influenced by the blocking of the two essential SH-groups. However, the rate constant of the reaction of two equivalents of the SH-reagent, a maleimide spin probe, with the tubulin-colchicine complex was only 50% of the rate constant found with uncomplexed tubulin. As direct steric interaction of the essential SH-groups with the colchicine-binding site can be excluded, we can now definitively decide that binding of colchicine to tubulin induces a conformational change, which affects the accessibility of the most reactive SH-groups.
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003078:Colchicine / Q000031:analogs & derivatives* / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding; D013438:Sulfhydryl Compounds; D014404:Tubulin / Q000378:metabolism*
D003497:Cyclic N-Oxides; D013438:Sulfhydryl Compounds; D014404:Tubulin; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; C032782:allocolchicine; D003078:Colchicine