Reversible dimer dissociation of tubulin S and tubulin detected by fluorescence anisotropy.
Concentration-dependent dissociation of dimers of goat brain tubulin S and tubulin was studied by fluorescence anisotropy. Upon dilution, assembly-competent fluorescein 5'-maleimide labeled dimers of tubulin S and tubulin show a progressive decrease in fluorescence anisotropy. That this lowering of anisotropy results from the dissociation of tubulin S dimers into monomers was shown by dilution experiments with unlabeled homologous and heterologous proteins. A nonlinear least-squares fit of the data gave a dissociation constant of 7.1 x 10(-8) M for tubulin S compared to 7.2 x 10(-7) M for tubulin at 25 degrees C in 0.1 M PEM buffer, pH 7.0. van't Hoff plots of dimer-monomer dissociation of tubulin S and tubulin also show considerable differences in delta H and delta S. Effects of ionic strength and colchicine on the equilibrium constants are also substantially different for tubulin and tubulin S. The implications of these observations on the influence of C-terminal tails on tubulin structure are discussed.
Panda
D
D
Department of Biochemistry, Bose Institute, Calcutta, India.
Roy
S
S
Bhattacharyya
B
B
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003078:Colchicine / Q000378:metabolism; D003374:Coumarins; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006041:Goats; D008301:Maleimides; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry*; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry*
D003374:Coumarins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008301:Maleimides; D010446:Peptide Fragments; D014404:Tubulin; C046279:tubulin S; C037041:fluorescein 5-maleimide; C074193:N-(4-(7-(diethylamino)-4-methylcoumarin-3-yl))maleimide; D003078:Colchicine