Association states of tubulin in the presence and absence of microtubule-associated proteins. Analysis by electric birefringence.
Electric birefringence has been used to examine the states of association of tubulin in phosphocellulose-purified tubulin or depolymerized microtubule protein solutions at low temperature. In a high electric field (1000-4000 V/cm), tubulin could be orientated (owing to the existence of a permanent and/or induced dipole) and exhibited a positive birefringence (delta n), related to its intrinsic optical anisotropy. The analysis of the relaxation process (depending on hydrodynamic properties of molecules), by measurement of the time decay of delta n, revealed the existence of a multicomponent or polydisperse system, whatever the tubulin solution. Two relaxation times, representative of the smallest and the largest orientated species, were obtained by computer-fitting analysis. The mean values of relaxation time for phosphocellulose-purified tubulin were 0.8 and 8 microseconds. In microtubule protein solutions, large-sized macromolecular species with relaxation time up to 450 microseconds were detected. The largest species (relaxation times ranging from 50 to 450 microseconds) could be eliminated by centrifugation at 3000000 X g for 1 h. Addition of microtubule-associated protein to either pure tubulin or high-speed centrifuged microtubule protein led to a rapid formation of large species analogous to those present in microtubule protein. Molecular dimensions of the relaxing structures were estimated using simple hydrodynamic models and values of rotational diffusion constants calculated from the relaxation times, and compared to those of the structures described in the literature. In conclusion, we have found that (a) phosphocellulose-purified tubulin is not only composed of elementary species (dimers) but also contains tubulin-associated forms of limited size (up to 7-10 dimers), (b) depolymerized microtubule protein solutions contain ring oligomers and structures very much larger, the formation of which is dependent on the presence of microtubule-associated protein.
D000818:Animals; D001718:Birefringence; D001921:Brain / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D051381:Rats; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D014404:Tubulin