Characterization of microtubule protein oligomers by analytical ultracentrifugation

Abstract

Samples of microtubule protein prepared by repeated cycles of assembly-disassembly were examined at low temperatures by sedimentation velocity ultracentrifugation. Sedimenting boundaries corresponding to the 6 S tubulin subunit as well as to two oligomeric species with s020,w values of 18.6 S and 30.6 S were observed. The 30 S to 6 S mass ratio varied with total protein concentration, suggesting that a concentration-dependent equilibrium exists between 6 S and oligomeric species of tubulin. A study of the effects of pH on the mass distribution among the species demonstrated that the 6 S species was favored at low pH values (5.8 to 6.5), the 30 S oligomer was favored at moderate pH values (6.5 to 7.4), and the 18 S oligomer was formed in increasing proportions at higher pH values (7.4 to 8.2). Incubation of purified microtubule protein solutions with increasing concentrations of NaCl initially favored conversion of the 30 S oligomer to the 18 S species with further increases in salt concentration resulting in the dissolution of both the 30 S and 18 S oligomers. The depolymerizing effects of high salt concentrations were substantially reversible, providing further evidence for 6 S-oligomer equilibria. The manipulation of the solution variables of pH and ionic strength in a systematic fashion led to the construction of a "phase diagram" for the microtubule protein species which provided a relatively complete description of the mass distribution among the 6 S, 18 S, and 30 S species over a range of physiological and near-physiological solution conditions.