Characterization of oligomers of tubulin by two-dimensional native electrophoresis.

We and others [Lee et al. (1973) J. Biol. Chem. 248, 7253-7262; Kravit et al. (1982) J. Cell Biol. 95, 344a; Kravit et al. (1984) J. Cell Biol. 99, 188-198] have observed oligomers of tubulin by native polyacrylamide gel electrophoresis (PAGE), even when they were not evident in sedimentation velocity or gel-exclusion chromatography experiments under comparable conditions. Aggregates of tubulin are also seen on native starch gels. Tubulins purified from calf brain, sea urchin egg (Strongylocentrotus purpuratus), and antarctic fish brain (Pagothenia borchgrevinki) give rise to similar distributions of aggregates. Unlike microtubules, these oligomers are relatively insensitive to temperature (5-25 degrees C), pH (6.1-8.8), the absence of excess GTP and/or Mg+2, stoichiometric concentrations of colchicine, and a variety of electrophoresis buffers. These aggregates, once formed during electrophoresis, associate and dissociate slowly. Depending upon the incubation conditions, they give rise to kinetically controlled distributions that appear in two-dimensional native PAGE as a square array of discrete polymeric species. The fastest migrating species (monomers) are often observed to reequilibrate preferentially into the second band. The second band reequilibrates into the fourth, the third band into the sixth, the fourth into the eighth, etc. (The assignment of molecular weights to these species by Ferguson analysis is tentative due to their slow reequilibration.) Thus, a feature of the reequilibration is that association occurs more rapidly than dissociation and each species is occasionally observed to "dimerize." This behavior is suggestive of irreversible aggregation (possibly crosslinking) or of the formation of slowly dissociating aggregates. Although they may be related to the protofilaments of microtubules, these oligomers appear to be another example of nonmicrotubular, polymorphic aggregates of tubulin.