Selection of a nucleation-promoting element following chemical modification of tubulin

Following a 16-h incubation with a large excess of 2,5-hexanedione (2,5-HD) while in the assembled state, bovine brain tubulin contained a powerful nucleating component, the presence of which lowered the dissociation rate from 83 s-1 for untreated tubulin to 13 s-1 for 2,5-HD-treated tubulin. This nucleating component could be selectively concentrated by sequential stringent (conditions of low temperature and low tubulin concentration) cycles of assembly and disassembly. In 2-(N-morpholino)ethanesulfonic acid buffer without glycerol, the critical concentration of assembly of untreated tubulin (2.4 mg/mL) was 19 times higher than that of 2,5-HD-treated tubulin subjected to three sequential stringent cycles of assembly and disassembly (0.13 mg/mL). This highly nucleating 2,5-HD-treated tubulin preparation could both copolymerize with untreated tubulin and seed subcritical concentration assembly of untreated tubulin. Experiments to define the assembly-altering component have identified structural alterations to the alpha-tubulin monomer. While the alpha-tubulin subunit of native untreated tubulin dimer contained no chymotryptic cleavage sites, the native 2,5-HD-treated alpha-tubulin subunit was cleaved by chymotrypsin to yield a 37-kDa C-terminal fragment.