Tubulin structure and biochemical properties after in-vitro reaction with 2,5-hexanedione (110134) (HD) or 3,4-dimethyl-2,5-hexanedione (25234791) (DMHD) were examined. Bovine or rat brain tubulin and rat testis tubulin were incubated with sodium-glutamate and guanosine-5'-triphosphate (GTP) alone, or in the presence of either HD or DMHD. Initial experiments were performed with bovine brain tubulin and HD. Electrophoretic analysis of the tubulin preparation after incubation with HD showed the development of covalently crosslinked tubulin dimers that were composed of similar amounts of alpha- and beta-tubulin. Changes in the assembly parameters varied depending on the tubulin concentration, GTP concentration and temperature. The major effect of HD treatment was to reduce the time of the nucleation phase of assembly up to 10 fold. The results of using HD incubation conditions which inhibit crosslinking indicated a correlation between crosslinking and assembly enhancement. Electron microscopy of control and treated tubulin showed treated polymers to be shorter than the controls, but otherwise similar. The effects on assembly of rat brain or testis tubulin after incubation with HD, and of rat brain tubulin after incubation with DMHD, were the same as those described above for bovine brain tubulin. Mixing experiments showed that only a small amount of HD derivatized rat brain tubulin was required to be mixed with control tubulin to induce altered assembly properties. The author concludes that both the kinetics of tubulin polymerization and the morphology of the final assembly product were modified by gamma-diketone incubation.
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