Mammalian sperm tubulin: an exceptionally large number of variants based on several posttranslational modifications.

Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The alpha- and beta-tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in alpha and 0 to 15 in beta. Corresponding values for side-chain glutamyl residues were 0-6 in alpha and 0-3 in beta. Additional alpha variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in alpha- and beta-tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.