Brain tubulin preparations contain an abundant type of tubulin which does not undergo the normal cycle of tyrosination-detyrosination, and whose nature is still unknown. We have used peptide sequence analysis and mass spectrometry combined with immunological procedures to show that this non-tyrosinatable tubulin has a specific primary structure. It differs from the tyrosinated isotype in that it lacks a carboxy-terminal glutamyl-tyrosine group on its alpha-subunit. Thus, non-tyrosinatable tubulin originates from a well-defined posttranslational modification of the tubulin primary structure which is located at the expected site of activity of tubulin tyrosine ligase. This probably accounts for the reason why it cannot be tyrosinated. The significance of this abundant brain isotubulin and the metabolic pathway involved in its formation remain to be elucidated. This should shed light on the relation between the structural diversity of the carboxy terminus of alpha-tubulin and the regulation of functional properties of microtubules.

Chicken gizzard telokin was purified to apparent homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This preparation yielded upon mass spectrometry analysis seven mass peaks spanning from 15 858 to 17 100 Da. Anion exchange-high performance liquid chromatography of the purified telokin revealed a high diversity of telokin molecules. By combining protein chemistry to chromatography and mass spectrometry, the telokin heterogeneity was analyzed. Three acetylated N-termini were found, AMI, MIS, and SGR. Cyanogen bromide cleavage of telokin yielded six different C-terminal peptides corresponding to the removal of one to six C-terminal glutamyl residues from the protein sequence deduced from the cDNA. Phosphorylation of telokin was detected, thus increasing the heterogeneity of the telokin preparation. In addition, peptide sequencing has shown that telokin contained either an aspartyl or a glutamyl residue at position 27, probably resulting from chicken polymorphism.