Limited subtilisin digestion of the tubulin α, β heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin-cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C-terminus of the molecule. The C-terminal peptides of both α and β subunits of tubulin were purified by anion-exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp-438 and His-406 of α and Gln-433 and His-396 of β tubulin chains. Quantitative analysis of our data show that cleavage at positions His-406 (α) and His-396 (β) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu-445 or −435 of α and β tubulin, respectively.
