Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2.

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Abstract
We have characterized the epitope of the rat monoclonal antibody YL 1/2 in detail using synthetic peptides and several alpha‐tubulin derivatives. The epitope seems to be provided by the linear sequence spanning the carboxy‐terminal residues of tyrosinated alpha‐tubulin. By competitive ELISA, dipeptides covering the carboxyl end could be antigenically recognized. Three sites were deduced at the dipeptide level: a negatively charged side chain in the penultimate position followed by an aromatic residue which must carry the free carboxylate group. Experiments with longer peptides point to a further negative charge provided by a carboxylate group on the third residue from the end. Thus the tripeptide Glu‐Glu‐Tyr was only 5‐fold less active than the octapeptide spanning the carboxy‐terminal alpha‐tubulin sequence. The octapeptide itself showed only a 40‐fold lower activity than tyrosinated alpha‐tubulin. In line with the emerging epitope requirements of YL 1/2, the Escherichia coli rec A protein, the catalytic subunit of the cyclic AMP‐dependent muscle protein kinase as well as performic acid‐oxidized actin were recognized by YL 1/2 in immunoblots. These results thus define the sequence requirements within a probably linear epitope and give rise to some general questions concerning experiments where monoclonal antibodies are microinjected into cells in order to assess the contribution of a known antigen to cellular physiology.

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