Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2.
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.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D009842:Oligopeptides / Q000276:immunology; D051381:Rats; D014404:Tubulin / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009842:Oligopeptides; D014404:Tubulin