A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.