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 (2S,3S,4S)-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 mM CNQX. When photoaffinity labeling was performed in the presence of 2 mM kainic acid, incorporation of [3H]ABCPA was blocked by approximately 70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by approximately 30% with 10 mM glutamate.