A NOVEL PHOTOAFFINITY LIGAND FOR KAINATE SITES LABELS 2 POLYPEPTIDES WITH MOLECULAR-MASS 45 AND 33.5 KDA IN CHICK CEREBELLUM

The synthesis of amidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic ac id (ABCPA) is described. This novel kainic acid analogue, bearing a ph otolabile functionality on the isopropenyl side chain, was proven to b e a good inhibitor of [H-3]CNQX and [H-3]kainic acid binding on chick cerebellar membranes. [H-3]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. [H-3]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 [H-3]ABCPA was blocked by simila r to 70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorpora tion of radioactivity in both bands was blocked by similar to 30% with 10 mM glutamate.