A novel photoaffinity ligand for kainate sites labels two polypeptides with molecular mass 45 and 33.5 kDa in chick cerebellum.
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.
Sivvas
E
E
Department of Physiology, Medical School, University of Patras, Greece.
Voukelatou
G
G
Papaioannou
D
D
Aletras
A J
AJ
Kouvelas
E D
ED
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000378:metabolism*; D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism*; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
C086289:4-(1-(4-azidobenzamidomethyl)ethenyl)-2-carboxypyrrolidin-3-ylacetic acid; D000345:Affinity Labels; D001386:Azides; D010455:Peptides; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D007608:Kainic Acid