1. Purification and biochemical characterization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive l-glutamate receptors of pig brain
- Author
-
Yuo-Sheng Chang, L H Gao, Tzong-Yuan Wu, B F Li, C I Liu, and Chia-Lin Wu
- Subjects
Octoxynol ,Swine ,Synaptic Membranes ,Glutamic Acid ,Kainate receptor ,AMPA receptor ,Biology ,Biochemistry ,Chromatography, Affinity ,Polyethylene Glycols ,Potassium Chloride ,Agglutinin ,Receptors, Kainic Acid ,Affinity chromatography ,Centrifugation, Density Gradient ,Animals ,Electrophoresis, Gel, Two-Dimensional ,Receptors, AMPA ,Receptor ,alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ,Molecular Biology ,Brain Chemistry ,Gel electrophoresis ,Kainic Acid ,Glutamate receptor ,Glutamate binding ,Cell Biology ,Molecular biology ,Rats ,Molecular Weight ,Solubility ,Chromatography, Gel ,Research Article - Abstract
Two preparations of glutamate receptors were purified from the synaptic junctions of pig brain by a combination of detergent solubilization, anion-exchange chromatography, wheat-germ agglutinin affinity chromatography and sedimentation through sucrose gradients. These preparations were enriched in specific L-[3H]glutamate binding activity (> 5000 pmol of glutamate binding sites/mg of protein), and the rank order of ligand affinity for binding to these preparations was: quisqualate > 6-cyano-7-nitroquinoxaline-2,3-dione > α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) > L-glutamate > kainate ≫ N-methyl-D-aspartate≈ L-2-amino-4-phosphonobutyrate. SDS/PAGE analysis revealed that more than 80% of the protein in either of these preparations appeared as a single protein band of 106 kDa. Two-dimensional gel electrophoresis further revealed that these 106 kDa proteins consisted of a series of acidic proteins which were recognized by antibodies against rat AMPA receptor subunits. These 106 kDa proteins were also recognized by wheat-germ agglutinin and concanavalin A; in addition, peptide N-glycosidase F treatment of these preparations decreased their size to 99 kDa. Our results suggest that the putative glutamate receptors isolated here are likely to belong to the AMPA subtype of glutamate receptors in pig brain. Using the purification procedure reported here, 5 µg of AMPA receptor proteins can be isolated from 250 g of pig brain tissue.
- Published
- 1996