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Purification and biochemical characterization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive l-glutamate receptors of pig brain
- Source :
- Biochemical Journal. 319:49-57
- Publication Year :
- 1996
- Publisher :
- Portland Press Ltd., 1996.
-
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.
- 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
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 319
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....0c1345b73abc7a007dea8c2b3ca55217