Purification and biochemical characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors of pig brain.
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 > alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) > L-glutamate > kainate > > N-methyl-D-aspartate approximately 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 wheatgerm 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 micrograms of AMPA receptor proteins can be isolated from 250 g of pig brain tissue.
Chang
Y C
YC
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Wu
T Y
TY
Li
B F
BF
Gao
L H
LH
Liu
C I
CI
Wu
C L
CL
D000818:Animals; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D018698:Glutamic Acid / Q000378:metabolism; D007608:Kainic Acid / Q000378:metabolism; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D018091:Receptors, AMPA / Q000302:isolation & purification* / Q000378:metabolism; D018092:Kainic Acid Receptors / Q000378:metabolism; D012995:Solubility; D013552:Swine; D013570:Synaptic Membranes / Q000737:chemistry; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D018698:Glutamic Acid; D007608:Kainic Acid; D017830:Octoxynol; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C010615:Nonidet P-40