Solubilization, characterization, and partial purification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, quisqualate-, kainate-sensitive L-glutamate binding sites from porcine brain synaptic junctions.
L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.
Chang
Y C
YC
Institute of Life Science, National Tsing Hua University, Taiwan, Republic of China.
Lin
Y H
YH
Lee
Y H
YH
Leng
C H
CH
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002845:Chromatography; D003902:Detergents; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006358:Hot Temperature; D007051:Ibotenic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D007608:Kainic Acid / Q000494:pharmacology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D016318:Quisqualic Acid / Q000494:pharmacology*; D012995:Solubility; D013569:Synapses / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
D003902:Detergents; D005971:Glutamates; D007051:Ibotenic Acid; D018698:Glutamic Acid; D011092:Polyethylene Glycols; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D016318:Quisqualic Acid; D017830:Octoxynol; C010615:Nonidet P-40; D007608:Kainic Acid