Solubilization, Characterization, and Partial Purification of α‐Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid‐, Quisqualate‐, Kainate‐Sensitive l‐Glutamate Binding Sites from Porcine Brain Synaptic Junctions

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Abstract
L‐[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X‐114 in the presence of KC1. The solubilized binding sites bound l‐[3H]glutamate reversibly with Kd and Bmax values of 1.48 ± 0.18 μM 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‐as‐partate (NMDA) l‐glutamate receptors but not by NMDA or α‐amino‐4‐phosphonobutyrate. The rank order for the competitive ligands in displacing l‐[3H]glutamate was: quisqualate > α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid > l‐glutamate > kainate.