1378171

Purification of AMPA type glutamate receptor by a spider toxin.

Article date: 1992/5/1

PubMed ID: 1378171

Journal name: Brain research. Molecular brain research (ISSN: 0169-328X)

ABSTRACT

A glutamate receptor was purified from Triton X-100-solubilized bovine cerebellum membranes. The purification was carried out in two steps: affinity chromatography using a spider toxin (Joro spider toxin; JSTX) immobilized on a lysine-agarose column, and a Mono Q anion exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified active fraction showed a single band with Coomassie Blue staining, which migrated with a M® = 130,000. The specific [3H]amino-3-hydroxy-5-methyl-isoxazole propionate ([3H]AMPA) binding activity of the affinity-purified fraction was 2095-fold higher than that of the crude soluble fraction. Lineweaver-Burk plot analysis showed a Kd of 12.7 nM [3H]AMPA in the purified fraction. The purified fraction was examined with patch-clamp recording methods in reconstituted liposomes. A glutamate-activated channel was observed and was inhibited with JSTX. The rank order of potency of agonists inducing channel currents was AMPA = glutamate greater than quisqualate much greater than kainate greater than NMDA. Thus, there is strong evidence that the 130 kDa protein is a purified component of the native AMPA type glutamate channel of bovine cerebellum.

Author List: Shimazaki K, Robinson H P, Nakajima T, Kawai N, Takenawa T

Publication Types: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't

Substances mentioned in the article: Chloride Channels; Glutamates; JSTX spider toxin; Liposomes; Membrane Proteins; Nerve Tissue Proteins; Receptors, AMPA; Receptors, Neurotransmitter; Spider Venoms; Ibotenic Acid; Glutamic Acid; N-Methylaspartate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Quisqualic Acid; Kainic Acid;

Mesh terms: Animals; Cattle; Cerebellum/chemistry; Chloride Channels; Chromatography, Affinity; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Glutamates/pharmacology; Glutamic Acid; Ibotenic Acid/analogs & derivatives; Ion Channel Gating/drug effects; Kainic Acid/pharmacology; Kinetics; Liposomes; Membrane Proteins/drug effects; N-Methylaspartate/pharmacology; Nerve Tissue Proteins/isolation & purification; Quisqualic Acid/pharmacology; Receptors, AMPA; Receptors, Neurotransmitter/drug effects; Spider Venoms; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid;

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