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Solubilization and Purification of an α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid Binding Protein from Bovine Brain
Author(s) -
Hunter Chyren,
Wenthold Robert J.
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb11353.x
Subject(s) - ampa receptor , kainate receptor , biochemistry , chemistry , binding site , binding protein , amino acid , dissociation constant , gel electrophoresis , glutamate receptor , receptor , gene
α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) is a selective ligand for an excitatory amino acid receptor subtype in mammalian brain. We have solubilized an AMPA binding protein from bovine brain membranes with 1% Triton X‐100 in 0.5 M phosphate buffer and 20% glycerol at 37°C and purified the stable binding sites using a series of chromatographic steps. Scatchard analysis of the purified preparation showed a curvilinear plot with dissociation constants of 10.6 and 323 n M and B max values of 670 and 1,073 pmol/mg of protein for the high‐ and low‐affinity sites, respectively. Inhibition constants for several excitatory amino acid analogues were similar to those obtained for other membrane and solubilized preparations. Gel filtration of the soluble AMPA binding protein showed a single peak of [ 3 H]AMPA binding activity at M r ∼ 500,000. With sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the purified AMPA binding protein showed a single major band at M r = 110,000. Previously, we have shown that a monoclonal antibody (KAR‐B1) against a frog brain kainate binding protein selectively recognizes an unknown protein in mammalian brain migrating at M r ∼ 100,000. We now show that this antibody recognizes the major component of the purified AMPA binding protein, supporting a structural similarity between the frog brain kainate binding protein and the mammalian AMPA binding protein.