SOLUBILIZATION AND PURIFICATION OF AN ALPHA-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID BINDING-PROTEIN FROM BOVINE BRAIN

被引:20
作者
HUNTER, C
WENTHOLD, RJ
机构
[1] Section on Neurotransmitter Receptor Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland
关键词
EXCITATORY AMINO ACIDS; ALPHA-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID (AMPA) RECEPTOR; QUISQUALATE RECEPTOR; DETERGENT SOLUBILIZATION; BOVINE CORTEX;
D O I
10.1111/j.1471-4159.1992.tb11353.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
alpha-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-degrees-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 nM 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 [H-3]AMPA binding activity at M(r) approximately 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) approximately 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.
引用
收藏
页码:1379 / 1385
页数:7
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