Hydrodynamic and pharmacological characterization of putative alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors solubilized from pig brain.
L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins.
Wu
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TY
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chang
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YC
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002498:Centrifugation; D002850:Chromatography, Gel; D003902:Detergents; D007608:Kainic Acid / Q000494:pharmacology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D017470:Receptors, Glutamate / Q000187:drug effects* / Q000378:metabolism; D012995:Solubility; D013552:Swine; D013861:Thiocyanates / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism / Q000494:pharmacology*
D003902:Detergents; D017470:Receptors, Glutamate; D013861:Thiocyanates; D011092:Polyethylene Glycols; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D017830:Octoxynol; C010615:Nonidet P-40; D007608:Kainic Acid; C009941:potassium thiocyanate