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Title: | Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate. | Austin Authors: | Cincotta, M;Summers, R J;Beart, P M | Affiliation: | University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospital, Heidelberg, Victoria, Australia | Issue Date: | 15-Feb-1989 | Publication information: | Analytical Biochemistry; 177(1): 150-5 | Abstract: | A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-[3H]glutamate. Effective labeling of glutamate receptors in slide-mounted 10-microns sections was obtained using a low incubation volume (0.15 ml) and rapid washing: a procedure where high ligand concentrations were achieved with minimal waste. Saturation experiments using [3H]glutamate revealed a single binding site of micromolar affinity. The Bmax was trebled in the presence of Ca2+ (2.5 mM) and Cl- (20 mM) with no change in the Kd. Binding was rapid, saturable, stereospecific, and sensitive to glutamate receptor agonists. The proportions of [3H]glutamate binding sensitive to N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were 34, 54, and 51%, respectively. NMDA inhibited binding at a distinct subset of L-[3H]glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-[3H]glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue. | Gov't Doc #: | 2568102 | URI: | https://ahro.austin.org.au/austinjspui/handle/1/12631 | Journal: | Analytical biochemistry | URL: | https://pubmed.ncbi.nlm.nih.gov/2568102 | Type: | Journal Article | Subjects: | Animals Aspartic Acid.analogs & derivatives.metabolism Autoradiography Binding, Competitive Brain.metabolism Glutamates.diagnostic use Glutamic Acid Ibotenic Acid.analogs & derivatives.metabolism Kainic Acid.metabolism Kinetics Male N-Methylaspartate Radioligand Assay Rats Rats, Inbred Strains Receptors, Glutamate Receptors, Neurotransmitter.analysis Software Tritium.diagnostic use alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid |
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