Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-(/sup 3/H)glutamate

Journal Article · Wed Feb 15 00:00:00 EST 1989 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(89)90030-4· OSTI ID:5703138

Cincotta, M; Summers, R J; Beart, P M

A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-(/sup 3/H)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 (/sup 3/H)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 (/sup 3/H)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-(/sup 3/H)glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-(/sup 3/H)glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue.

• Research Organization: Univ. of Melbourne, Victoria (Australia)
• OSTI ID: 5703138
• Journal Information: Anal. Biochem.; (United States), Vol. 177:1
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
RECEPTORS
CHEMICAL COMPOSITION
ASPARTIC ACID
AUTORADIOGRAPHY
BIOCHEMICAL REACTION KINETICS
BRAIN
GLUTAMIC ACID
RATS
TRITIUM COMPOUNDS
ANIMALS
BODY
CARBOXYLIC ACIDS
CENTRAL NERVOUS SYSTEM
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
NERVOUS SYSTEM
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PROTEINS
REACTION KINETICS
RODENTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques