Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes

Journal Article · Tue Jan 05 04:00:00 EST 1988 · J. Biol. Chem.; (United States)

OSTI ID:6837957

Chen, J W; Cunningham, M D; Galton, N; Michaelis, E K

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approx. 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger M/sub r/ protein in synaptic membranes. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 /sup +/M, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L(/sup 3/H)glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.

• Research Organization: Univ. of Kansas, Lawrence (USA)
• OSTI ID: 6837957
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:1; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
BODY
BRAIN
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
CENTRAL NERVOUS SYSTEM
GLUTAMIC ACID
LABELLED COMPOUNDS
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MEMBRANES
NERVE CELLS
NERVOUS SYSTEM
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PROTEINS
PURIFICATION
RECEPTORS
SOMATIC CELLS
TRITIUM COMPOUNDS