Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes
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), Vol. 263:1
- Country of Publication:
- United States
- Language:
- English
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GLUTAMIC ACID
MEMBRANE TRANSPORT
PROTEINS
PURIFICATION
TRITIUM COMPOUNDS
BRAIN
CELL MEMBRANES
NERVE CELLS
RECEPTORS
AMINO ACIDS
ANIMAL CELLS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM
LABELLED COMPOUNDS
MEMBRANE PROTEINS
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ORGANIC ACIDS
ORGANIC COMPOUNDS
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550201* - Biochemistry- Tracer Techniques