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Title: 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 inhibitmore » 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.« less
Authors:
; ; ;
Publication Date:
OSTI Identifier:
6837957
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Biol. Chem.; (United States); Journal Volume: 263:1
Research Org:
Univ. of Kansas, Lawrence (USA)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 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; MEMBRANES; NERVOUS SYSTEM; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANS; SOMATIC CELLS 550201* -- Biochemistry-- Tracer Techniques