Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. Possible relationship of this protein to physiologic glutamate receptors.
Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approximately 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 Mr protein in synaptic membranes. Protease activity during protein purification was prevented by introducing five protease inhibitors, and a three-step purification procedure was developed that yielded a high degree of purification of glutamate-binding proteins. 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 microM, 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-[3H]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.
Chen
J W
JW
Department of Biochemistry, University of Kansas, Lawrence 66045.
Cunningham
M D
MD
Galton
N
N
Michaelis
E K
EK
D000818:Animals; D001921:Brain / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism*
D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter