Isolation of glutamate-binding proteins from rat and bovine brain synaptic membranes and immunochemical and immunocytochemical characterization.
We previously isolated two glutamate-binding proteins from rat brain synaptic membranes of 71,000 and 63,000 mol. wt [Chen et al. (1988) J. biol. Chem. 263, 417-426]. In the present study, the 71,000 and 63,000 mol. wt glutamate-binding proteins were purified from rat and bovine brain synaptic membranes by affinity chromatographic separation on an L-glutamate-derived Trisacryl matrix. The major protein component in the purified fractions was a 71,000 mol. wt protein as judged by sodium dodecyl sulfate gel electrophoresis. This fraction represented enrichment of bovine brain glutamate-binding proteins by a factor of 7000-8000 when compared with brain homogenates. The ligand binding characteristics of the proteins purified by this new procedure are very similar to those of the proteins purified by the procedures we described in previous studies. Polyclonal antibodies were raised in mice against the purified, native rat and bovine brain glutamate-binding proteins. These two sets of antibodies interacted specifically with the glutamate-binding proteins but not with any glutamate-metabolizing enzymes. Both sets of antibodies labeled a 71,000 mol. wt protein in Western blots of synaptic membranes obtained from either rat or bovine brain, an indication of homology between these proteins. Both sets of antibodies produced immunoprecipitation of approximately 70-75% of all glutamate-binding proteins from solubilized synaptic membrane proteins. These observations are an indication that the glutamate-binding proteins described above are the most common glutamate-binding entities in both bovine and rat brain synaptic membranes and that they can be easily purified in a one-step chromatographic procedure.
Wang
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Center for Biomedical Research, University of Kansas, Lawrence 66045.
Kumar
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KN
Michaelis
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EK
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005971:Glutamates / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism* / Q000648:ultrastructure
D005971:Glutamates; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter