Immunochemical characterization of the non-NMDA glutamate receptor using subunit-specific antibodies. Evidence for a hetero-oligomeric structure in rat brain.

摘要: Antibodies were made to synthetic peptides corresponding sequences specific the glutamate receptor (GluR) subunits, GluR1-4. The specificity of antibodies was established by Western blotting using membranes simian kidney cells (COS-7) transfected with GluR subunit DNA. Four found be selective for each four and a fifth antibody recognized both GluR2 3. All five immunoadsorbed Triton X-100-solubilized rat brain [3H]AMPA binding activity labeled an Mr = 108,000 band in samples brain. structure studied subunit-specific covalently attached protein A-agarose analyzing subunits bound blotting. Each its respective as well other three forms GluR, showing that detergent solubilized exists hetero-oligomers composed two or more subunits. Evidence supporting similar membrane obtained synaptic chemically cross-linked dithiobis(succinimidylpropionate). immunoaffinity-purified 3-selective antibody. This antibody, A-agarose, adsorbed 55% activity, after elution 1 M KSCN, 22-37% recovered. Analysis purified product showed major immunoreactive at 108,000, silver staining identified same no additional polypeptides. complex, therefore, appears up exclusively In preparation, addition band, higher molecular weight components also detected. These bands migrated 325,000, 470,000, 590,000. Similar sized proteins seen sample, 590,000 component being substantially enriched cross-linking. is largest detected, it has size consistent pentamer protein.