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Keywords:

  • Glutamate decarboxylase;
  • Cerebellum;
  • Immunoblotting;
  • Subunit structure

Abstract: The subunit structure of brain glutamate decarboxylase in cerebellum was investigated by using gel electrophoresis and antisera that specifically recognize the individual isoforms of brain glutamate decarboxylase (termed GAD65 and GAD67). The antisera were prepared against peptides that corresponded to amino acid sequences specific to each isoform. Each antiserum reacted specifically with the appropriate peptide in an ELISA and with the appropriate form of GAD on immunoblots. Nondenaturing gradient gel electrophoresis indicated that GAD is principally multimeric with monomeric forms comprising <3% of the total. Immunoprecipitation and immunoaffinity chromatography experiments were performed with antisera W624 and W883, which were prepared against peptides specific to GAD65 and GAD67, respectively. Immunoprecipitates prepared from cerebellar supernatants with W624 contained both GAD65 and GAD67, whereas some GAD67 was left in the supernatant. In a similar manner, immunoprecipitates prepared with W883 contained both GAD65 and GAD67, whereas some GAD65 remained in the supernatant. In addition, immunoaffinity columns prepared with either W624 or W883 retained both GAD65 and GAD67 even after extensive washing. These results are consistent with the presence of heteromultimers of GAD65 and GAD67 in cerebellum in addition to homomers of each form.