Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase.
Immunoaffinity columns are prepared from the monoclonal antibody (MAb) GAD-1. These columns are used to enrich glutamic acid decarboxylase (GAD) from the cytosolic fraction of rat brain homogenates and from Triton X-100 extracts of the brain membrane fraction. In each case enzyme activity is enriched over 400-fold. The immunopurified fractions were analyzed by SDS-PAGE. Fractions purified from the cytosol consisted of a quantitatively major band of 59 kDa, and one band of 63 kDa, as well as a group centered around 55 kDa. Fractions purified from membranes consisted primarily of the 59 and 63 kDa components; only traces of the lower-molecular-weight components were present. The entire set of proteins purified on GAD-1 immunoaffinity columns is strongly recognized by 2 widely used antisera to GAD, those described in Saito et al. (1974) and Oertel et al. (1981). The 59 kDa protein from the cytosolic fraction was purified to homogeneity by preparative SDS-PAGE; a partial amino acid sequence of this protein was obtained. The 59 kDa protein has a high degree of sequence homology with the deduced amino acid sequence of the protein that was coded for by a cDNA for feline GAD (Kaufman et al., 1986; Kobayashi et al., 1987). Thus, these proteins are either products of a single gene that diverged during the evolution of rat and cat from a common ancestor, or are members of a closely related set of genes found in both species. The MAb GAD-6 recognizes the 59 kDa band and the group of bands centered around 55 kDa on Western blots. Therefore, these proteins are immunochemically related. GAD-6 does not recognize the 63 kDa band. In Western blots of unfractionated homogenates of the whole brain, the only band recognized by GAD-6 is a 59 kDa band.(ABSTRACT TRUNCATED AT 250 WORDS)
Chang
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YC
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Gottlieb
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001923:Brain Chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D005968:Glutamate Decarboxylase / Q000276:immunology* / Q000302:isolation & purification; D007106:Immune Sera / Q000276:immunology; D008566:Membranes / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D009419:Nerve Tissue Proteins; D005968:Glutamate Decarboxylase