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Journal of Neuroscience, Vol 8, 2123-2130, Copyright © 1988 by Society for Neuroscience
Characterization of the proteins purified with monoclonal antibodies to glutamic acid decarboxylase
YC Chang and DI Gottlieb
Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110.
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)
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