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Journal of Neuroscience, Vol 11, 2725-2731, Copyright © 1991 by Society for Neuroscience
Regulatory properties of brain glutamate decarboxylase (GAD): the apoenzyme of GAD is present principally as the smaller of two molecular forms of GAD in brain
DL Martin, SB Martin, SJ Wu and N Espina
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
The apoenzyme of glutamate decarboxylase [enzyme without bound cofactor,
pyridoxal 5'-phosphate (pyridoxal-P)] serves as a reservoir of inactive
glutamate decarboxylase (GAD) that can be activated when additional GABA
synthesis is required. We have investigated which of two molecular forms of
GAD is present as apoenzyme in synaptosomes and in cortex, caudate nucleus,
hippocampus, and cerebellum of rat brain. Endogenous glutamate
apodecarboxylase (apoGAD) was labeled by incubating extracts of
synaptosomes or punches of each region with 32P- pyridoxal-P, followed by
reduction with NaBH4, to link covalently the 32P-pyridoxal-P to GAD.
Proteins were separated by SDS-PAGE. Punches from all four brain regions
and forebrain synaptosomes contained two forms of GAD with apparent Mrs of
63 and 65 kDa as identified by immunoblotting with four antiGAD sera.
Punches and synaptosomes contained a major 32P-pyridoxal-P-labeled band
with an apparent Mr of 63 kDa that was stained on immunoblots by the
antiGAD serum 1440 and the monoclonal antibody GAD-6, and a minor labeled
band at 65 kDa that was stained by the 1440, 6799, and K2 antisera.
Synaptosomes contained remarkably few other strongly labeled proteins, but
punches contained several other labeled bands. Three additional lines of
evidence indicate that the labeled 63-kDa protein is apoGAD: (1) it was
purified by immunoaffinity chromatography with the GAD-1 monoclonal
antibody; (2) it yielded one major labeled peptide when digested with
chymotrypsin, and that peptide appeared identical in peptide-mapping
experiments to the labeled active-site peptide isolated from
chromatographically prepared rat brain GAD; and (3) its labeling was
selectively blocked by 4-deoxypyridoxine 5'-phosphate, a competitive
inhibitor of the binding of pyridoxal-P to GAD.(ABSTRACT TRUNCATED AT 250
WORDS)
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