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.
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)
Martin
D L
DL
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201.
Martin
S B
SB
Wu
S J
SJ
Espina
N
N
D000818:Animals; D001051:Apoenzymes / Q000032:analysis* / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002421:Caudate Nucleus / Q000201:enzymology; D002531:Cerebellum / Q000201:enzymology; D002540:Cerebral Cortex / Q000201:enzymology; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000378:metabolism; D006624:Hippocampus / Q000201:enzymology; D015151:Immunoblotting; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping; D011732:Pyridoxal Phosphate / Q000378:metabolism; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology
D001051:Apoenzymes; D011732:Pyridoxal Phosphate; D002918:Chymotrypsin; D005968:Glutamate Decarboxylase