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

Abstract

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)

GABAergic Interneurons Implications for Understanding Schizophrenia and Bipolar Disorder

“…In contrast, expression of mRNA for GAD 65 has been found to be relatively stable (Ding et al 1998;Feldblum et al 1998). Similarly, expression of GAD 65 protein also appears to be relatively stable (Martin et al 1993) and appears to be controlled primarily through post-translational mechanisms (Miller et al 1991).…”

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

“…Whereas GAD67 is a cytosolic enzyme and distributed throughout the cell body, GAD65 is preferentially located in nerve termini, and is reversibly anchored to the membrane of synaptic vesicles (3)(4)(5)(6). At least 50% of GAD in brain is present as the pyridoxal 5Ј phosphate (PLP)-free apo-enzyme (7,8); GAD65 has been suggested to constitute the majority of this enzyme reservoir (3,8), which can be activated by influx of cofactor. In contrast to GAD65, the majority of GAD67 has been reported to be saturated with PLP (1,3,8).…”

“…At least 50% of GAD in brain is present as the pyridoxal 5Ј phosphate (PLP)-free apo-enzyme (7,8); GAD65 has been suggested to constitute the majority of this enzyme reservoir (3,8), which can be activated by influx of cofactor. In contrast to GAD65, the majority of GAD67 has been reported to be saturated with PLP (1,3,8). The functional implication of these differences for GABA-ergic mechanisms has remained elusive.…”

The role and the mechanism of γ-aminobutyric acid during central nervous system development

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