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Recently we have reported that the membrane-associated form of the ␥-aminobutyric acid-synthesizing enzyme, L-glutamate decarboxylase (MGAD), is regulated by the vesicular proton gradient (Hsu, C. C., Thomas, C., Chen, W., Davis, K. M., Foos, T., Chen, J. L., Wu, E., Floor, E., Schloss, J. V., and Wu, J. Y. (1999) J. Biol. Chem. 274, 24366 -24371). In this report, several lines of evidence are presented to indicate that L-glutamate decarboxylase (GAD) can become membrane-associated to synaptic vesicles first through complex formation with the heat shock protein 70 family, specifically heat shock cognate 70 (HSC70), followed by interaction with cysteine string protein (CSP), an integral protein of the synaptic vesicle. The first line of evidence comes from purification of MGAD in which HSC70, as identified from amino acid sequencing, co-purified with GAD. Second, in reconstitution studies, HSC70 was found to form complex with GAD 65 as shown by gel mobility shift in non-denaturing gradient gel electrophoresis. Third, in immunoprecipitation studies, again, HSC70 was co-immunoprecipitated with GAD by a GAD 65 -specific monoclonal antibody. Fourth, HSC70 and CSP were co-purified with GAD by specific anti-GAD immunoaffinity columns. Furthermore, studies here suggest that both GAD 65 and GAD 67 are associated with synaptic vesicles along with HSC70 and CSP. Based on these findings, a model is proposed to link anchorage of MGAD to synaptic vesicles in relation to its role in ␥-aminobutyric acid neurotransmission.L-Glutamate decarboxylase (GAD 1 ; EC 4.1.1.15) is the ratelimiting enzyme involved in the synthesis of ␥-aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian brain (1). There are two well characterized GAD isoforms in the brain, namely GAD 65 and GAD 67 , referring to GAD with molecular masses of 65 and 67 kDa, respectively (for review, see Ref.2). GAD 67 is mostly soluble and is distributed evenly throughout the cell, whereas GAD 65 is concentrated at the nerve terminals (3) and constitutes the majority of the membrane-associated GAD (MGAD) (4, 5). Despite its importance, our knowledge regarding the regulation of GAD activity is quite limited. Recently, we have shown that soluble GAD (SGAD) is activated by dephosphorylation, mediated by a Ca 2ϩ -dependent phosphatase, calcineurin, and is inhibited by phosphorylation, mediated by a cAMP-dependent protein kinase A (6, 7). Conversely, MGAD is activated by protein phosphorylation, which depends on the integrity of the electrochemical gradient of synaptic vesicles (5). Hence, GAD activity appears to be regulated differently depending on whether it exists as a soluble or membrane-anchored protein. Judging from the amino acid sequences, it is unlikely that GAD 65 and/or GAD 67 can be integral membrane components, since neither contains a stretch of hydrophobic amino acids long enough to span the membrane (greater than 20 residues), a typical feature for integral membrane proteins. Furthermore, both isoforms lack the appropriate consensus se...

No abstract

The effects of taurine in the mammalian nervous system are numerous and varied. There has been great difficulty in determining the specific targets of taurine action. The authors present a review of accepted taurine action and highlight recent discoveries regarding taurine and calcium homeostasis in neurons. In general there is a consensus that taurine is a powerful agent in regulating and reducing the intracellular calcium levels in neurons. After prolonged L-glutamate stimulation, neurons lose the ability to effectively regulate intracellular calcium. This condition can lead to acute swelling and lysis of the cell, or culminate in apoptosis. Under these conditions, significant amounts of taurine (mM range) are released from the excited neuron. This extracellular taurine acts to slow the influx of calcium into the cytosol through both transmembrane ion transporters and intracellular storage pools. Two specific targets of taurine action are discussed: Na(+)-Ca2+ exchangers, and metabotropic receptors mediating phospholipase-C.

The recombinant forms of the two human isozymes of glutamate decarboxylase, GAD65 and GAD67, are potently and reversibly inhibited by molecular oxygen (Ki = 0.46 and 0.29 mM, respectively). Inhibition of the vesicle-associated glutamate decarboxylase (GAD65) by molecular oxygen is likely to result in incomplete filling of synaptic vesicles with gamma-aminobutyric acid (GABA) and may be a contributing factor in the genesis of oxygen-induced seizures. Under anaerobic conditions, nitric oxide inhibits both GAD65 and GAD67 with comparable potency to molecular oxygen (Ki = 0.5 mM). Two forms of porcine cysteine sulfinic acid decarboxylase (CSADI and CSADII) are also sensitive to inhibition by molecular oxygen (Ki = 0.30 and 0.22 mM, respectively) and nitric oxide (Ki = 0.3 and 0.2 mM, respectively). Similar inhibition of glutamate decarboxylase and cysteine sulfinic acid decarboxylase by two different radical-containing compounds (O2 and NO) is consistent with the notion that these reactions proceed via radical mechanisms.