To continue the purification of the rate-limiting enzymes involved in the synthesis of gamma-aminobutyric acid (GABA), glycine, aspartate/glutamate, and taurine, namely L-glutamate decarboxylase (GAD), serine transhydroxymethylase (serine-THM), cysteinesulfinic and cyteic acids decarboxylase (CSAD/CAD) and aspartate amino transferase (AAT) and glutaminase (GLNase), respectively. The purified enzyme preparations will be used as antigens for the production of specific polyclonal antibodies and for the screening of the production of monoclonal antibodies as previously described. To further identify the synaptic connectivities and the neuronal projections which involve GABA, glycine, taurine or aspartate/glutamate as their neurotransmitters by immunocytochemical localization of their synthetic enzymes, namely GAD, serine-THM, CSAD/CAD, and AAT/GLNase, respectively. In addition, double-labeling techniques will be employed to identify two antigens on the same brain section so that the synaptic connectivity and the nature of co-existence of amino acid transmitters with other neuroactive substances, e.g., neuropeptides, can be elucidated. To elucidate the mechanism by which the steady-state level of GABA is regulated. We propose to study the effect of PLP- and Zn++-deficiency on the levels of GABA, GAD activity and mRNA for GAD. In addition, we also propose to isolate and characterize Zn++-binding protein and to determine whether this Zn++-binding protein may function as a regulator for Zn++-mediated processes, similar to Ca++-binding protein, calmodulin, which mediates many Ca++-dependent cellular processes. To purify GAD mRNA by polysome immunoprecipitation and to use the purified mRNA for synthesis and cloning of its cDNA. A rat brain cDNA library will be constructed and the clone with the longest DNA insert will be selected for sequence analysis. We also plan to use (H3)cDNA probes to localize specific mRNA for GAD in situ by nucleic acid hybridization and GAD by immunocytochemistry. Recently, we have successfully localized a low molecular weight apolipoprotein, apoVLDL-II and its mRNA, by a combination of immunohistochemistry and in situ hybridization to cloned (H3)apoVLDL-II cDNA. To elucidate the nature of glial GAD and its relationship to neuronal GAD with a view to determining the functional role of glia in neurotransmission. We plan to use hybridoma technique which we have successfully developed for neuronal GAD to obtain monoclonal antibody to glial GAD.
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