We plan to perform the following studies: 1. To purify both benzodiazepines (BDZP) and GABA receptors to homogeneity by benzodiazepine-Sepharose affinity columns and by methods similar to those we have successfully developed for the purification of various transmitter enzymes and proteins. 2. To characterize BDZP and GABA receptors in terms of their physical and chemical properties, with a special emphasis on their subunit structures which are essential for the understanding of the mechanism of allosteric interaction between BDZP, GABA and these receptors. To determine whether one type of subunit is for ligand binding and another one is as regulatory subunit, similar to an allosteric enzyme, aspartate transcarbamylase. Also, to elucidate the role of each subunit in binding to agonists, antagonists, and ions similar to what have been done for acetylcholine receptor. 3. To obtain specific polyclonal and monoclonal antibodies with the same procedures that we have developed for GABA-synthesizing enzyme, L-glutamate decarboxylase (GAD) and to develop sensitive immunochemical techniques such as radioimmunassay for both BDZP and GABA receptors in order to determine the amount of receptor proteins in addition to their binding activities. 4. To elucidate the inter-relationship between GABA and BDZP systems by localizing GAD, GABA-receptor and BDZP receptor by a combination of immunocytochemical and autoradiographic methods and by double-staining techniques. 5. To isolate and identify the nature of endogenous BDZP-like substances.
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