Pharmacological manipulation of neurotransmitter metabolism has been a powerful tool in the treatment of nervous and mental diseases. Many insights in psychopharmacology have resulted from our basic knowledge of neurotransmitter systems. The long-term goal of this research is to elucidate the control mechanisms that regulate the synthesis of gamma-aminobutyric acid (GABA), a major neurotransmitter in brain. Current evidence suggests that glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis, is regulated at two levels. At one level, the amount of holoGAD (the active form containing bound cofactor, pyridoxal-5'-phosphate) appears to be controlled by a cycle of inactivation (apoGAD formation) and reactivation (holoGAD formation) which is under allosteric control by ATP and Pi. This mechanism is supported by enzymatic and neurochemical studies. At the second level, GAD activity may be regulated by the interconversion of different forms of the enzyme each of which has different regulatory properties. We have recently discovered multiple forms of GAD that appear to differ in the extent to which they undergo the cycle of inactivation and reactivation (level one regulation). These forms could result from several mechanisms; for example, from interconversion of the forms (eg. by phosphorylation) in response to changes in neuronal activity, or from maturation of GAD as it moves from the ribosome to the synapse. They could also result from the combination of non-identical subunits as does lactate dehydrogenase or could be different forms found in different cell types or brain regions. We plan to examine these possibilities by studies of the purified enzyme and studies with brain slices, synaptosomes and in vivo. We plan a structural comparison of the forms (e.g. to determine subunit structure, possibility of phosphorylation, etc.). Such studies will show how the forms differ and thus provide important information on how they could be interconverted. We also plan studies with synaptosomes, and brain slices to determine if depolarization alters that inactivation/reactivation cycle of each form or causes the conversion of one form to another. We plan to determine if the relative amounts of the forms differ among brain regions or during development. Finally, we plan to compare the regulatory and structural differences among the forms from human brain.
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