The study of GABA synthesis regulation of GABA biosensor

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammal brain. Glutamic acid decarboxylase (GAD) catalyses alpha-decarboxylation of L-glutamate to GABA. The amino acid sequence of GAD isoforms, GAD 65 and GAD67, shows two distinct domains, a conserved carboxy domain and a divergent short amino-terminal domain. GAD65 activity is regulated by cofactor, pyridoxal 5'-phosphate (PLP), and GAD67 is relatively non-responsive to PLP. Despite this difference, catalytic PLP binding sites in both GAD65 and GAD67 are located in conserved carboxy domains. In order to identify region that regulates GABA synthesis, chimeric forms of rat GAD by domain swapping were engineered and expressed in CHO cells. Chimeric GAD activity was assayed with new fluorometric GABA detection method. The results suggest that GAD amino-terminal domain regulates GABA synthesis. The mechanism may explain the regulation of metabolic and neurotransmitter GABA synthesis.;New fluorometric GABA detection method is safe and convenient as well as applicable to studying the relationship between genetic information and enzymatic function of GAD isoforms. New fluorometric GABA detection method was applied to develop two novel biosensors, spectrophotometric and fluorometric biosensor. Both biosensors are made by the sol-gel method that encapsulates enzyme in porous silica glass. Recombinant enzymes were encapsulated in porous silica glass and detected GABA with two methods. Furthermore, GABA biosensor detected GABA, which was generated in CHO cells. Therefore, GABA biosensors will be useful for studies of both metabolic GABA production and neuronal GABA release. The applications based on GABA biosensor technology are very diverse and include such as in vivo GABA detection or robotic GABA detection.;The study of GAD isoform regulation and GABA biosensor is useful tools to screen drugs for diverse neuronal and non-neuronal diseases.