Gamma - Aminobutyric Acid
3. Sources and Properties of Glutamate Decarboxylase
Glutamate decarboxylase (GAD; EC 4.1.1.15) is the pyridoxal - 5 ′ - L - phosphate (PLP) - dependent enzyme that synthesizes GABA (Sardana et al. 2006 ). In higher plants, GAD has been purifi ed and character- ized from cowpea (Johnson et al. 1997 ), squash (Matsumoto et al. 1986 ), potato (Satyanarayan and Nair 1985 ), and rice germ (Zhang et al. 2007 ). The optimum synthesis pH is between 5.5 and 6.5, and the optimum temperatures of most plants ’ GAD activity is between 37 and 40 ° C (with the exception of squash GAD, which has an optimal temperature of 60 ° C). A unique feature of plant and yeast GAD is the ability to bind calmodulin (CaM), an acidic, bilobed protein (molecular mass of 16.7 kDa) that can activate around 40 distinct proteins in a calcium - dependent manner. Most CaM - binding domain regions are basic, hydrophobic, and devoid of mul-
Figure 9.1. Schematic representation of neutral (a) and zwitterionic (b) GABA structures.
(1989) also indicates that glutamate and GABA are produced during protein storage and mobilization as a means of recycling arginine - derived nitrogen and carbon.
4.1.3. Plant Defense Phytophagous activity by insects and other invertebrates destroys vacuolar compartmentalization, increases H + levels in the cytosol, and stimulates GABA synthesis. Ramputh and Bowm (1996) investigated the hypothesis that mechanical stimulation or damage resulting from phytophagous insects stimulated GABA accumula- tion, which in turn deterred insect growth and devel- opment. Results showed that increasing GABA levels in a synthetic diet from 1.6 to 2.6 μ mol · g − 1 fresh weight reduced the growth, developmental, and survival rates of cultured Harris larvae.
4.1.4. Plant Development GAD is a soluble protein with a Ca 2+ /calmodulin binding domain, and its expression level is regulated during development by transcription and posttranscriptional processes.
Genetic studies show that changing GABA concen- tration had severe consequences on plant develop- ment (Bouch é et al. 2003 ). Transgenic plants that ectopically expressed a constitutively active GAD enzyme have a higher concentration of GABA and a lower concentration of glutamate, resulting in abnormal growth and development. Plant GADs carry a C - terminal extension that binds to Ca 2+ / calmodulin to modulate enzyme activity. Kazuhito and Fumio (2007) investigated rice, which possesses two distinct GADs, OsGAD1 and OsGAD2. Both have a C - terminal extension, but the former peptide 0.03 to 2.00 μ mol/g fresh weight (Steward 1949 ).
GABA can be accumulated rapidly under several environmental stress conditions, such as mechanical stimulation, damage, cold shock (Wallace et al.
1984 ), heat shock, hypoxia (Shelp et al. 1995 ), cyto- solic acidifi cation, darkness, water stress (Handa et al. 1983 ), phytohormones, and drought stress (Serraj et al. 1998 ). In plants, rapid, stress - induced GABA accumulation is well documented, with levels increasing 20 - to 40 - fold within 5 minutes. However, the role of GABA accumulation is contentious.
4.1.1. p H Regulation In vitro GAD from a variety of tissues has a sharp pH optimum around 5.8, with 10 – 40% of maximal activity at pH 7.0 (Bown and Shelp 1997 ). Because GAD activity increases as pH decreases and glutamate decarbox- ylation is a proton consuming reaction, it follows that GABA production could act as a sink for excess protons in the cytoplasm and, thus, regulate pH (Carroll et al. 1994 ; Crawford et al. 1994 ; Snedden et al. 1992 ). In vivo 15 N and 31 P NMR spectroscopy also shows that GABA synthesis is stimulated by a decrease in cytoplasmic pH.
4.1.2. Nitrogen Storage The conversion of glu- tamate to GABA increases under conditions that inhibit glutamine synthesis, reduce protein synthe- sis, or enhance protein degradation (Scott - Taggart et al. 1999 ). This prompts the hypothesis that GABA is a temporary nitrogen store. In addition, glutamate fl ux through the GABA shunt is comparable to the direct incorporation of glutamate into protein (Tuin and Shelp 1996 ). Evidence from Micallef and Shelp
Table 9.1. Properties of GAD from lactic acid bacteria.
Species
Subunit MW (kDa)
Optimum pH
Optimum Temperature
Km
(mM) Reference
Lactococcus lactis subsp. 54 4.7 — 0.51 Nomura et al. 1999
Lactobacillus brevis CGMCC 1306 62 4.4 37 8.22 Huang et al. 2007
Lactobacillus brevis IFO 2005 60 4.2 30 9.3 Ueno et al. 1997
Lactococcus lactis 1.009 65 4.7 50 1.9 Xu et al. 2004
Lactobacillus brevis OPK - 3 53.4 — — — Park and Oh 2007
receptors have been studied. A single injection of low doses of different antidepressants does not seem to affect brain GABA - A receptor binding in rat brains. Repeated or prolonged administration of antidepressants is either without effect or downregu- lates GABA - A receptor complexes. There are con- fl icting reports with regard to effects of antidepressants on GABA receptor binding sites. For example, several different classes of antidepressant agents including tricyclic antidepressants, monoamine oxidase inhibitors, and selective serotonin reuptake inhibitors induce an increase in GABA receptor binding sites in the rat frontal cortex and hippocam- pus following chronic administration.
4.2.2. Curing Neurological Disease Stiff - person syndrome (SPS) is a rare neurologic disease charac- terized by muscle rigidity and episodic painful spasms (Yamamoto et al. 2007 ). Although the etiol- ogy is not well understood, it is postulated that the pathophysiology of SPS is created by antibodies against GAD (Moersch and Woltman 1956 ). The loss of GABA - ergic input from inhibitory spinal interneurons and impaired supraspinal GABA - ergic neurons leads to the hyperexcitability of motor neurons and subsequent progressive muscle rigidity (Sandbrink et al. 2000 ).
In Parkinson ’ s, GABA is reduced in an area of the brain called the subthalamic nucleus. This region is working in “ overdrive ” in the disease process, and it is important to calm this hyper - reactive circuit because GABA is an inhibitory transmitter.
There is normally an increase in extracellular K + during a seizure. Along with the increase in intra- cellular Na + and depolarization, carrier - mediated GABA release would be greater during seizures and would reduce excitability. Indeed, the anticonvul- sant gabapentin can enhance nonvesicular GABA release induced via heteroexchange release by the GABA analog nipecotic acid. Along with the recently described effect of gabapentin on calcium currents, enhancing nonvesicular GABA release could inhibit seizures.
4.2.3. Preventing Brain Senescence Human cerebral cortical function degrades during aging.
contains an authentic CaM - binding domain, while the latter does not. The results suggested that the C - terminal extension of OsGAD2 is a strong auto- inhibitory domain and that truncation of this domain causes the enzyme to act constitutively with higher activity both in vitro and in vivo.
4.1.5. Potential Modulator of Ion Transport Kinnersley and Lin (2000) studied duckweed to investigate whether plants also possessed GABA receptors. Results supported the hypothesis that GABA activity in plants involved interaction with a receptor, an effect on mineral transport, and sug- gested a way that GABA could amplify stress per- ception in plants. The association between growth and mineral content of GABA - treated plants sup- ported the hypothesis that one of the functions of GABA in plants involved regulating ion transport.
4.2. Roles of GABA in Animals
In 1950, GABA was discovered in mammalian brain tissue (Roberts and Frankel 1950 ). The concentra- tion of GABA in these tissues is 1,000 times greater than amine. GABA is now recognized as the most important inhibitory neurotransmitter in the mam- malian CNS. From 30 – 50% of all central synapses are GABA - ergic (Paredes and Agmo 1992 ). GABA has several physiological functions, such as neuro- transmission, tranquilization, and hypotensive and diuretic effects.
4.2.1. Regulating Mood Several lines of preclini- cal and clinical evidence suggest that the amino acid neurotransmitter GABA has a role in mood disor- ders, particularly depression (Emrich et al. 1980 ; Shiahs and Yatham 1998 ). GABA - A receptors are heteropentameric ligand - gated chloride channels that mediate the majority of rapid inhibitory synap- tic transmission (Sieghart 1992 ). GABA - B recep- tors appear to be of major importance in synaptic processing within the brain and are present at both post - and presynaptic sites. Their activation can hyperpolarize neurons and diminish neurotransmit- ter release from presynaptic terminals (Bowery 1993 ). The effects of antidepressant drugs on GABA
detoxifi cation of ammonia to glutamine and decreas- ing diffusion of ammonia from the blood into the brain (Paul 2003 ).
Weight loss (Van den Pol 2003 ): Body weight is maintained via a homeostatic system involving the CNS. Recently, a study found that GABA and glu- tamate were present in key neurons that regulate body weight and, consequently, may represent important orexigenic/anorexigenic mediators that convey information to other neurons within the hypothalamus as well as from the hypothalamus to other brain regions that participate in energy regula- tion (Meister 2007 ).
Regulate sleep: Luppi et al. (2007) found that the tonic inhibition of locus coeruleus noradrenergic and dorsal raphe serotonergic neurons during sleep was due to a tonic GABA - ergic inhibition by neurons localized in the dorsal paragigantocellular reticular nucleus and the ventrolateral periaqueduc- tal gray.
5. Preparation of GABA