Succinic Semialdehyde as a Substrate for the Formation of γ-Aminobutyric Acid

The conversion of succinic semialdehyde into gamma-aminobutyric acid (GABA) by GABA-transaminase was measured in rat brain homogenate in the presence of different concentrations of the cosubstrate glutamate. The calculated kinetic parameters of succinic semialdehyde for GABA-transaminase were a limiting Km value of 168 microM and a limiting Vmax value of 38 mumol g-1 h-1. Combination with previously obtained data for the conversion of GABA into succinic semialdehyde revealed a kEq value of 0.04, indicating that equilibrium of GABA-transaminase is biased toward the formation of GABA. The increased formation of GABA in the presence of succinic semialdehyde was not due to an increased conversion of glutamate into GABA by glutamic acid decarboxylase. Therefore these results indicate that succinic semialdehyde can act as a precursor for GABA synthesis.     

Effects of Anticonvulsants on the Formation of γ-Hydroxybutyrate from γ-Aminobutyrate in Rat Brain

Abstract: The conversion of γ-aminobutyrate (GABA) via succinic semialdehyde to γ-hydroxybutyrate has been examined in rat brain homogenates. A number of anticonvulsants, including sodium valproate and phenobarbitone, inhibited this metabolic pathway. These results are interpreted in the light of the characteristics of aldehyde reductases known to reduce succinic semialdehyde.     

ASSAY AND PROPERTIES OF 4-AMINOBUTYRIC-2-OXOGLUTARIC ACID TRANSAMINASE AND SUCCINIC SEMIALDEHYDE DEHYDROGENASE IN RAT BRAIN TISSUE

Abstract— The activity of 4-aminobutyric-2-oxoglutaric acid transaminase (GABA transaminase) and succinic semialdehyde dehydrogenase was determined in total rat brain homogenate. GABA transaminase activity was measured using a coupled enzyme method which utilizes endogenous succinic semialdehyde dehydrogenase to convert the formed succinic semialdehyde into succinate. The concurrently produced NADH was used as an estimate of GABA transaminase activity. This method could be used since it was shown that the dehydrogenase was about twice as active as the transaminase and because no significant accumulation of the intermediate succinic semialdehyde could be detected. GABA transaminase was inhibited by high ionic strength. In contrast NaCl decreased the apparent K _m and increased V _max for succinic semialdehyde dehydrogenase at high but not al low tissue concentrations. Increasing tissue concentration also resulted in a decrease of the apparent K _m, but did not change the V_max of succinic semialdehyde dehydrogenase and it is suggested that this enzyme can exist in two distinct states of aggregation, one with a high and one with a low affinity for succinic semialdehyde. The high affinity form of the enzyme is thought to prevent succinic semialdehyde from accumulation in the GABA transaminase assay. It is concluded that within certain limits the coupled enzyme method described here can be used for the assay of GABA transaminase activity.     

4-aminobutyrate is not available to bacteroids of cowpea Rhizobium MNF2030 in snake bean nodules

Laboratory cultures of cowpea Rhizobium MNF2030 grew on 4-aminobutyrate (GABA) as sole source of carbon and nitrogen. GABA transport was active since it was inhibited by carbonyl cyanide mchlorophenyl hydrazone and 2,4-dinitrophenol and cells developed a 400-fold concentration gradient across the cell membrane. Arsenite treatment of GABA-grown cells revealed stoichiometric conversion of GABA to pyruvate, indicating that 2-oxoglutarate is not an intermediate in GABA catabolism. GABA catabolism by cells of strain MNF2030 grown on GABA appreared to involve GABA transaminase, succinic semialdehyde dehydrogenase and malic enzyme; the first two enzymes were specifically induced by growth on GABA. The deamination process and removal of NH₃ in cells catabolizing GABA involved GABA: 2-oxoglutarate transaminase; glutamate: oxaloacetate aminotransferase; asparate: pyruvate aminotransferase and alanine dehydrogenase.Isolated snakebean bacteroids of strain MNF2030 transported only small amounts of GABA and had uninduced levels of GABA catabolic enzymes, even though the nodules contained significant levels of GABA. The data suggest that GABA is not available to snakebean nodule bacteroids, presumably because of a control imposed by the peribacteroid membrane.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid - DTT dithiothreitol - SSAD succinic semialdehyde dehydrogenase - GABAT 4-aminobutyrate transaminase - GABA 4-aminobutyrate     

Time Course of Changes in Immunoreactivities of GABA Degradation Enzymes in the Hippocampal CA1 Region after Adrenalectomy in Gerbils

Adrenalectomy (ADX) has been useful for a good in vivo model for apoptosis in the hippocampus by the absence of corticosteroids following ADX. In some neurodegenerative diseases, GABAergic neurons are more resistant to neuronal damage as compared with glutamatergic neurons. In the present study, we observed chronological changes in three GABA degradation enzymes, e.g., GABA transaminase (GABA-T), succinic semialdehyde dehydrogenase (SSADH) and succinic semialdehyde reductase (SSAR) immunoreactivity and protein levels in the gerbil hippocampal CA1 region after ADX. Changes in their immunoreactivities were distinct in the stratum pyramidale of the CA1 region. GABA-T immunoreactivity and protein level were significantly increased in the CA1 region 3 h after ADX, in contrast, SSAR and SSADH immunoreactivity and protein level were increased 12 h and 3–12 h, respectively, after ADX. These results suggest that the increases of GABA-T, SSADH and SSAR immunoreactivity and protein levels in the hippocampal CA1 region in ADX gerbils may be associated with the control of GABA levels in this region.     

Compartmentation of GABA metabolism raises intriguing questions

This synopsis covers the compartmentation of γ-aminobutyrate (GABA) metabolism, highlighting recent progress with Arabidopsis (Arabidopsis thaliana) and raising questions about mitochondrial GABA and succinic semialdehyde (SSA) transport, the fate of succinic semialdehyde once it exits mitochondria, and biochemical interactions between GABA metabolism and related processes such as photorespiration.     

Mechanism of action of an anticonvulsant, sodium dipropylacetate

The hypothesis that the brain GABA level increase which is induced by a sodium dipropyl acetate treatment arises either through inhibition of succinic semialdehyde dehydrogenase (SSADH), or through inhibition of GABA transaminase by succinic semialdehyde (SSA), has been considered. It appeared that in vivo brain GABA level increase cannot be attributed to SSADH inhibition, and that SSA is not a GABA precursor. It has been shown that SSA is neither in vivo nor in vitro a GABA-transaminase inhibitor. 4-hydroxybenzaldehyde, a potent SSADH inhibitor did not increase GABA level at a dosage which induces a 99% inhibition of SSADH.     

Purification of rat brain succinate-semialdehyde dehydrogenase and study of its inhibition by branched chain fatty acids]

Rat brain succinic semialdehyde deshydrogenase has been purified 1300 fold. This enzyme is inhibited non competitively by the same branched chain fatty acids which inhibit GABA-transaminase competitively with respect to GABA. The respective activities of GABA-T and SSADH found in rat brain indicate that at anticonvulsant doses, the acids dipropylacetic and 2-methyl 2-ethyl caproic preferentially inhibit GABA-transaminase thus inducing a rise in cerebral GABA level. This increase is therefore not due to metabolism of the succinic semialdehyde by GABA-T.     

Endogenous γ-Hydroxybutyrate in Rat Brain Areas: Postmortem Changes and Effects of Drugs Interfering with γ-Aminobutyric Acid Metabolism

Abstract: The possibility that γ-hydroxybutyrate (GHB), a metabolite of γ-aminobutyric acid (GABA), may play a role in the CNS has recently come to attention. We describe here a sensitive and specific mass fragmento-graphic technique that allows the measurement of picomole amounts of GHB in single rat brain areas. Moreover, we show that GHB can accumulate postmortem, an effect that is blocked by the use of microwave irradiation to kill the animals. To understand further the relationship between GABA and GHB formation, we treated rats with drugs known to inferfere with GABA metabolism at different levels and concomitantly measured GABA and GHB in cerebral cortex and cerebellum. Isoniazide, which blocks the formation of GABA, also decreases GHB. Blockers of the catabolism of GABA, such as aminooxyacetic acid and γ-acetylenic GABA, increase GABA levels and decrease those of GHB. Sodium dipropylacetate increases both GABA and GHB, supporting the hypothesis that this effective antiepileptic drug also blocks in vivo the enzyme that converts succinic semialdehyde to succinic acid.     

Production and Characterization of Monoclonal Antibodies to Bovine Brain Succinic Semialdehyde Reductase

Abstract: Monoclonal antibodies against bovine brain succinic semialdehyde reductase were produced and characterized. A total of nine monoclonal antibodies recognizing different epitopes of the enzyme were obtained, of which two inhibited the enzyme activity and three stained cytosol of rat spinal cord neurons as observed by indirect immunofluorescence microscopy. When unfractionated total proteins of bovine brain homogenate were separated by gel electrophoresis and immunoblotted, the antibodies specifically recognized a single protein band of 34 kDa, which comigrates with purified bovine succinic semialdehyde reductase. Using the antisuccinic semialdehyde reductase antibodies as probes, we investigated the cross-reactivities of brain succinic semialdehyde reductases from some mammalian and an avian species. The immunoreactive bands on western blots appeared to be the same in molecular mass—34 kDa—in all animal species tested, including humans. The result indicates that brain succinic semialdehyde reductase is distinct from other aldehyde reductases and that mammalian brains contain only one succinic semialdehyde reductase. Moreover, the enzymes among the species are immunologically very similar, although some properties of the enzymes reported previously were different from one another.     

PREPARATION OF SUCCINIC SEMIALDEHYDE: ITS COLORIMETRIC ESTIMATION AS AN ASSAY FOR GABA AMINOTRANSFERASE

Abstract— A simple method is described for the preparation of succinic semialdehyde by the oxidation of 4-hydroxybutyric acid with dimethyl sulphoxide in the presence of phosphorus pentoxide. The colorimetric estimation of succinic semialdehyde by condensation with 3-methyl-2-benzothiazolinone-2-hydrazone is used as an assay for GABA aminotransferase activity in mouse brain. The values obtained for the specific activity of this enzyme in mouse brain using the colorimetric method agree with those obtained by the more conventional isotopic method.     

Enzymatic preparation of radiolabeled succinic semialdehyde

[U-14C]Succinic semialdehyde was prepared with yields of 30-40% by oxidation of purified [U-14C]4-aminobutyric acid with commercially available bovine plasma monoamine oxidase. [U-14C]Succinic semialdehyde was purified by cation-exchange chromatography and quantified as the oxime and methoxime derivatives using liquid partition chromatography on silicic acid. The availability of [U-14C]succinic semialdehyde permits the reliable assay of succinic semialdehyde dehydrogenase in crude cell extracts of lymphocytes isolated from human blood, cultured human lymphoblasts, and other tissues where 4-aminobutyric acid metabolism is known to occur.     

GABA shunt deficiencies and accumulation of reactive oxygen intermediates: insight from Arabidopsis mutants

In plants, succinic semialdehyde dehydrogenase (SSADH)-deficiency results in the accumulation of reactive oxygen intermediates (ROI), necrotic lesions, dwarfism, and hypersensitivity to environmental stresses. We report that Arabidopsis ssadh knockout mutants contain five times the normal level of gamma-hydroxybutyrate (GHB), which in SSADH-deficient mammals accounts for phenotypic abnormalities. Moreover, the level of GHB in Arabidopsis is light dependent. Treatment with gamma-vinyl-gamma-aminobutyrate, a specific gamma-aminobutyrate (GABA)-transaminase inhibitor, prevents the accumulation of ROI and GHB in ssadh mutants, inhibits cell death, and improves growth. These results provide novel evidence for the relationship between the GABA shunt and ROI, which may, in part, explain the phenotype of SSADH-deficient plants and animals.     

Succinic semialdehyde couples stress response to quorum-sensing signal decay in Agrobacterium tumefaciens

Quorum sensing (QS) signal decay in Agrobacterium tumefaciens occurs in response to starvation or host signals. We have demonstrated that the gamma-aminobutyric acid (GABA) shunt metabolite links stress response to QS signal decay. Mutation of the aldH gene encoding a succinic semialdehyde dehydrogenase (SSADH) that converts succinic semialdehyde (SSA) to succinic acid results in early expression of the signal degrading enzyme, AttM. Exogenous addition of SSA or its precursor GABA induces AttM expression and abolishes Ti plasmid conjugative transfer. SSA acts by binding to the repressor AttJ that regulates the attKLM operon. attK encodes another SSADH. The stress alarmone ppGpp and SSA modulates separately the expression of the two SSADH enzymes, which might control the intracellular SSA level and hence to switch on/off the QS signal decay system in response to environmental changes. These findings document for the first time a sophisticated signalling mechanism of the widely conserved GABA degradation pathway in prokaryotes.     

Kinetic and Structural Characterization for Cofactor Preference of Succinic Semialdehyde Dehydrogenase from Streptococcus pyogenes

The γ-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers NADP⁺ over NAD⁺ as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with NADP⁺ at 1.6 Å and 2.1 Å resolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2’-phosphate group of adenine-side ribose in NADP⁺. Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.     

Virus‐induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ‐aminobutyric acid metabolic pathway

γ‐Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA‐Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus‐induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA‐Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs‐ and SlGABA‐Ts‐silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH‐silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ‐hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ‐hydroxybutyrate and subsequent ROS accumulation under salt stress.     

GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform

GABA (γ-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.     

Succinic semialdehyde dehydrogenase activity in bovine adrenal medulla and blood platelets: a comparative study with the brain enzyme

In the present paper we report the presence of succinic semialdehyde dehydrogenase (SSADH) in bovine adrenal medulla and blood platelets. Both enzymes present some analogies with the brain enzyme in terms of cofactor requirements, optimal pH, mitochondrial localizaton and inhibition by AMP. However, the activity of the platelet enzyme is 100 times lower than that of the brain and affinities of both enzymes for their specific substrate succinic semialdehyde and NAD are different. The presence of SSADH in adrenal medulla and blood platelets allows us to confirm the presence of a complete GABA bypass in these tissues, where the neurotransmitter could have important regulator functions.     

Transaminase encoded by NCgl2515 gene of Corynebacterium glutamicum ATCC13032 is involved in γ-aminobutyric acid decomposition

Corynebacterium glutamicum that expresses an exogenous l-glutamate decarboxylase (GAD) gene can synthesize γ-aminobutyric acid (GABA). GABA is decomposed to succinic semialdehyde (SSA) by GABA transaminase (GABA-T) and to succinate thereafter by SSA dehydrogenase (SSADH). However, deletion of the gabT gene encoding GABA-T could not prevent GABA from decomposing at neutral pH. In this study, an additional transaminase gene, NCgl2515, was deleted in a gabT-deleted GAD strain, and GABA fermentation in this gabT NCgl2515-deleted GAD strain was investigated. GABA concentration remained at 22.5–24.0 g/L when pH was maintained at 7.5–8.0, demonstrating that GABA decomposition was reduced. Activity assay indicated that unlike GabT, which exhibits high GABA-T activity (1.34 ± 0.06 U/mg) and utilizes only α-ketoglutarate as amino acceptor, the purified NCgl2515 protein exhibits very low GABA-T activity (approximately 0.03 U/mg) only when coupled with the SSADH, GabD, but can utilize both α-ketoglutarate and pyruvate as amino acceptor. The optimum pH for coupled NCgl2515–GabD was 8.0, similar to that of GabT (7.8). Therefore, NCgl2515 has weak GABA-T activity and is involved in GABA decomposition in C. glutamicum. Deletion of gabT and NCgl2515 could effectively reduce GABA decomposition at neutral pH.