Role of transamination in the mobilization of respiratory substrates in germinating seeds of castor oil plants

The metabolism of 1,4-14C-succinate and 2,3-14C-succinate and the activity of succinic semialdehyde dehydrogenase (EC 1.2.1.16) were studied in germinating seeds of castor oil plants (Ricinus communis L.). Succinate metabolism involved succinate dehydrogenase and was sensitive to metabolites of the gamma-aminobutyric acid shunt. Considerable accumulation of the label in amino acids reflected the progression of transamination reactions. Succinic semialdehyde dehydrogenase was purified from the endosperm of castor oil plants. Kinetic characteristics of the enzyme were evaluated. Our study indicates that the mobilization of respiratory substrates during germination of castor oil plants is related to active transamination of ketoacids in the Krebs cycle and involves the gamma-aminobutyric acid shunt.     

Biosynthesis of γ-aminobutyric acid from spermine in maize seedlings

The administration of labelled spermine [tetramethylene-1,4-¹⁴C] to Zea mays shoots resulted in the formation of radioactive γ-aminobutyric acid (GABA). A chemical degradation of radioactive GABA suggested that its radioactivity was located on C-1 and C-4, indicating that GABA is a product of spermine metabolism in maize seedlings.     

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.     

Unraveling the function of paralogs of the aldehyde dehydrogenase super family from Sulfolobus solfataricus

Aldehyde dehydrogenases (ALDHs) have been well established in all three domains of life and were shown to play essential roles, e.g., in intermediary metabolism and detoxification. In the genome of Sulfolobus solfataricus, five paralogs of the aldehyde dehydrogenases superfamily were identified, however, so far only the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) and α-ketoglutaric semialdehyde dehydrogenase (α-KGSADH) have been characterized. Detailed biochemical analyses of the remaining three ALDHs revealed the presence of two succinic semialdehyde dehydrogenase (SSADH) isoenzymes catalyzing the NAD(P)⁺-dependent oxidation of succinic semialdehyde. Whereas SSO1629 (SSADH-I) is specific for NAD⁺, SSO1842 (SSADH-II) exhibits dual cosubstrate specificity (NAD(P)⁺). Physiological significant activity for both SSO-SSADHs was only detected with succinic semialdehyde and α-ketoglutarate semialdehyde. Bioinformatic reconstructions suggest a major function of both enzymes in γ-aminobutyrate, polyamine as well as nitrogen metabolism and they might additionally also function in pentose metabolism. Phylogenetic studies indicated a close relationship of SSO-SSALDHs to GAPNs and also a convergent evolution with the SSADHs from E. coli. Furthermore, for SSO1218, methylmalonate semialdehyde dehydrogenase (MSDH) activity was demonstrated. The enzyme catalyzes the NAD⁺- and CoA-dependent oxidation of methylmalonate semialdehyde, malonate semialdehyde as well as propionaldehyde (PA). For MSDH, a major function in the degradation of branched chain amino acids is proposed which is supported by the high sequence homology with characterized MSDHs from bacteria. This is the first report of MSDH as well as SSADH isoenzymes in Archaea.     

Devenir de la putrescine-1,4-14C chez Glycine max

Fate of Putrescine-1,4-¹⁴C in Glycine max Putrescine-1,4-¹⁴C was supplied to young decotylized Glycine max (L.) Merr. cv. Chippewa plants growing under aseptic conditions on a liquid medium with nitrogen supplied either as ammonium chloride or nitrates. Whatever the source of nitrogen the diamine was quickly transformed to γ-aminobutyric acid, succinic acid and malic acid; only a very minor part was utilized for the synthesis of polyamines. In the presence of ammonium chloride the putrescine catabolism may be slower than in the presence of nitrates. The results are explained by a weak isotopic dilution or by a diamine oxidase activity lower in “ammonium” plants than in “nitrate” plants; the two causes might co-exist. The possibility of a diamine compartmentation has to be considered.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Succinic semialdehyde dehydrogenase of wheat grain

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6–9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K _m for SSA and for NAD are 7.4x10^-6 M and 2x10^-4 M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.Abbreviations GABA -aminobutyric acid - GABA-T -aminobutyric acid transaminase - ME mercaptoethanol - SSA succinic semialdehyde - SSA-DH succinic semialdehyde dehydrogenase     

Metabolism of the alpha-Pyridone Ring of Ricinine in Ricinus communis L

Chemically synthesized ricinine-3,5-¹⁴C was used to study the metabolism of this alkaloid in the plant which produces it, Ricinus communis L. In a time course study, ricinine-3,5-¹⁴C was administered to a series of castor plants (Ricinus communis L.) and the radioactivity recovered in the ricinine samples showed a decrease with increase in time. It was also observed that the alkaloid was translocated to the seed. The in vivo conversion of ricinine-3,5-¹⁴C to respiratory ¹⁴CO₂ occurred in both light and dark and indicated that the α-pyridone ring of ricinine could be degraded.     

Distribution and oxidation of malondialdehyde in mice

The metabolism of melondialdehyde (MDA) by male and female Swiss mice was investigated. Distribution of an i.p. dose of MDA is rapid and uniform throughout the body. Conversion of ¹⁴C-labeled MDA to CO₂ is complete 4 hours after an i.p. dose of 5 μmol to 200 μmol with no signs of short term toxicity. The yields of CO₂ from [1-¹⁴C]-β-alanine, [3-¹⁴C]-β-alanine, [1-¹⁴C]-sodium acetate, and [2-¹⁴C]-sodium acetate were also determined. Comparison of the yields of CO₂ from this series of compounds suggests the intermediacy of malonic semialdehyde in the metabolism of MDA. High doses (600 μmol) of β-alanine or acetate given prior to ¹⁴C-MDA reduced the yield of ¹⁴CO₂. Ethanol and disulfiram were both inhibitors of MDA metabolism, indicating the involvement of aldehyde dehydrogenase in the oxidation of MDA.These data demonstrate the ability of animal tissues to rapidly remove exogeneously administered MDA. They also have implications with respect to the possible pathological consequences of MDA generation.     

Efflux of γ-aminobutyric acid from and appearance of free arachidonic acid inside synaptosomes

When synaptosomes were depolarized in the presence of Ca²⁺, or when Ca²⁺ was added to synaptosomes pretreated with Ca²⁺ ionophore (A23187), free arachidonic acid was clearly increased within synaptosomes, and at the same time an efflux of γ-aminobutyric acid from synaptosomes was observed. Moreover, when synaptosomes labelled with [¹⁴C]arachidonic acid were depolarized in the presence of Ca²⁺, there was a significant decrease in the radioactivity of the fatty acid of phosphatidylinositol and phosphatidylcholine. Exogenously added arachidonic acid, but not other fatty acids, stimulated the efflux of γ-aminobutyric acid in the absence of Ca²⁺. These observations suggest that the release of arachidonic acid from phospholipids is an intrinsic part of the biochemical mechanism that modulates the γ-aminobutyric acid efflux.     

The γ‐aminobutyric acid shunt contributes to closing the tricarboxylic acid cycle in Synechocystis sp. PCC 6803

A traditional 2‐oxoglutarate dehydrogenase complex is missing in the cyanobacterial tricarboxylic acid cycle. To determine pathways that convert 2‐oxoglutarate into succinate in the cyanobacterium Synechocystis sp. PCC 6803, a series of mutant strains, Δsll1981, Δslr0370, Δslr1022 and combinations thereof, deficient in 2‐oxoglutarate decarboxylase (Sll1981), succinate semialdehyde dehydrogenase (Slr0370), and/or in γ‐aminobutyrate metabolism (Slr1022) were constructed. Like in Pseudomonas aeruginosa, N‐acetylornithine aminotransferase, encoded by slr1022, was shown to also function as γ‐aminobutyrate aminotransferase, catalysing γ‐aminobutyrate conversion to succinic semialdehyde. As succinic semialdehyde dehydrogenase converts succinic semialdehyde to succinate, an intact γ‐aminobutyrate shunt is present in Synechocystis. The Δsll1981 strain, lacking 2‐oxoglutarate decarboxylase, exhibited a succinate level that was 60% of that in wild type. However, the succinate level in the Δslr1022 and Δslr0370 strains and the Δsll1981/Δslr1022 and Δsll1981/Δslr0370 double mutants was reduced to 20–40% of that in wild type, suggesting that the γ‐aminobutyrate shunt has a larger impact on metabolite flux to succinate than the pathway via 2‐oxoglutarate decarboxylase. ¹³C‐stable isotope analysis indicated that the γ‐aminobutyrate shunt catalysed conversion of glutamate to succinate. Independent of the 2‐oxoglutarate decarboxylase bypass, the γ‐aminobutyrate shunt is a major contributor to flux from 2‐oxoglutarate and glutamate to succinate in Synechocystis sp. PCC 6803.     

Characterising the Role of GABA and Its Metabolism in the Wheat Pathogen Stagonospora nodorum

A reverse genetics approach was used to investigate the role of γ-aminobutyric acid metabolism in the wheat pathogenic fungus Stagonospora nodorum. The creation of mutants lacking Sdh1, the gene encoding succinic semialdehyde dehydrogenase, resulted in strains that grew poorly on γ-aminobutyric acid as a nitrogen source. The sdh1 mutants were more susceptible to reactive oxygen stress but were less affected by increased growth temperatures. Pathogenicity assays revealed that the metabolism of γ-aminobutyric acid is required for complete pathogenicity. Growth assays of the wild-type and mutant strains showed that the inclusion of γ-aminobutyric acid as a supplement in minimal media (i.e., not as a nitrogen or carbon source) resulted in restricted growth but increased sporulation. The addition of glutamate, the precursor to GABA, had no effect on either growth or sporulation. The γ-aminobutyric acid effect on sporulation was found to be dose dependent and not restricted to Stagonospora nodorum with a similar effect observed in the dothideomycete Botryosphaeria sp. The positive effect on sporulation was assayed using isomers of γ-aminobutyric acid and other metabolites known to influence asexual development in Stagonospora nodorum but no effect was observed. These data demonstrate that γ-aminobutyric acid plays an important role in Stagonospora nodorum in responding to environmental stresses while also having a positive effect on asexual development.     

Inborn errors of GABA metabolism

Defects in man in four steps of 4-aminobutyric acid (GABA) metabolism may interefere with the function of this major inhibitory neurotransmitter. Glutamic acid decarboxylase, 4-aminobutyric acid aminotransferase, succinic semialdehyde dehydrogenase, and homocarnosinase are closely identified with the brain, but two of these enzymes are expressed in cultured peripheral cells, which may permit novel approaches to the study of the metabolism and regulation of GABA.     

Metabolism of alpha-Ketoglutarate by Roots of Woody Plants

The uptake and metabolism of α-ketoglutarate-5-¹⁴C by peach, apple, and privet root tissues were studied over various time intervals. As much as 80% of the absorbed ¹⁴C appeared as ¹⁴CO₂ in 320 minutes in peach roots. Apple and privet roots were less effective in this conversion with the bulk of the ¹⁴C found in the organic acid fraction. This indicates differences in organic acid metabolism among species of woody plants.

The ¹⁴C accumulated in malate earlier and in larger quantities than in citrate. Both glutamate and aspartate were labeled in 10 minutes and glutamate was labeled as early as 3 minutes. The labeling pattern does not clearly distinguish between the synthesis of glutamate by glutamic dehydrogenase or by transamination with oxaloacetate.

The rapid metabolism of α-ketoglutarate to glutamate by the 3 species studied indicates the presence of enzyme systems important in amino acid synthesis in the roots of woody plants.

     

γ-Aminobutyric acid-stimulated chloride permeability in crayfish muscle

γ-Aminobutyric acid selectively increased Cl^? permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in VAn Harreveld's solution at room temperature took up ³⁶Cl^? to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-s incubations, the control ³⁶Cl^? uptake space was 131 ± 4 ml/kg (n = 60) and this was significantly increased by γ-aminobutyric acid at 200 μM or higher concentrations to 177 ± 4 ml/kg (n = 48, P < 0.05). This effect was specific for chloride since γ-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. γ-Aminobutyric acid stimulation of ³⁶Cl^? uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The γ-aminobutyric acid stimulation of Cl^? permeability is dose dependent with 50% of the maximal effect at 40 μM γ-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The γ-aminobutyric acid agonist muscimol causes the same maximal effect on Cl^? uptake as γ-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist γ-amino, β-hydroxybutyric acid produced little or no stimulation of ³⁶Cl^? flux. The response to γ-aminobutyric acid was blocked by 2 mM β-guanidinopropionate or γ-guanidinobutyrate, 0.5 mM bicuculline, and 10 μM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 μM. Antagonists did not affect control ³⁶Cl^? uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter γ-aminobutyric acid involves a rapid increase in membrane permeability to Cl^?     

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD⁺/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.     

The ALDH21 gene found in lower plants and some vascular plants codes for a NADP+‐dependent succinic semialdehyde dehydrogenase

Lower plant species including some green algae, non‐vascular plants (bryophytes) as well as the oldest vascular plants (lycopods) and ferns (monilophytes) possess a unique aldehyde dehydrogenase (ALDH) gene named ALDH21, which is upregulated during dehydration. However, the gene is absent in flowering plants. Here, we show that ALDH21 from the moss Physcomitrella patens codes for a tetrameric NADP⁺‐dependent succinic semialdehyde dehydrogenase (SSALDH), which converts succinic semialdehyde, an intermediate of the γ‐aminobutyric acid (GABA) shunt pathway, into succinate in the cytosol. NAD⁺ is a very poor coenzyme for ALDH21 unlike for mitochondrial SSALDHs (ALDH5), which are the closest related ALDH members. Structural comparison between the apoform and the coenzyme complex reveal that NADP⁺ binding induces a conformational change of the loop carrying Arg‐228, which seals the NADP⁺ in the coenzyme cavity via its 2′‐phosphate and α‐phosphate groups. The crystal structure with the bound product succinate shows that its carboxylate group establishes salt bridges with both Arg‐121 and Arg‐457, and a hydrogen bond with Tyr‐296. While both arginine residues are pre‐formed for substrate/product binding, Tyr‐296 moves by more than 1 Å. Both R121A and R457A variants are almost inactive, demonstrating a key role of each arginine in catalysis. Our study implies that bryophytes but presumably also some green algae, lycopods and ferns, which carry both ALDH21 and ALDH5 genes, can oxidize SSAL to succinate in both cytosol and mitochondria, indicating a more diverse GABA shunt pathway compared with higher plants carrying only the mitochondrial ALDH5.     

Distribution of Three Enzymes of γ-Aminobutyric Acid Metabolism in Monkey Retina

Abstract: The distributions of glutamate decarboxylase (EC 4.1.1.15), γ-aminobutyric acid transaminase (EC 2.6.1.19), and succinate semialdehyde dehydrogenase (EC 1.2.1.24) were determined in monkey retina. The decarboxylase was almost restricted to the inner plexiform layer. The transaminase was also highest in this layer, but activities were 40% as high in the adjacent third of the inner nuclear layer and in the ganglion cell and fiber layers. Succinate semialdehyde dehydrogenase was distributed very differently. Although it also showed a peak of activity in the inner plexiform layer, there was a second equal peak in the photoreceptor inner segment layer and a smaller peak in the outer plexiform layer, regions where both γ-aminobutyric acid transaminase and glutamate decarboxylase were essentially absent.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

Biosynthesis of azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.