Succinic Semialdehyde Dehydrogenase Activity Research Articles

Inhibition of succinic semialdehyde dehydrogenase activity by alkenal products of lipid peroxidation

Lipid peroxidation causes the generation of the neurotoxic aldehydes acrolein and 4-hydroxy- trans-2-nonenal (HNE). These products are elevated in neurodegenerative diseases and acute CNS trauma. Previous studies demonstrate that mitochondrial class 2 aldehyde dehydrogenase (ALDH2) is susceptible to inactivation by these alkenals. In the liver and brain another mitochondrial aldehyde dehydrogenase, succinic semialdehyde dehydrogenase (SSADH/ALDH5A1), is present. In this study, we tested the hypothesis that aldehyde products of lipid peroxidation inhibit SSADH activity using the endogenous substrate, succinic semialdehyde (SSA, 50 μM). Acrolein potently inhibited SSADH activity (IC 50=15 μM) in rat brain mitochondrial preparations. This inhibition was of an irreversible and noncompetitive nature. HNE inhibited activity with an IC 50 of 110 μM. Trans-2-hexenal (HEX) and crotonaldehyde (100 μM each) did not inhibit activity. These data suggest that acrolein and HNE disrupt SSA metabolism and may have subsequent effects on CNS neurochemistry.

Brain GABA metabolism after chronic ethanol consumption and withdrawal in rats.

We have studied the activities of the GABA metabolizing enzymes-GABA aminotransferase (GABA-AT), succinic semialdehyde dehydrogenase (SSA-DH) and SSA reductase (SSA-R) and the levels of GABA, glutamine and glutamate in rats preferring water (WP) or ethanol (EP) after 6 months of ethanol consumption and 12 hours to 7 days after withdrawal. We showed decreased GABA levels in the brain stem, decreased GABA-AT activity in the hemispheres and brain stem, and enhanced GABA-AT activity in the striatum of EP rats compared with the control or WP animals following chronic consumption of ethanol. We found decreased activity of SSA-R and SSA-DH in the hemispheres and brain stems of alcohol-treated rats compared to the control rats. Withdrawal (0.5-7 days) induced numerous profound changes in GABA metabolism.

Enzymatic and immunologic identification of succinic semialdehyde dehydrogenase in rat and human neural and nonneural tissues.

We have identified succinic semialdehyde dehydrogenase protein in rat and human neural and nonneural tissues. Tissue localization was determined by enzymatic assay and by western immunoblotting using polyclonal antibodies raised in rabbit against the purified rat brain protein. Although brain shows the highest level of succinic semialdehyde dehydrogenase activity, substantial amounts of enzyme activity occur in mammalian liver, pituitary, heart, and ovary. We further demonstrate the absence of succinic semialdehyde dehydrogenase enzyme activity and protein in brain, liver, and kidney tissue samples from an individual affected with succinic semialdehyde dehydrogenase deficiency, thereby verifying the specificity of our antibodies.

Pre- and postnatal diagnosis of succinic semialdehyde dehydrogenase deficiency using enzyme and metabolite assays.

We report our cumulative experience for the prenatal diagnosis of succinic semialdehyde dehydrogenase (SSADH) deficiency in seven 'at-risk' pregnancies from four unrelated families. Prenatal diagnosis was performed by determination of 4-hydroxybutyric acid (4-HBA) concentration in amniotic fluid using isotope-dilution gas chromatography-mass spectrometry in conjunction with assay of SSADH activity in biopsied chorionic villus and/or cultured amniocytes. In three of four pregnancies predicted as affected, confirmation was obtained by demonstration of deficient SSADH activity in fetal tissues. Our results suggest that determination of 4-HBA concentration in amniotic fluid combined with enzyme determination in cultured or biopsied tissue represents a reliable method for the prenatal diagnosis of SSADH deficiency.,

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.

A Rhizobium meliloti mutant, lacking a functional γ-aminobutyrate (GABA) bypass, is defective in glutamate catabolism and symbiotic nitrogen fixation

A Rhizobium meliloti mutant, CMF1 2:38, was isolated which was specifically defective in the degradation of glutamate as sole carbon and nitrogen source. Biochemical analysis of CMF1 2:38 revealed a reduction in succinic semialdehyde dehydrogenase (SSDH) activity, the third enzyme of the γ-aminobutyrate (GABA) bypass. Evidence is presented which suggests that the Tn 5-induced mutation in CMF1 2:38 exists in a regulatory gene governing the expression of both NAD and NADP-linked SSDH activity. CMF1 2:38 nodulated alfalfa plants, but was reduced in its nitrogen fixation activity and biomass accumulating ability relative to the wild-type strain. The results presented in this study indicate that the GABA bypass is a major mechanism of glutamate degradation in R. meliloti CMF1 and that glutamate catabolism via this pathway may play an important role in the symbiotic nitrogen fixation process.

A fluorimetric assay for succinic semialdehyde dehydrogenase activity suitable for prenatal diagnosis of the enzyme deficiency.

Succinic semialdehyde dehydrogenase (SSAD) is an enzyme involved in the turnover of the neurotransmitter 4-aminobutyrate (GABA). Deficiency of SSAD results in developmental delay, ataxia, seizures and 4-hydroxybutyric aciduria. We have developed a simple fluorimetric assay for the enzyme and applied it to measurement of SSAD activity in a range of cell types often used for prenatal and postnatal diagnosis of enzyme defects. Lymphocytes from children with SSAD deficiency were found to have < 5% of the activity found in lymphocytes from normal children. Heterozygotes are asymptomatic and have intermediate enzyme activities. Although SSAD activity has been detected previously in uncultured chorionic villi, we found that SSAD was not expressed in cultured chorionic villus cells nor in some fibroblast-like amniocytes from control fetuses. Lymphocytes from fetal blood and non-fibroblastic amniocytes have high SSAD activities, and should be suitable for prenatal diagnosis of SSAD deficiency.

4-Hydroxybutyric aciduria: Further clinical heterogeneity in a new case

A 2.5-year-old girl presented with severely delayed speech development and mild motor retardation. Urinary organic acid analysis showed the presence of 4-hydroxybutyric acid and other metabolites consistent with the diagnosis 4-hydroxybutyric aciduria. Succinic semialdehyde dehydrogenase activity was absent in lymphocyte lysates. The clinical symptoms in this case were unusually mild compared to previously reported patients. No correlation was found between the mild symptoms and the levels of metabolite excretion or the residual succinic semialdehyde dehydrogenase activity in this patient compared to more severely affected cases.

Purification and properties of two succinic semialdehyde dehydrogenases from Klebsiella pneumoniae

Two forms of succinic semialdehyde dehydrogenase have been isolated in Klebsiella pneumoniae M5a1. The two enzymes could be separated by filtration on Sephacryl S-300 and their apparent molecular weights were approx. 275,000 and 300,000. The large enzyme is specific for NADP. The smaller enzyme, which is induced by growth on 3-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid and gamma-aminobutyrate, has been purified to 96% homogeneity by affinity chromatography. The NAD-linked succinic semialdehyde dehydrogenase was able to use NADP as cofactor. Its induction is coordinated with 3- and 4-hydroxylase, the enzymes which initiate degradation of 3- and 4-hydroxyphenylacetic acid. The NAD-linked form is also induced by exogenous succinic semialdehyde. The large enzyme is specific for NADP and has been isolated from a defective mutant which lacked the activity of the NAD-linked succinic semialdehyde dehydrogenase. Activity and stability conditions and true K m values for substrates and cosubstrates of the two enzymes were determined. Some aspects of the induction of the NAD-linked enzyme participating in the metabolism of 4-hydroxyphenylacetic and gamma-aminobutyrate were studied.

Oxidation of [U-14C]Succinic Semialdehyde in Cultured Human Lymphoblasts: Measurement of Residual Succinic Semialdehyde Dehydrogenase Activity in 11 Patients with 4-Hydroxybutyric Aciduria

The oxidation of [U-14C]succinic semialdehyde to 14CO2 has been investigated in cultured lymphoblasts to develop a whole cell assay for succinic semialdehyde dehydrogenase. We have previously demonstrated deficiency of this enzyme in extracts of white cells derived from 13 patients with 4-hydroxybutyric aciduria. Major goals were the demonstration of greater residual succinic semialdehyde dehydrogenase activity in patient cell lines and the better representation of physiology in vivo. In 18 control lymphoblast lines, the conversion of [U-14C]succinic semialdehyde to 14CO2 was 1579 +/- 310 dpm. The mean value in lymphoblasts derived from 11 patients with deficiency of succinic semialdehyde dehydrogenase was 112 +/- 36 dpm approximating 7% of the mean control value. Analysis of organic acids produced from [U-14C]succinic semialdehyde in control lymphoblasts indicated that 14CO2 emanated from the tricarboxylic acid cycle; the major metabolic products were succinic and lactic acids. In the presence of 5mM malonic and 2-propylpentanoic (valproic) acids, 14CO2 production in a control lymphoblast line was decreased by 68 and 45%, respectively. The whole cell assay is less laborious than our previously described assay employing cell extracts, and the general trend was the demonstration of higher residual levels of activity for lymphoblasts derived from patients.

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

1. 1. In the present paper we report the presence of succinic semialdehyde dehydrogenase (SSADH) in bovine adrenal medulla and blood platelets. 2. 2. Both enzymes present some analogies with the brain enzyme in terms of cofactor requirements, optimal pH, mitochondrial localization and inhibition by AMP. 3. 3. 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. 4. 4. 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.

Electron-cytochemical localization of succinic semialdehyde dehydrogenase activity in Purkinje neurons and hepatocytes of the rat

A method is presented for the ultrastructural demonstration of succinate semialdehyde dehydrogenase (SSADH) activity in cerebellar Purkinje neurons and liver hepatocytes; SSADH is an enzyme involved in the degradation of γ-aminobutyric acid (GABA). Incubation media originally used for light microscopy were considered. Reaction products were mainly detected when fresh tissue was used. In Purkinje cells, grains ascribable to SSADH activity were localized on the mitochondria (especially on the outer membrane); some extramitochondrial formazan deposits were also found. After brief fixation by immersion or perfusion, only a few formazan granules were detected in the cytoplasm. A similar distribution pattern was observed in hepatocytes, in which extramitochondrial grains and grains on the nuclear membrane were frequent. The actual existence and the possible meaning of extramitochondrial SSADH activity is critically discussed on the basis of the data in the literature.

Properties of succinic semialdehyde dehydrogenase in cultured human lymphoblasts.

A direct assay has been developed for succinic semialdehyde dehydrogenase in sonicates of human lymphocytes and Epstein-Barr Virus transformed cultured lymphoblasts. Enzyme activity was quantified by incubating cell extracts with uniformly labeled [14C]succinic semialdehyde and monitoring the conversion to [14C]succinic acid. Radiolabeled products were separated by liquid partition chromatography on hydrated silicic acid. Kinetic properties and requirements of succinic semialdehyde dehydrogenase in lymphoblast sonicates were investigated in order to determine optimal conditions for the direct assay. Enzyme activity was stimulated by dithiothreitol, ammonium and potassium ions and 0.1% Triton X-100. The concentrations for half maximal activation by ammonium and potassium were 5.2 and 13.7 mM respectively. The mean activity of succinic semialdehyde dehydrogenase in assays in which equimolar NADP+ had been substituted for NAD+ was 19% of the activity of assays which contained NAD+. Substrate Michaelis constants were 21 and 30 microM for NAD+, and 26, 42 and 70 microM for succinic semialdehyde. The enzyme displayed a pH optimum between 8 and 9 and demonstrated a slight temperature activation between 37 degrees and 45 degrees C. A deficiency of succinic semialdehyde dehydrogenase activity was documented in cultured lymphoblasts derived from a patient with gamma-hydroxybutyric aciduria.

Defective succinic semialdehyde dehydrogenase activity in 4-hydroxybutyric aciduria.

Succinic semialdehyde dehydrogenase deficiency has been demonstrated in a fourth patient with 4-hydroxybutyric aciduria. Lysates of freshly isolated lymphocytes and cultured lymphoblasts of the patient had much lower than control activity in the conversion of U-14C-4-aminobutyric acid to 14C-succinic acid in an assay designed to estimate succinic semialdehyde dehydrogenase utilizing endogenous 4-aminobutyrate transaminase. Lymphocyte and lymphoblast lysates of the patient accumulated U-14C-succinic semialdehyde when incubated with U-14C-4-aminobutyric acid and NAD+ whereas none could be detected in controls. Assays using U-14C-succinic semialdehyde as substrate for succinic semialdehyde dehydrogenase in lysates of cultured lymphoblasts characterized the patient as having a severe deficiency of succinic semialdehyde dehydrogenase. The data indicate that defective activity of succinic semialdehyde dehydrogenase is responsible for 4-hydroxybutyric aciduria.

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity.

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.

Succinic semialdehyde dehydrogenase deficiency: an inborn error of gamma-aminobutyric acid metabolism

Gamma-hydroxybutyric aciduria is a disorder of gamma-aminobutyric acid metabolism in which a compound of known neuropharmacologic activity accumulates. We have studied two patients in whom high levels of gamma-hydroxybutyric acid were found in blood, urine and cerebrospinal fluid. A coupled assay has been developed which estimates succinic semialdehyde dehydrogenase activity in isolated human lymphocytes. The mean activity of succinic semialdehyde dehydrogenase in a control and the four parents and two healthy siblings of these patients was 8.8 ±1.9 pmol · min −1 · mg −1 protein. In the patients the activities were 0.8 and 1.1 pmol · min −1 · mg −1 protein, approximately 9–13% of control. In the presence of saturating amounts of NAD +, lymphocyte sonicates, derived from the patients accumulated a significant amount of 14C-succinic semialdehyde from 14C-gamma aminobutyric acid, whereas none could be detected in controls. The data suggest a deficiency of succinic semialdehyde dehydrogenase in these patients, the first documented defect of the metabolism of gamma-aminobutyric acid in man.

Histochemical patterns of succinic semialdehyde dehydrogenase activity within the Purkinje cell population during rat cerebellar histogenesis

The histochemical detection of SSADH activity corroborates some crucial steps of the maturation of Purkinje neurons in rat cerebellum. The SSADH-specific activity seems to appear early, within the first week of postnatal life, in a few Purkinje cells. Around the 12th day most cells were SSADH positive, even if some were still negative. This period is an important step of postnatal histogenesis when many histological and physiological data show important neuronal changes. At the end of cerebellar histogenesis the SSADH activity showed the same pattern as in adult rats. Our findings suggest that in the Purkinje cell population the maturation does not occur simultaneously thus confirming the concept of "heterogeneity" (different functional states).

Inhibition of Wheat Succinic Semialdehyde Dehydrogenase by Oxaloacetate

The effect of Krebs' cycle intermediates, nucleotides, aminoacids and polyamines on succinic semialdehyde dehydrogenase activity was studied. The enzyme is noncompetitively inhibited by oxaloacetate whereas other tested compounds have no effect. The Ki in the presence of succinic semialdehyde and NAD were determined. The results are discussed in relation to the γ-aminobutyrate shunt.

Changes in γ-aminobutyric acid-shunt enzymes in regions of rat brain with ketamine anaesthesia

The in vivo effects of ketamine hydrochloride (100 mg/kg) on the activity of glutamic acid decarboxylase (GAD), 4-aminobutyrate-2-oxoglutarate aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSA-DH) were studied in homogenates and subcellular fractions (synaptosomal and mitochondrial) from 3 regions (cerebral hemispheres, cerebellum and brain stem) of rat brain. The activity of the enzymes was determined at 15, 30, 45 and 60 min after ketamine administration. The results of the study indicate that ketamine has different effects on different brain regions. In synaptosomal fractions from both brain stem and cerebellum, ketamine increased GAD activity but it had no effect on GAD activity in the cerebral hemispheres. In brain stem, ketamine inhibited both GABA-T as well as SSA-DH activity (mitochondrial fractions), whereas in cerebral hemispheres it caused only transient increases in GABA-T and SSA-DH activity. In cerebellum (mitochondrial fractions), ketamine had no effect on GABA-T activity whereas SSA-DH activity was inhibited except for the first 15 min of ketamine administration. The data are of relevance to the mechanism of the anticonvulsant effect of ketamine.

Interconnection of GABA-ergic neurons in rat extrapyramidal tract: Analysis using intracerebral microinjection of kainic acid

The interconnection of γ-aminobutyric acid (GABA)-ergic neurons in the rat extrapyramidal tract was analyzed neurochemically using the technique of intracerebral microinjection of kainic acid. Two days after the stereotaxic microinjection of kainic acid (2 μg/μl) into the left striatum and thereafter, the rats exhibited an ipsilateral rotational movement which persisted until they were killed, and the striatal, pallidal, and nigral GABA concentrations and 1-glutamate decarboxylase (GAD) activities of the ipsilateral side in these animals were significantly reduced. On the other hand, a significant reduction of GABA-transaminase·succinic semialdehyde dehydrogenase activity was found only in the left striatum. The intrapallidal injection of kainic acid induced a significant decrease in the ipsilateral pallidal and nigral GABA concentrations without affecting that in the striatum. In contrast, the unilateral nigral injection of kainic acid induced no significant alteration of GABA concentrations in any central nervous system regions examined. The striatal kainic acid treatment not only selectively abolished the high-affinity uptake system for [ 3H]GABA, but also exhibited a selective suppression of the high K +-evoked release of [ 3H]GABA from striatal slices. Histological examination of kainic acid-treated striatal, pallidal, and nigral tissues revealed that the degeneration of neurons and subsequent gliosis occurred only at each injection site. Our results indicate that in the extrapyramidal system the striatopallidal, striatonigral, and pallidonigral GABA-ergic neurons are present, and may be involved in the occurrence of behavioral abnormalities observed in kainic acid treated animals. We also suggest that the high-affinity uptake and evoked release systems for GABA indeed reside within neurons which are susceptible to kainic acid.