Inhibition of Succinic Semialdehyde Dehydrogenase by N-Formylglycine

Abstract

N-formylglycine was developed as a dead-end inhibitor of the succinic semialdehyde dehydro-genase reaction. At 4mM, it inhibited Aspergillus niger succinic semialdehyde dehydrogenase by 40%. N-formylglycine is a reversible, complete inhibitor; the inhibition is competitive with succinic semialdehyde and uncompetitive with respect to NAD⁺ and the K_i values are 4.9 and 10.4 mM respectively. Potato succinic semialdehyde dehydrogenase is also inhibited by N-formylglycine to a similar extent, the nature of the inhibition being identical to that observed with the A. niger enzyme.     

Enzymatic and Immunologic Identification of Succinic Semialdehyde Dehydrogenase in Rat and Human Neural and Nonneural Tissues

Abstract: 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.     

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.     

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.     

Archaeal Aldehyde Dehydrogenase ST0064 from Sulfolobus tokodaii,a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase,Is a Succinate Semialdehyde Dehydrogenase

Aldehyde dehydrogenase ST0064, the closest paralog of previously characterized allosteric non-phosphorylating glyceraldehyde-3-phosphate (GAP) dehydrogenase (GAPN, ST2477) from a thermoacidophilic archaeon, Sulfolobus tokodaii, was expressed heterologously and characterized in detail. ST0064 showed remarkable activity toward succinate semialdehyde (SSA) (K _m of 0.0029 mM and k _cat of 30.0 s^?1) with no allosteric regulation. Activity toward GAP was lower (K _m of 4.6 mM and k _cat of 4.77 s^?1), and previously predicted succinyl-CoA reductase activity was not detected, suggesting that the enzyme functions practically as succinate semialdehyde dehydrogenase (SSADH). Phylogenetic analysis indicated that archaeal SSADHs and GAPNs are closely related within the aldehyde dehydrogenase superfamily, suggesting that they are of the same origin.     

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.     

Partial Inhibition of Brain Succinate Dehydrogenase by 3-Nitropropionic Acid Is Sufficient to Initiate Striatal Degeneration in Rat

Abstract: Chronic inhibition of succinate dehydrogenase (SDH) by systemic injection of the selective inhibitor 3-nitropropionic acid (3NP) has been used as an animal model for Huntington's disease (HD). However, the mechanisms by which 3NP produces lesions in the striatum are not fully characterized. A quantitative histochemical method was developed to study the level of regional SDH inhibition resulting from intraperitoneal injection of 3NP or chronic intoxication using osmotic pumps. The results showed that (a) 3NP was an irreversible SDH inhibitor in vivo, (b) the level of SDH inhibition in the striatum (the brain region most vulnerable to 3NP) was similar to that observed in other brain regions not affected by the toxin, such as the cerebral cortex, and (c) the neurotoxic threshold of SDH inhibition in the brain was 50–60% of control levels. The present study demonstrates that the selective degeneration in the striatum observed after chronic 3NP administration cannot be ascribed to a preferential inhibition of SDH in this particular brain region. This work also suggests that the partial decrease in the activity of the respiratory chain complex II–III reported in HD patients may be sufficient to induce the selective striatal degeneration observed in this disorder.     

Effect of valproic acid on the urinary metabolic profile of a patient with succinic semialdehyde dehydrogenase deficiency

The metabolic changes in a patient with succinic semialdehyde dehydrogenase deficiency were investigated following valproate administration using urease pretreatment and gas chromatography-mass spectrometry. A stable isotope dilution technique was used for quantification of urinary 4-hydroxybutyrate. Urinary levels of 4-hydroxybutyrate were 4-fold higher after 1-month valproate therapy. 4,5-Dihydrohexanoate, 2-deoxytetronate and 3-deoxytetronate were also 1.7-2.7-fold higher. The urinary excretions of 4-hydroxybutyrate in valproate non-medicated controls were age dependence and decreased with age. Relationships between 4-hydroxybutyrate excretion and 4-hydroxyvalproate or 5-hydroxyvalproate excretion were observed in valproate medicated controls. It seems that 4-hydroxyvalproate and 5-hydroxyvalproate as well as valproate are involved with increased excretion of 4-hydroxybutyrate following valproate administrations.     

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.     

琥珀酸对大鼠海马CA1区神经元电压依赖性钙电流的影响

目的：观察不同浓度的琥珀酸对大鼠海马CA1区神经元电压依赖性钙通道（voltage—dependent calcium channels,VDCC）电流的作用,初步探讨琥珀酸对神经元保护的电生理学基础。方法：采用传统全细胞膜片钳技术和制霉菌素（nystatin）穿孔膜片钳技术观察琥珀酸对海马CA1区神经元VDCC电流的影响。结果：不同浓度的琥珀酸（10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1）在海马CA1区对低电压激活（low—voltage activated,LVA）钙通道电流未见任何影响,而对高电压激活（high—voltage activated,HVA）钙通道电流的抑制呈浓度依赖性。对照组HVA钙电流为580．05±17．32pA,分别给予10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1。的琥珀酸后,HVA钙电流依次为563．74±16．65,517．99±15．24,444．66±13．26,405．32±19．11,269．03±9．96和86．41±3．25pA,同对照组相比差异有统计学意义（n=8,P〈0．01）。结论：琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响。由此推测琥珀酸可能通过抑制HVA钙电流减少Ca^2＋内流而影响海马CA1区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关。     

Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Glutamate dehydrogenase (GDH) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low-speed pellet (480 g for 10 min). Approximately 60% of the high-speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates alpha-ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L-leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 microM GTP for the soluble and particulate fractions, respectively. The Hill plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45 degrees C and pH 7.4 in 50 mM phosphate buffer.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Effect of Electroconvulsive Shock on Mitochondrial Respiratory Chain in Rat Brain

It is well described that impairment of energy production has been implicated in the pathogenesis of a number of diseases. Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy (ECT) to minimize its side effects, little progress has been made in understanding its mechanism of action. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase from rat brain after acute and chronic electroconvulsive shock (ECS). Our results showed that mitochondrial respiratory chain enzymes activities were increased after acute ECS in hippocampus, striatum and cortex of rats. Besides, we also demonstrated that complex II activity was increased after chronic ECS in cortex, while hippocampus and striatum were not affected. Succinate dehydrogenase, however, was inhibited after chronic ECS in striatum, activated in cortex and not affected in hippocampus. Finally, complex IV was not affected by chronic ECS in hippocampus, striatum and cortex. Our findings demonstrated that brain metabolism is altered by ECS.     

Immunochemical Characterization of the Deficiency of the α-Ketoglutarate Dehydrogenase Complex in Thiamine-Deficient Rat Brain

Abstract: Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate-dependent, mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD-induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.     

Suppression of Mitochondrial Succinate Dehydrogenase, a Primary Target of β-Amyloid, and Its Derivative Racemized at Ser Residue

Abstract: β-Amyloid cores contain considerable amounts of d -Ser and d -Asp residues in Alzheimer's disease. We investigated the cytotoxic effects of various synthetic β-amyloids, including d -Ser-substituted derivatives, on primary cultured neurons and nonneuronal HeLa cells. β25–35, its d -Ser²⁶-substituted derivative, and β1–40 in 10–100 n M specifically suppressed mitochondrial succinate dehydrogenase activity [MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction] in HeLa cells, which are dependent on ATP production mainly from glycolysis, but did not exert detectable cytotoxicity, assessed by dye exclusion test, NADH levels, and uptake of [³H]Leu and [³H]Tdr. The β-amyloids, on the other hand, did exert neurodegenerative effects on rat hippocampal cultured neurons in which ATP is mostly synthesized by the mitochondrion. The activities of β25–35 and [ d -Ser²⁶]β25–35 are dependent on their having β-structures and not random forms. Although β25–35 was degraded rapidly by proteinase(s) in brain extract or leucine aminopeptidase, [ d -Ser²⁶]β25–35 is fairly resistant. These results indicate that one of the primary targets of β-amyloids is suppression of mitochondrial succinate dehydrogenase, and the vulnerability of the brain to β-amyloids can be explained by its large dependence on mitochondrial energy production. Moreover, racemization of serine residues of β-amyloids may be involved in neurodegeneration and formation of senile plaques through escaping from the degradation process by brain proteinases.     

Regional Distribution in Rat Brain of 1-Pyrroline-5-Carboxylate Dehydrogenase and Its Localization to Specific Glial Cells

A possible alternative route for production of a small glutamate pool in brain is from proline or ornithine to 1-pyrroline-5-carboxylate (P5C) and thence to glutamate. The conversion from ornithine to P5C is catalyzed by ornithine delta-aminotransferase (OrnT) whereas that from proline is catalyzed by proline oxidase (PrO). The conversion of P5C to glutamate is catalyzed by 1-pyrroline-5-carboxylate dehydrogenase (PDH). Biochemical assays of PDH and PrO in various rat brain regions indicate no positive correlation between the two enzymes nor between either activity and high-affinity glutamate uptake or the regional distribution of OrnT. We have localized PDH and PrO histochemically by modifications of the Van Gelder [J. Neurochem. 12, 231-237, (1965)] method for gamma-aminobutyric acid (GABA) transaminase. The enzymes were found only in certain types of glial cells; the best stained were the Bergmann glial cells of the cerebellum but, for PDH, there was also good staining of astrocytes in the dentate area of the hippocampus. Since both these areas are believed to have heavy glutamate innervation and numerous GABA interneurons, these findings may reflect an alternative route of glutamate production in glial cells near some glutamate and/or GABA tracts but they do not support this as a possible route for glutamate formation in most brain regions. The findings do, however, provide further evidence for chemical specialization of glial cells.     

Inhibitors and Dipeptide Substrates for a Microsomal Tyrosylsulfotransferase from rat Brain

Abstract

The sulfotransferase associated with a microsomal fraction from rat brain was previously shown to transfer sulfate groups from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to peptides derived from the chole cystokinin (CCK) molecule. Three tyrosine-containing dipeptide derivatives, i.e., Cbz-Glu-Tyr, Cbz-Gly-Tyr and Ac-Phe-Tyr are shown here to accept the [³⁵S] sulfate group from [³⁵S] PAPS under the action of this sulfotransferase. The sulfotransferase activity evaluated with either any of these dipeptide derivatives or CCK-8 as acceptors is similarly inhibited by a series of compounds, i.e., lipophilic polycyclic compounds like fluphenazine, tyrosine derivatives like Boc-O-benzyl-tyrosine and phenolsulfotransferase inhibitors like 4,4-di-isothiocyano 2′,2′-disulfonic acid stilbene.     

The Effects of Nano-anatase TiO2 on the Activation of Lactate Dehydrogenase from Rat Heart

Lactate dehydrogenase (LDH, EC1.1.1.27), widely expressed in the heart, liver, and other tissues, plays an important role in glycolysis and glyconeogenesis. The activity of LDH is often altered upon inflammatory responses in animals. Nano-TiO₂ was shown to provoke various inflammatory responses both in rats and mice; however, the molecular mechanism by which TiO₂ exerts its toxicity has not been completely understood. In this report, we investigated the mechanisms of nano-anatase TiO₂ (5 nm) on LDH activity in vitro. Our results showed that LDH activity was greatly increased by low concentration of nano-anatase TiO₂, while it was decreased by high concentration of nano-anatase TiO₂. The spectroscopic assays revealed that the nano-anatase TiO₂ particles were directly bound to LDH with mole ratio of [nano-anatase TiO₂] to [LDH] was 0.12, indicating that each Ti atom was coordinated with five oxygen/nitrogen atoms and a sulfur atoms of amino acid residues with the Ti–O(N) and Ti–S bond lengths of 1.79 and 2.41 Å. We postulated that the bound nano-anatase TiO₂ altered the secondary structure of LDH, created a new metal ion-active site for LDH, and thereby enhanced LDH activity.     

珊瑚Acropora pulchra (brook)的化学成分研究

首次对来自中国北海涠洲岛的珊瑚Acropora pulchra(brook)的化学成分进行研究,从乙醇浸提液的乙酸乙酯部分分离鉴定出7个化合物,并确立结构为十五酸十七酯(I)、正十六碳醇(Ⅱ)、鲨肝醇(Ⅲ)、1-正十六酸甘油酯(Ⅳ)、N—l—羟基甲基-2-羟基-(E,E)-3,7-十七碳二烯基十六酸酰胺(V)、胸腺嘧啶(Ⅵ)、尿嘧啶(Ⅶ)。