Succinic semialdehyde dehydrogenase: Purification and properties of the enzyme from monkey brain

doi:10.1016/0006-3002(61)90900-3

Biochimica et Biophysica Acta

Volume 52, Issue 1, 2 September 1961, Pages 29-35

https://doi.org/10.1016/0006-3002(61)90900-3 Get rights and content

Abstract

DPN-dependent succinic semialdehyde dehydrogenase from rhesus monkey brain retains full activity when purified by fractionation in the absence of reducing agents. In this state, the enzyme is relatively insensitive to arsenite. Addition of mercaptoethanol markedly increases the arsenite sensitivity. Both DPN and mercaptoethanol are effective in protecting the enzyme from auto-oxidation. Additional experiments are presented in support of a role of DPN in stabilizing the tertiary structure of the enzyme in the vicinity of the aldehyde binding site.

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Cited by (27)

Hypoxia and GABA shunt activation in the pathogenesis of Alzheimer's disease
2016, Neurochemistry International
Citation Excerpt :
SSADH is a NAD+-dependent oxidoreductase and a member of the aldehyde dehydrogenase family (family 5, member A1) (ALDH5A1) (Kim et al., 2011). SSADH was purified and characterized from monkey brain by Albers and Koval (1961) and the gene was cloned much later by Chambliss et al. (1995). Given that SSADH has an important role in the function of the GABA shunt, the regulation of its expression, especially its transcriptional control, is poorly understood.
We have previously observed that the conversion of mild cognitive impairment to definitive Alzheimer's disease (AD) is associated with a significant increase in the serum level of 2,4-dihydroxybutyrate (2,4-DHBA). The metabolic generation of 2,4-DHBA is linked to the activation of the γ-aminobutyric acid (GABA) shunt, an alternative energy production pathway activated during cellular stress, when the function of Krebs cycle is compromised. The GABA shunt can be triggered by local hypoperfusion and subsequent hypoxia in AD brains caused by cerebral amyloid angiopathy. Succinic semialdehyde dehydrogenase (SSADH) is a key enzyme in the GABA shunt, converting succinic semialdehyde (SSA) into succinate, a Krebs cycle intermediate. A deficiency of SSADH activity stimulates the conversion of SSA into γ-hydroxybutyrate (GHB), an alternative route from the GABA shunt. GHB can exert not only acute neuroprotective activities but unfortunately also chronic detrimental effects which may lead to cognitive impairment. Subsequently, GHB can be metabolized to 2,4-DHBA and secreted from the brain. Thus, the activation of the GABA shunt and the generation of GHB and 2,4-DHBA can have an important role in the early phase of AD pathogenesis.
Succinic semialdehyde dehydrogenase from mammalian brain: Subunit analysis using polyclonal antiserum
1992, International Journal of Biochemistry
- 1.
  1. NAD⁺-dependent succinic semialdehyde dehydrogenase was purified to apparent homogeneity from rat brain and highly purified from human brain.
- 2.
  2. Molecular exclusion chromatography of the purified enzymes on Sephadex G-150 and G-200 revealed M_r values of 203,000 and 191,000 for rat and human, respectively.
- 3.
  3. Electrophoresis on sodium dodecylsulfate polyacrylamide gels revealed a single subunit of M_r 54,000 for rat and 58,000 for human. Isoelectric focusing of the purified rat enzyme yielded a pI of 6.1.
- 4.
  4. For both proteins, K_m values for short-chain aldehydes acetaldehyde and propionaldehyde ranged from 0.33 to 2.5 mM; K_m values for succinic semialdehyde were in the 2–4 μM range.
- 5.
  5. The subunit structure of both enzymes was investigated in brain extracts and purified preparations by immunoblotting, using a polyclonal rabbit antiserum against the purified rat brain enzyme.
- 6.
  6. For rat and human extracts, single bands were detected at M_r, 54,000 and 58,000, comparable to findings in the purified preparations. Immunoblotting analyses in other species (guinea pig, hamster, mouse and rabbit) revealed single subunits of M_r, 54,000–56,500.
Kinetic mechanism of potato tuber succinate semialdehyde dehydrogenase
1990, Plant Science
The steady state kinetic mechanism of purified succinate semialdehyde dehydrogenase enzyme from potato (Solanum tuberosum L. cv Kufri Chandramukhi) tubers was investigated. The potato enzyme exhibited Michaelis-Menten kinetics with respect to both the substrates, succinate semialdehyde and NAD⁺. Initial velocity studies suggested a sequential mechanism for the enzyme. The enzyme had a relatively low $K_{m}$ value for succinate semialdehyde (4.65 μM) and was inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD⁺ ( $K_{i} = 215 μ M$ ). NADH, one of the products of the reaction, inhibited competitively with respect to NAD⁺ with a $K_{i}$ value of 53.8 μM and non-competitively with respect to succinate semialdehyde. $p- Hydroxybenzaldehyde$ , as a dead-end inhibitor, gave competitive inhibition with respect to succinate semialdehyde and uncompetitive inhibition with respect to NAD⁺. The results of these studies, together with analysis of the dead-end inhibition by AMP, were consistent with an ordered bi, bi sequential mechanism for the potato enzyme in which NAD⁺ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it. The mechanism obtained here was compared with the mechanisms proposed for this enzyme from other sources.
Metabolism, enzymology and possible roles of 4-aminobutyrate in higher plants
1990, Phytochemistry
4-Aminobutyrate is a widely distributed non-protein amino acid and is found in many plants in high amounts. It is formed by the decarboxylation of glutamate by the enzyme, glutamate decarboxylase. Increase in 4-aminobutyrate content and glutamate decarboxylase activity have been observed in many plants under a variety of environmental stress conditions. 4-Aminobutyrate is catabolized in plants by the operation of the 4-aminobutyrate shunt in which it is converted to succinate via succinate semialdehyde by the enzymes, 4-aminobutyrate transaminase and succinate semialdehyde dehydrogenase. Although universal distribution of glutamate decarboxylase among plants is well known, the occurrence of catabolic enzymes is not so well established. Possible physiological roles of 4-aminobutyrate in higher plants are documented and discussed here.
Potato tuber succinate semialdehyde dehydrogenase: Purification and characterization
1989, Archives of Biochemistry and Biophysics
Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (M_r) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of M_r 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mm sodium pyrophosphate buffer. In 100 mm Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD⁺ as cofactor and NADP⁺ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a K_m of 4.6 μm for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 μm. The K_m for NAD⁺ was found to be 31 μm. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethylmaleimide, by heavy metal ions Hg²⁺, Cu²⁺, Cd²⁺, and Zn²⁺, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.
Human brain "high K<inf>m</inf>" aldehyde dehydrogenase: Purification, characterization, and identification as NAD<sup>+</sup>-dependent succinic semialdehyde dehydrogenase
1988, Archives of Biochemistry and Biophysics
NAD-dependent succinic semialdehyde dehydrogenase (EC 1.2.1.24) has been purified to homogeneity from human brain via ion-exchange chromatography and affinity chromatography employing Blue Sepharose and 5′-AMP Sepharose. Succinic semialdehyde dehydrogenase was never previously purified to homogeneity from any species; this preparation therefore allows the determination of its molecular weight, subunit molecular weight, subunit composition, isoelectric points, and substrate specificity for the first time. The enzyme is a tetramer of M_r 230,000 to 245,000 and consists of weight-nonidentical subunits (M_r 61,000 and 63,000). On isoelectric focusing the enzyme separates into five bands with the following isoelectric points: 6.3, 6.6, 6.8, 6.95, and 7.15. Its substrates include glutaric semialdehyde, nitrobenzaldehyde, and short chain aliphatic aldehydes in addition to succinic semialdehyde which is the best substrate. The K_m values for succinic semialdehyde, acetaldehyde, and propionaldehyde are 1, 875, and 580 μm, respectively. The enzyme is inactive with 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde as substrates. Its subcellular localization is in the mitochondrial fraction. Succinic semialdehyde dehydrogenase is sensitive to inhibition by disulfiram (a drug used therapeutically to produce alcohol aversion) resembling, in this respect, aldehyde dehydrogenase (EC 1.2.1.3). It does not, however, interact with the antibody developed in the rabbit vs aldehyde dehydrogenase, suggesting that the two enzymes are structurally distinct.

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Succinic semialdehyde dehydrogenase: Purification and properties of the enzyme from monkey brain

Abstract

J. Biol. Chem.

J. Biol. Chem.