Purification and characterization of aldehyde dehydrogenase from human brain

doi:10.1016/0003-9861(87)90409-7

Archives of Biochemistry and Biophysics

Volume 255, Issue 2, June 1987, Pages 409-418

https://doi.org/10.1016/0003-9861(87)90409-7 Get rights and content

Abstract

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified from human brain; this constitutes the first purification to homogeneity from the brain of any mammalian species. Of the three isozymes purified two are mitochondrial in origin (Peak I and Peak II) and one is cytoplasmic (Peak III). By comparison of properties, the cytoplasmic Peak III enzyme could be identified as the same as the liver cytoplasmic E1 isozyme (N. J. Greenfield and R. Pietruszko (1977) Biochim. Biophys. Acta483, 35–45). The Peak I and Peak II enzymes resemble the liver mitochondrial E2 isozyme, but both have properties that differ from those of the liver enzyme. The Peak I enzyme is extremely sensitive to disulfiram while the Peak II enzyme is totally insensitive; liver mitochondrial E2 isozyme is partially sensitive to disulfiram. The specific activity is 0.3 μmol/mg/min for the Peak I and 3.0 μmol/mg/min for the Peak II enzyme; the specific activity of the liver mitochondrial E2 isozyme is 1.6 μmol/min/mg under the same conditions. The Peak I enzyme is also inhibited by acetaldehyde at low concentrations, while the Peak II enzyme and the liver mitochondrial E2 isozyme are not inhibited under the same conditions. The precise relationship of brain Peak I and II enzymes to the liver E2 isozyme is not clear but it cannot be excluded at the present time that the two brain mitochondrial enzymes are brain specific.

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Aldehyde dehydrogenases (ALDH) play a key role in neuronal protection. They exert this function by metabolizing biogenic amine-related aldehydes, e.g. 3,4-dihydroxyphenylacetaldehyde (DOPAL), and by protecting neurons against aldehyde- and oxidative stress-related neurotoxicity. The role of these different isoenzymes has been discussed in other neurodegenerative disorders before. It is somewhat surprising that only few studies have investigated their role in the aetiology of Parkinson's disease (PD), in both the degeneration of dopaminergic neurons and the formation of Lewy bodies. Earlier studies report severe alterations of the cytosolic isoform of ALDH expression (ALDH 1A1) in the substantia nigra of patients with PD. However, there are no data regarding the activity of ALDH 2 located at the inner mitochondrial membrane. Since mitochondrial dysfunctions are hypothesized to be of importance in the aetiology of PD we have examined the enzymatic activity of mitochondrial ALDH 2 in post-mortem putamen and frontal cortex of patients with PD and controls. We found that mitochondrial ALDH 2 activity in contrast to the frontal cortex was significantly increased in the putamen of patients with PD compared to controls.
The conserved R166 residue of ALDH5A (succinic semialdehyde dehydrogenase) has multiple functional roles
2009, Chemico-Biological Interactions
Citation Excerpt :
These data suggest that the electronegative group undergoes hydrogen bonding with a positively charged amino acid. ALDH2 has over 4500-fold greater catalytic efficiency towards acetaldehyde over succinic semialdehyde and utilizes propionaldehyde, lipophilic aldehydes and aromatic aldehydes with high efficiency [10,19]. These data suggest that ALDH2 has a more lipophilic binding pocket than ALDH5A.
ALDH5 (aka succinic semialdehyde dehydrogenase) is a NAD⁺-dependent aldehyde dehydrogenase crucial for the proper removal of the GABA metabolite succinic semialdehyde (SSA). All known ALDH5 family members contain the conserved amino acid sequence “MITRK”. Our studies of rat ALDH5A indicate that residue R166 in this sequence may play a role in the substrate specificity of ALDH5A for the γ-carboxylated succinic semialdehyde versus other aliphatic and aromatic aldehydes including acetaldehyde and benzaldehyde. We tested the hypothesis that the R166 residue regulates aldehyde specificity by utilizing rat ALDH5A wild-type (R166wt) and R166K, R166H, R166A, and R166E mutants. The V_MAX using SSA fell whereas the K_M for SSA increased for all mutants analyzed yielding k_cat/K_M (s⁻¹/μM) ratios of 52.3 (R166wt), 5.5 (R166K), 0.01 (R166H), 0.008 (R166E), and 0.004 (R166A). Utilization of acetaldehyde by the R166H mutant was similar to R166wt with k_cat/K_M's of 0.003 and 0.002, respectively. Almost no activity towards acetaldehyde was noted for the R166E and R166A mutants. Unexpectedly, the K_M for NAD⁺ changed: 21 μM (R166wt), 81 μM (R166K), 63 μM (R166H), 35 μM (R166E) and 44 μM (R166A). As release of NADH can be a rate-limiting step for ALDH activity, NADH binding was evaluated for R166wt and R166H enzymes. The K_D of NADH for R166H (0.9 μM) was 11-fold less than that of ALDH5A wt (10.3 μM) and possibly explains the increase in the K_M for NAD⁺. Furthermore, data using R166K and R166H mutants demonstrate that inhibition of enzyme activity by low pH is regulated in part by the R166 residue. Our data indicate that the R166 residue of ALDH5A regulates multiple enzymatic functions.
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Recent evidence indicates a role for oxidative stress and resulting products, e.g. 4-hydroxy-2-nonenal (4HNE) in the pathogenesis of Parkinson's disease (PD). 4HNE is a known inhibitor of mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme very important to the dopamine (DA) metabolic pathway. DA undergoes monoamine oxidase-catalyzed oxidative deamination to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is metabolized primarily to 3,4-dihydroxyphenylacetic acid (DOPAC) via ALDH2. The biotransformation of DOPAL is critical as previous studies have demonstrated this DA-derived aldehyde to be a reactive electrophile and toxic to dopaminergic cells. Therefore, 4HNE produced via oxidative stress may inhibit ALDH2-mediated oxidation of the endogenous neurotoxin DOPAL. To test this hypothesis, ALDH2 in various model systems was treated with 4HNE and activity toward DOPAL measured. Incubation of human recombinant ALDH2 with 4HNE (1.5–30 μM) yielded inhibition of activity toward DOPAL. Furthermore, ALDH2 in rat brain mitochondrial lysate as well as isolated rat brain mitochondria was also sensitive to the lipid peroxidation product at low micromolar, as evident by a decrease in the rate of DOPAL to DOPAC conversion measured using HPLC. Taken together, these data indicate that 4HNE at low micromolar inhibits mitochondrial biotransformation of DOPAL to DOPAC, and generation of the lipid peroxidation product may represent a mechanism yielding aberrant levels of DOPAL, thus linking oxidative stress to the uncontrolled production of an endogenous neurotoxin relevant to PD.
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Aldehyde dehydrogenase (EC 1.2.1.3) in the liver of skipjack tuna (Katsuwonus pelamis) was extracted from mitochondrial fractions by Triton X-100. The enzyme was purified by ammonium sulfate fractionation, Toyopearl HW-55F, DEAE-Toyopearl 650M, and 5′-AMP-Sepharose 4B column chromatography. The molecular mass of skipjack ALDH (200 kDa) was similar to those of mammals and yeast. The optimum pH was around 10.0 and the enzyme was stable at pH 9.0 and 10.5, but it gradually became unstable when the pH was lower than 7.0. The optimum temperature was around 40°C. The enzyme was stable at 30°C for 60 min, but only 10% of the original activity remained at 40°C for 60 min. The enzyme was activated by Mg²⁺ and Mn²⁺, and inhibited by Li⁺, Ba²⁺, Cu²⁺ and Fe³⁺. The K_m values for skipjack ALDH were: 15.0 M for acetaldehyde, 32.1 μM for propionaldehyde, 25.9 μM for formaldehyde, 58.7 μM for octylaldehyde, 24.6 μM for benzaldehyde and 40.2 μM for 5-hydroxyindoleacetaldehyde. Skipjack ALDH showed different affinities for substrates than mammalian ALDHs: high affinity against formaldehyde, low affinity against benzaldehyde and medium affinity towards acetaldehyde and propionaldehyde.
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Purification and characterization of aldehyde dehydrogenase from human brain

Abstract

J. Biol. Chem

Biochim. Biophys. Acta

J. Biol. Chem

Essays Neurochem. Neuropharmacol

Alcoh. Clin. Exp. Res

J. Neurochem

Mol. Pharmacol

Canad. J. Biochem