Induction of mitochondrial aldehyde dehydrogenase by submergence facilitates oxidation of acetaldehyde during re-aeration in rice

Post-hypoxic injuries in plants are primarily caused by bursts of reactive oxygen species and acetaldehyde. In agreement with previous studies, we found accumulations of acetaldehyde in rice during re-aeration following submergence. During re-aeration, acetaldehyde-oxidizing aldehyde dehydrogenase (ALDH) activity increased, thereby causing the acetaldehyde content to decrease in rice. Interestingly, re-aerated rice plants showed an intense mitochondrial ALDH2a protein induction, even though ALDH2a mRNA was submergence induced and declined upon re-aeration. This suggests that rice ALDH2a mRNA is accumulated in order to quickly metabolize acetaldehyde that is produced upon re-aeration.     

Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix

An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague–Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.     

Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix

An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague-Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.     

Oxidation of lactaldehyde by cytosolic aldehyde dehydrogenase and inhibition of cytosolic and mitochondrial aldehyde dehydrogenase by metabolites

An enzyme fraction which oxidizes lactaldehyde to lactic acid has been purified from goat liver. This enzyme was found to be identical with the cytosolic aldehyde dehydrogenase. Lactaldehyde was found to be primarily oxidized by this enzyme. Almost 90% of the total lactaldehyde-oxidizing activity is located in the cytosol. Methylglyoxal and glyceraldehyde 3-phosphate were found to be strong competitive inhibitors of this enzyme. Aldehyde dehydrogenase from goat liver mitochondria has also been partially purified and found to be strongly inhibited by these metabolites. The inhibitory effects of these metabolites on both these enzymes are highly pH dependent. The inhibitory effects of both the metabolites have been found to be stronger for the cytosolic enzyme at pH values higher than the physiological pH. For the mitochondrial enzyme, the inhibition with methylglyoxal was more pronounced at higher pH values, whereas stronger inhibition was observed with glyceraldehyde 3-phosphate at physiological pH.     

Induction of aldehyde dehydrogenase by polycyclic aromatic hydrocarbons in rats

Short-term intragastric administration of selected polycyclic aromatic hydrocarbons (100 mg/kg daily for 4 days) to male Wistar rats resulted in marked changes in liver cytosolic aldehyde dehydrogenase activity. Non-carcinogenic anthracene, phenanthrene and chrysene produced a 2.5–3-fold increase in the activity assayed with propionaldehyde as substrate and NAD as coenzyme. Weakly carcinogenic 1,2-benzanthracene enhanced aldehyde dehydrogenase activity 9-fold and the potent carcinogens 3,4-benzpyrene and 3-methylcholanthrene 30-fold. With benzaldehyde as substrate and NADP as coenzyme the differences between the groups were even more pronounced. Somewhat similar but less manifest effects on aldehyde dehydrogenase activity were detected also in the liver microsomes and in the postmitochondrial fractions of the small intestinal mucosa. On the basis of their ability to induce aldehyde dehydrogenase activity the compounds could be divided into three groups. This classification was found to correlate well with the carcinogenic potency of the compounds. It appeared that the exposure to polycyclic aromatic hydrocarbons, especially the carcinogenic ones, was followed by synthesis of a new aldehyde dehydrogenase form. This new form was differentiated from the normally existing cytosolic aldehyde dehydrogenase by its ability to oxidize benzaldehyde in the presence of NADP.     

Purification and characterization of grass carp mitochondrial aldehyde dehydrogenase

The molecular biology and enzymology of aldehyde dehydrogenase (ALDH) have been extensively investigated. However, most of the studies have been confined to the mammalian forms, while the sub-mammalian vertebrate ALDHs are relatively unexplored. In the present investigation, an ALDH was purified from the hepatopancreas of grass carp (Ctenopharygodon idellus) by affinity chromatographies on α-cyanocinnamate-Sepharose and Affi-gel Blue agarose. The 800-fold purified enzyme had a specific activity of 4.46 U/mg toward the oxidation of acetaldehyde at pH 9.5. It had a subunit molecular weight of 55 000. Isoelectric focusing showed a single band with a pI of 5.3. N-terminal amino acid sequencing of 30 residues revealed a positional identity of ∼70% with mammalian mitochondrial ALDH2. The kinetic properties of grass carp ALDH resembled those of mammalian ALDH2. The optimal pH for the oxidation of acetaldehyde was 9.5. The K_m values for acetaldehyde were 0.36 and 0.31 μM at pH 7.5 and 9.5, respectively. Grass carp ALDH also possessed esterase activity which could be activated in the presence of NAD⁺.     

Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications

Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.     

Purification and characterization of grass carp mitochondrial aldehyde dehydrogenase

The molecular biology and enzymology of aldehyde dehydrogenase (ALDH) have been extensively investigated. However, most of the studies have been confined to the mammalian forms, while the sub-mammalian vertebrate ALDHs are relatively unexplored. In the present investigation, an ALDH was purified from the hepatopancreas of grass carp (Ctenopharygodon idellus) by affinity chromatographies on alpha-cyanocinnamate-Sepharose and Affi-gel Blue agarose. The 800-fold purified enzyme had a specific activity of 4.46 U/mg toward the oxidation of acetaldehyde at pH 9.5. It had a subunit molecular weight of 55000. Isoelectric focusing showed a single band with a pI of 5.3. N-terminal amino acid sequencing of 30 residues revealed a positional identity of approximately 70% with mammalian mitochondrial ALDH2. The kinetic properties of grass carp ALDH resembled those of mammalian ALDH2. The optimal pH for the oxidation of acetaldehyde was 9.5. The K(m) values for acetaldehyde were 0.36 and 0.31 microM at pH 7.5 and 9.5, respectively. Grass carp ALDH also possessed esterase activity which could be activated in the presence of NAD(+).     

Inhibition of mitochondrial aldehyde dehydrogenase by nitric oxide-mediated S-nitrosylation

Mitochondrial aldehyde dehydrogenase (ALDH2) is responsible for the metabolism of acetaldehyde and other toxic lipid aldehydes. Despite many reports about the inhibition of ALDH2 by toxic chemicals, it is unknown whether nitric oxide (NO) can alter the ALDH2 activity in intact cells or in vivo animals. The aim of this study was to investigate the effects of NO on ALDH2 activity in H4IIE-C3 rat hepatoma cells. NO donors such as S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, and 3-morpholinosydnonimine significantly increased the nitrite concentration while they inhibited the ALDH2 activity. Addition of GSH-ethylester (GSH-EE) completely blocked the GSNO-mediated ALDH2 inhibition and increased nitrite concentration. To directly demonstrate the NO-mediated S-nitrosylation and inactivation, ALDH2 was immunopurified from control or GSNO-treated cells and subjected to immunoblot analysis. The anti-nitrosocysteine antibody recognized the immunopurified ALDH2 only from the GSNO-treated samples. All these results indicate that S-nitrosylation of ALDH2 in intact cells leads to reversible inhibition of ALDH2 activity.     

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. J?rnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.     

Lactate dehydrogenase associated with the mitochondrial fraction and with a mitochondrial inhibitor--I. Enzyme binding to the mitochondrial fraction

Rabbit skeletal muscle mitochondrial fraction shows LDH activity (212 +/- 43 U/g pellet). The majority of the mitochondrial enzyme was solubilized by washing with 0.15 M NaCl, pH 6, or by ultrasonic treatment in the same medium. It was also solubilized on increasing the ionic strength and the pH of the medium. Cytosoluble LDH was observed to bind in vitro to the particulate fraction and the enzyme bound was a sigmoidal function of the amount of soluble enzyme added. The bound enzyme is less active than the soluble one. Kinetically, active mitochondrial fraction or in vitro bound enzyme showed non-hyperbolic behavior which is different from the bi-bi sequential-ordered type mechanism of the soluble enzyme.     

Partially irreversible inactivation of mitochondrial aldehyde dehydrogenase by nitroglycerin

Mitochondrial aldehyde dehydrogenase (ALDH2) may be involved in the biotransformation of glyceryl trinitrate (GTN), and the inactivation of ALDH2 by GTN may contribute to the phenomenon of nitrate tolerance. We studied the GTN-induced inactivation of ALDH2 by UV/visible absorption spectroscopy. Dehydrogenation of acetaldehyde and hydrolysis of p-nitrophenylacetate (p-NPA) were both inhibited by GTN. The rate of inhibition increased with the GTN concentration and decreased with the substrate concentration, indicative of competition between GTN and the substrates. Inactivation of p-NPA hydrolysis was greatly enhanced in the presence of NAD(+), and, to a lesser extent, in the presence of NADH. In the presence of dithiothreitol (DTT) inactivation of ALDH2 was much slower. Dihydrolipoic acid (LPA-H(2)) was less effective than DTT, whereas glutathione, cysteine, and ascorbate did not protect against inactivation. When DTT was added after complete inactivation, dehydrogenase reactivation was quite modest (< or =16%). The restored dehydrogenase activity correlated inversely with the GTN concentration but was hardly affected by the concentrations of acetaldehyde or DTT. Partial reactivation of dehydrogenation was also accomplished by LPA-H(2) but not by GSH. We conclude that, in addition to the previously documented reversible inhibition by GTN that can be ascribed to the oxidation of the active site thiol, there is an irreversible component to ALDH inactivation. Importantly, ALDH2-catalyzed GTN reduction was partly inactivated by preincubation with GTN, suggesting that the inactivation of GTN reduction is also partly irreversible. These observations are consistent with a significant role for irreversible inactivation of ALDH2 in the development of nitrate tolerance.     

Rates of induction of mitochondrial aldehyde dehydrogenase in rat liver.

Aldehyde dehydrogenase activities in liver mitochondria isolated from rats given ethanol at hourly intervals by gastric intubation showed a brief lag period followed by a rapid increase in specific activities until a maximum was attained at about 3h.     

The removal of cytosolic-type aldehyde dehydrogenase from preparations of sheep liver mitochondrial aldehyde dehydrogenase and the unusual properties of the purified mitochondrial enzyme in assays.

The pI approximately 5.2 isoenzymes of mitochondrial aldehyde dehydrogenase were separated from the other isoenzymes by pH-gradient chromatography on DEAE-Sephacel. The pI approximately 5.2 material is immunologically identical with cytosolic aldehyde dehydrogenase. It also shows sensitivity to 20 microM-disulfiram and insensitivity to 4M-urea in assays. These and other criteria seem to establish that the material is identical with the cytosolic enzyme. Mitochondrial enzyme that had been purified to remove pI approximately 5.2 isoenzymes shows concentration-dependent lag phases in assays. These effects are possibly due to the slow establishment of equilibrium between tetramer and either dimers or monomers, with the dissociated species being intrinsically more active than the tetramer.     

Expression of a gene encoding mitochondrial aldehyde dehydrogenase in rice increases under submerged conditions

It is known that alcoholic fermentation is important for survival of plants under anaerobic conditions. Acetaldehyde, one of the intermediates of alcoholic fermentation, is not only reduced by alcohol dehydrogenase but also can be oxidized by aldehyde dehydrogenase (ALDH). To determine whether ALDH plays a role in anaerobic metabolism in rice (Oryza sativa L. cv Nipponbare), we characterized a cDNA clone encoding mitochondrial ALDH from rice (Aldh2a). Analysis of sub-cellular localization of ALDH2a protein using green fluorescent protein and an in vitro ALDH assay using protein extracts from Escherichia coli cells that overexpressed ALDH2a indicated that ALDH2a functions in the oxidation of acetaldehyde in mitochondria. A Southern-blot analysis indicated that mitochondrial ALDH is encoded by at least two genes in rice. We found that the Aldh2a mRNA was present at high levels in leaves of dark-grown seedlings, mature leaf sheaths, and panicles. It is interesting that expression of the rice Aldh2a gene, unlike the expression of the tobacco (Nicotiana tabacum) Aldh2a gene, was induced in rice seedlings by submergence. Experiments with ruthenium red, which is a blocker of Ca(2+) fluxes in rice as well as maize (Zea mays), suggest that the induction of expression of Adh1 and Pdc1 by low oxygen stress is regulated by elevation of the cytosolic Ca(2+) level. However, the induction of Aldh2a gene expression may not be controlled by the cytosolic Ca(2+) level elevation. A possible involvement of ALDH2a in the submergence tolerance of rice is discussed.     

Site-selective labeling at Cys302 of aldehyde dehydrogenase unveils a selective mitochondrial stain

A mitochondria-trackable fluorophore, CDy2, selectively labels Cys302 in the active site of mitochondrial aldehyde dehydrogenase (ALDH2).     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

Mouse mitochondrial aldehyde dehydrogenase isozymes: purification and molecular properties

Aldehyde dehydrogenase isozymes (AHD-1 and AHD-5) have been isolated in a highly purified state from extracts of mouse liver mitochondria. The enzymes have distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: AHD-1, 63,000; AHD-5, 49,000. Gel exclusion chromatography, using sephadex G-200, indicated that both isozymes are dimers, although AHD-1 may also exist as a monomeric form as well. The enzymes exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-1, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate, 1.4 mM and 0.78 mM respectively, whereas AHD-5 exhibited a low Km value with acetaldehyde of 0.2 microM. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-1, pH range 6.5-7.5; AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50% inhibition occurring 13 and 0.1 microM for the AHD-1 and AHD-5 isozyme respectively. Based upon the kinetic characteristics, it is suggested that AHD-5 may be the primary enzyme for oxidizing mitochondrial acetaldehyde during ethanol oxidation in vivo.     

Effects of alcohol consumption on mitochondrial aldehyde dehydrogenase isoenzymes in rat liver

The effects of long-term and short-term exposure of rats to ethanol on aldehyde dehydrogenase (ALDH) activity in the liver mitochondria were investigated. The specific activities of mitochondrial high Km ALDH and low Km ALDH after the prolonged administration of ethanol were both increased to levels about 2.5 times that of the control group. In contrast, high Km and low Km ALDH showed maximum activity 12 h after administration of a single large dose of ethanol, increasing 21 and 4.4 times, respectively, over the level in the control group. When ethanol was administered for a long time, the two ALDH isoenzyme levels showed approximately the same increase, while the high Km ALDH level was more significantly increased than the low Km ALDH level after a single large dose. These results suggest that the high Km ALDH level of the outer membrane was increased as a result of a transient increase in the level of acetaldehyde around the liver mitochondria after a single large dose of ethanol, and that high Km ALDH plays an important role in acetaldehyde metabolism. However, when ethanol was administered for a long time, the mitochondria were exposed to low concentrations of acetaldehyde over a long time, leading to an increase in levels of low and high Km ALDH in the matrix.