Dependent Aldehyde Dehydrogenase Research Articles

Identification of human liver aldehyde dehydrogenases that catalyze the oxidation of aldophosphamide and retinaldehyde

Biotransformation of the biologically and pharmacologically important aldehydes, retinaldehyde and aldophosphamide, is mediated, in part, by NAD(P)-dependent aldehyde dehydrogenases catalyze the oxidation of the aldehydes to their respective acids, retinoic acid and carboxyphosphamide. Not known at the onset of this investigation was which of the several known human aldehyde dehydrogenases (ALDHs) catalyze these reactions. Thus, human liver aldehyde dehydrogenases were chromatographically resolved and the ability of each to catalyze the oxidation of retinaldehyde and aldophosphamide was assessed. Only one, namely ALDH-1, catalyzed the oxidation of retinaldehyde; the K + m value was 0.3 μM. Three, namely ALDH-1, ALDH-2 and succinic Semialdehyde dehydrogenase, catalyzed the oxidation of aldophosphamide; K m values were 52, 1193, and 560 μM, respectively. ALDH-4, ALDH-5 and betaine aldehyde dehydrogenase did not catalyze the oxidation of either aldophosphamide or retinaldehyde. ALDH-1 and succinic Semialdehyde dehydrogenase accounted for 64 and 30%, respectively, of the total hepatic aldehyde dehydrogenase-catalyzed aldophosphamide (160 μM) oxidation. ALDH-1-catalyzed oxidation of aldophosphamide was noncompetitively inhibited by chloral hydrate; the K i value was 13μM. ALDH-2- and succinic semialdehyde dehydrogenase-catalyzed oxidation of aldophosphamide was relatively insensitive to inhibition by chloral hydrate. These observations strongly suggest an important in vivo role for ALDH-1 in the catalysis of retinaldehyde and aldophosphamide biotransformation. Succinic semialdehyde dehydrogenase-catalyzed biotransformation of aldophosphamide may also be of some in vivo importance.

Lactic Acid Formation by Free and Immobilized Cells of an Obligate Methylotroph

AbstractCells of the Gram‐negative obligate methylotrophic bacterium Methylobacillus flagellation KT, which can only use methanol and methylamine as sole carbon and energy source, oxidize 1,2‐propanediol to lactaldehyde and lactate by using a pyrroloquinoline quinone (PQQ) containing cytochrome c dependent methanol dehydrogenase and an NAD+‐dependent aldehyde dehydrogenase, both of which have a broad substrate specificity. The kinetics of 1,2‐propanediol oxidation and lactic acid appearance in the buffered medium were followed, and Km for substrate, pH, and temperature optima were determined. M. flagellatum KT cells immobilized in polyacrylamide, agarose, or carrageenan gels maintain the ability to oxidize 1,2‐propanediol to lactic acid. The half‐life of the process with the cells in carrageenan was increased 5–6 times compared with free cell suspensions.

Cloning an Escherichia coli gene encoding a protein remarkably similar to mammalian aldehyde dehydrogenases

The nucleotide (nt) sequence of 2.9 kb of Escherichia coli genomic DNA that encompasses a gene encoding a putative aldehyde dehydrogenase (ALDH) has been determined. The presence of an open reading frame beginning 2 nt downstream from the ALDH-coding sequence indicates that this gene may be part of a larger operon. An extended upstream nt sequence displays striking similarity to sequences found upstream or in intergenic regions of a number of other bacterial genes. Crude cell extracts from E. coli grown under several different conditions show weak but measurable ALDH enzyme activity that prefers NADP + over NAD + as coenzyme; however, aldH gene expression appears to be very low, since no specific transcripts derived from the novel gene can be detected on Northern blots of RNA isolated from these cells. The deduced E. coli protein contains 495 amino acid (aa) residues with a calculated M r of 53 418. Its aa sequence shows marked similarity to NAD + -dependent ALDHs of eukaryotes. About 40% aa sequence identity, and over 60% similarity, are detected between the E. coli protein and both the cytosolic (class-1) and the mitochondrial (class-2) forms of mammalian ALDHs. In contrast to the mammalian enzymes, which contain eight to eleven Cys residues, only four Cys are present in the E. coli protein, and of these only Cys 302, corresponding to the disulfiram-sensitive residue in the mammalian enzymes, is found at a conserved position in both the prokaryotic and the eukaryotic ALDHs. The availability of a bacterial ALDH with a high degree of similarity to mammalian ALDHs promises to facilitate future structural studies on these enzymes.

Oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. Role of aldehyde dehydrogenase and leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega) in leukotriene B4 omega-oxidation.

Leukotriene B4 (LTB4), a potent chemoattractant for leukocytes, is catabolized by human neutrophils via omega-oxidation. Neutrophil microsomes are known to oxidize 20-hydroxy-LTB4 (20-OH-LTB4) to its 20-oxo and 20-carboxy derivatives in the presence of NADPH. This activity has been ascribed to LTB4 omega-hydroxylase (cytochrome P-450LTB omega), a conclusion supported by our finding of the reversal of carbon monoxide inhibition by 450 nm light and by competitive inhibition studies. The oxidation of 20-oxo-LTB4 to 20-carboxy-LTB4 is also catalyzed by microsomes fortified with 1 mM NAD+, and this activity is not affected by cytochrome P-450LTB omega inhibitors. The evidence is compatible with involvement of a disulfiram-insensitive aldehyde dehydrogenase in this second oxidation pathway. Interaction of the two pathways is evidenced by facilitation of NADPH-dependent oxidation of 20-OH-LTB4 by the addition of NAD+. This synergism may be explained by removal of the aldehyde intermediate by the NAD(+)-dependent aldehyde dehydrogenase. Taken together with the finding that the NAD(+)-dependent activity is severalfold higher than the NADPH-dependent one, the dehydrogenase may be important in the oxidation of 20-OH-LTB4 to 20-carboxy-LTB4.

Metabolic mechanism of ethanol-isovaleric acid fermentation by a Clostridium saccharoperbutylacetonicum UV-mutant.

Mutant AS2-1, derived from Clostridium saccharoperbutylacetonicum N1-4 ATCC 13564, did not produce acetone, butanol, and acetic acid, but had enhanced ethanol production and newly produced isovaleric acid at the molar ratio of 1:1. The mutant was defective in thiolase (acetyl-CoA acetyltransferase) activity, while the parent N1-4 showed high thiolase activity (103.5 units/g protein). Addition of allyl alcohol into the mutant culture significantly decreased the production of both ethanol and isovaleric acid. AS2-1 mutant, therefore, had NAD+ — and NAD(P)+-dependent aldehyde dehydrogenase on the pathway to ethanol and isovaleric acid, respectively. It was suggested that an excess of electrons accumulated as the result of the deficiency in thiolase was consumed for the reductive formation of isovalerate from pyruvate. The equation of the novel ethanol–isovaleric acid fermentation was concluded as 1.5 mol glucose = 1 mol ethanol + 1 mol isovaleric acid + 2 mol CO2 + 2 mol H2O +1 mol H2.

Genetics of ocular NAD+-dependent alcohol dehydrogenase and aldehyde dehydrogenase in the mouse: evidence for genetic identity with stomach isozymes and localization of Ahd-4 on chromosome 11 near trembler.

Electrophoretic and activity variation of the stomach and ocular isozyme of aldehyde dehydrogenase (designated AHD-4) was observed between C57BL/6J and SWR/J inbred strains of mice. The phenotypes were inherited in a normal mendelian fashion, with two alleles at a single locus (Ahd-4) showing codominant expression. The alleles assorted independently of those at Adh-3 [encoding the stomach and ocular isozyme of alcohol dehydrogenase (ADH-C2)] on chromosome 3. Three chromosome 11 markers, hemoglobin alpha-chain (Hba), trembler (Tr), and rex (Re), were used in backcross analyses which established that Ahd-4 is closely linked to trembler. The distribution patterns for stomach and ocular AHD-4 phenotypes were examined among SWXL recombinant inbred mice, and those for stomach and ocular ADH-C2 among BXD recombinant inbred strains. The data provided evidence for the genetic identity of stomach and ocular ADH-C2 and of stomach and ocular AHD-4.

NADP+‐dependent aldehyde dehydrogenase from ‘Acetobacter rancens’ CCM 1774: Purification and properties

AbstractNADP+‐dependent aldehyde dehydrogenase from the cytosolic fraction of the alkane‐degradating ‘Acetobacter rancens’ CCM 1774 was purified 112‐fold (specific activity of 112 nkat mg−1 protein). After polyacrylamide gel electrophoresis of the purified enzyme only one band was visible. The relative molecular weight was estimated to be 82,000 by both gel filtration and disc gel electrophoresis. The substrate specifity of the purified enzyme within the straight chain aliphatic aldehyde series is confined to acetaldehyde and propionaldehyde. Butyraldehyde and formaldehyde are considerably less converted. NADP+ alone served as electron acceptor. Mg2+ stimulated the reaction velocity in a strong manner by ‘non‐competitive’ activation. The estimation of kinetic parameters and inhibition experiments of the irreversible oxidation of ethanal indicate a random kinetic mechanism at optimal aldehyde concentrations and saturating NADP+ concentrations at optimal pH 8.5 which, however, trends to become ordered with decreasing pH values. Properties described exclude the participation of the enzyme in degradation of n‐alkanes. One physiological function of this constitutive aldehyde dehydrogenase may be the intracellular detoxification of short chain aldehydes by conversion to corresponding fatty acids.

Biogenesis of microsomal aldehyde dehydrogenase in rat liver.

The biogenesis of a microsomal NAD(P)+-dependent aldehyde dehydrogenase (AlDH) in rat liver was studied using specific antibody raised against the purified enzyme prepared by the method of Nakayasu et al. (Nakayasu, H., Mihara, K., & Sato, R. (1978) Biochem. Biophys. Res. Commun. 83, 697-703). The antibody raised against purified AlDH inhibited the activity of microsome-bound AlDH as effectively as that of the detergent-solubilized enzyme, suggesting that AlDH molecules are present on the outer surface of microsomal vesicles and that a major portion of each molecule, including the active site, is exposed. The cell-free translation of RNA preparations extracted from free and membrane-bound polysomes showed that AlDH was exclusively synthesized on free polysomes. The in vitro synthesized AlDH had the same molecular weight as the authentic enzyme. When a single intravenous injection of [3H]leucine was given to rats, there was no detectable difference in the specific radioactivities of AlDH in rough and smooth microsomes even at the shortest time point, suggesting random incorporation of newly synthesized AlDH molecules into rough and smooth endoplasmic reticulum.

Wechselwirkungen der Aldehyddehydrogenase aus Acinetobacter calcoaceticus mit Membranlipiden I. Einfluß von Detergentien, Proteinasen und Phospholipasen auf die Solubilisierung und Aktivität des Enzyms

AbstractThe NADP+‐dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus was inducible by alkanes. It was located in the membranes surrounding the intracellular alkane inclusions. The enzyme could not be solubilized by changing of the pH or the ionic strength but by detergents at concentrations around the critical micelle concentration. High concentrations of detergents inactivated the enzyme; reactivation was possible by addition of natural membrane phospholipids. The enzyme in intact membranes gave lower Km values than the enzyme in the micelle form. The size of the particles of cell envelopes (including the inclusion membranes) and of micelles was controlled by measurements of the wavelength dependence of turbidity. Proteinases were unable to solubilize the enzyme; in the presence of detergent they inactivated it irreversibly. Phospholipase A2 and D inactivated the enzyme reversibly.

ACTIVITIES OF NAD+‐DEPENDENT ALDEHYDE DEHYDROGENASE AND ALCOHOL DEHYDROGENASE IN THE LIVER OF SPONTANEOUSLY HYPERTENSIVE RATS IN THE PROCESS OF DEVELOPMENT

The difference in the basal activities of NAD+-dependent aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) was investigated in the liver of age-matched spontaneously hypertensive (SH) and normotensive Wistar Kyoto (WK) rats. A significant difference between the SH and WK rats in the basal ALDH activity, ADH activity and the protein content of subcellular fractions was observed. The activities of mitochondrial low Km- and high Km-ALDH in the SH rats at 5-8 weeks of age were higher than those in the WK rats. The microsomal high Km-ALDH activity in the SH rats at 5 and 11 weeks of age was higher than that in the WK rats. The ADH activities in the SH rats at 5-14 weeks of age were lower than those in the WK rats. The mitochondrial protein content in the SH rats at 5-14 weeks of age was higher than those in the WK rats. At 14 weeks of age, an increase in the blood acetaldehyde level was observed after an intraperitoneal injection of 1.5 g/kg of ethanol in the SH rats. No difference in blood ethanol level was observed between the SH and WK rats.

The subcellular distribution and properties of aldehyde dehydrogenase of hepatoma AH-130

Aldehyde dehydrogenase subcellular distribution and activity were studied in the Yoshida hepatoma AH-130 and rat liver. NAD +- and NADP +-dependent dehydrogenase activities were lower in all hepatoma subfractions (except the cytosol) than in liver subfractions. In the presence of 0.025 mM substrate 78–80% of the liver NAD +- or NADP +-dependent aldehyde dehydrogenase was found in the mitochondria. With 10 mM substrate the enzyme activity was primarily in the mitochondria and microsomes. In the hepatoma a sharp increase of the soluble aldehyde dehydrogenase (either NAD +- or NADP + dependent) was observed at all substrate concentrations. The K m of the different isoenzymes (either identified by their localization or coenzyme dependency) were of the same order for liver and hepatoma. However, a high K m enzyme was present in liver mitochondria outer membranes but not in hepatoma. Hepatoma acetaldehyde dehydrogenase was inhibited, as was the liver enzyme, by diethyldithiocarbamate. The return of activity was slower for the hepatoma and neonatal liver than for the adult liver enzyme.

Acetaldehyde oxidation in Drosophila melanogaster and Drosophila simulans: Evidence for the presence of an NAD +-dependent dehydrogenase

1. 1. An NAD +-dependent oxidation of acetaldehyde has been found in two Drosophila species, D. melanogaster and D. simulans. 2. 2. The enzymatic activity was assessed in partially purified preparations from fly homogenates by monitoring spectrophotometrically NADH production at 340 nm at 25°C. 3. 3. Optimal activity was observed at pH values between 10 and 11 for both species. 4. 4. For both species the affinities ( K m ) for acetaldehyde were in the micromolar range whereas those for NAD + were in the 100 μM range. 5. 5. Catalytic activity ( V max ) was 2-fold higher in D. melanogaster, a species with high tolerance to ethanol, than in D. simulans, a species with a much lower tolerance to ethanol. 6. 6. Disulfiram proved to inhibit NADH production in preparations of both species. 7. 7. It is concluded that the enzyme involved is an NAD +-dependent aldehyde dehydrogenase (ALDH). Its possible biological significance in ethanol tolerance is discussed.

4-Aminobutyraldehyde dehydrogenase activity in rat brain.

An enzyme with NAD+-dependent 4-aminobutyraldehyde dehydrogenase activity was purified about 360-fold from rat brain extract. AMP-Sepharose chromatography was effective in separating the enzyme from other NAD+-dependent aldehyde dehydrogenases included in the extract. The KmS for the substrates NAD+ and 4-aminobutyraldehyde were 4.8 x 10(-4) and 8.3 x 10(-5) M, respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4-aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4-aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.

81] Aldehyde dehydrogenases from Pseudomonas aeruginosa

This chapter describes the assay method, purification, and properties of two aldehyde dehydrogenases—namely–, nicotinamide adenine dinucleotide (NAD + )-linked aldehyde dehydrogenase and nicotinamide adenine dinucleotide phosphate (NADP + )-dependent aldehyde dehydrogenase. In Pseudomonas aeruginosa, an aldehyde dehydrogenase induced by growth on ethanolhas also been studied. The reduction of NAD + is measured at 30°C with a recording spectrophotometer at 340 nm. The steps involved in the purification of NAD + are (1) the growth of the microorganism, (2) the preparation of extracts, (3) protamine sulfate treatment, (4) diethylaminoethyl (DEAE)-cellulose chromatography, (5) hydroxyapatite chromatography, and (6) Sephadex G-200 chromatography. The assay of NADP + -dependent aldehyde dehydrogenase enzyme activity is similar as for the NAD + -dependent enzyme, except that the reagents used are 4.3 m M pentanal dissolved in 54 m M potassium pyrophosphate buffer, pH 8.6, and 15 m M NADP + . The final concentrations in the assay are 4 m M for pentanal, 50 m M for potassium pyrophosphate, and 0.5 m M for NADP + .

Two Cytochromes c of Methylomonas J1

Two kinds of c-type cytochromes, cytochrome c-551 (I), and cytochrome c-551 (II), were highly purified and crystallized from cell-free extract of methanol-grown Methylomonas J (formerly Pseudomonas sp. J) and their physiochemical and biochemical properties were studied. Cytochrome c-551 (I) had an absorption peak at 409 nm in the oxidized form and peaks at 417, 523, 551 nm, and a shoulder at 532 nm in the reduced form. The millimolar extinction coefficient of the alpha-peak of the reduced form was 25.3. The isoelectric point was at pH 5.3 and its standard redox potential was 0.29 V at pH 7.0. The molecular weight was estimated to be 16,000. Cytochrome c-551 (II) had absorption maxima at 409 nm in the oxidized form, and at 416, 521, and 551 nm in the reduced form. The millimolar extinction coefficient of the alpha-peak of the reduced form was 22.4. The isoelectric point was at pH 4.3 and its standard redox form was 22.4. The isoelectric point was at pH 4.3 and its standard redox potential was 0.24 V at pH 7.0. The molecular weight was estimated to be 12,500. The two cytochromes were reduced by methanol dehydrogenase [EC 1.1.99.8] of this bacterium, and formaldehyde was detected as an oxidation product. Ammonium chloride was not essential for reduction of the cytochromes. No significant reduction of the cytochromes was observed by methylamine dehydrogenase isolated from methylamine-grown cells or by 2,6-dichlorophenol-indophenol (DCPIP)-dependent aldehyde dehydrogenase of the methanol-grown cells. The reduced forms of the cytochromes were oxidized by blue copper protein of the methanol-grown cells.

Subcellular distribution and properties of rabbit liver aldehyde dehydrogenases

In rabbit liver, both NAD +- and NADP +-dependent aldehyde dehydrogenases were identified. The activities were distributed among at least three major groups of isozymes identifiable by gel electrophoresis. These isozymes also differed in their substrate and coenzyme preferences, subcellular distributions, and/or responses to effectors. The NAD +-dependent aldehyde dehydrogenase activity was distributed among the mitochondrial, microsomal, and cytosolic fractions. The NADP +-dependent aldehyde dehydrogenase activity was largely microsomal, with little true cytosolic NADP +-dependent activity demonstrable. Aliphatic aldehydes were oxidized equally well by aldehyde dehydrogenases in all three fractions. Aromatic aldehydes, however, were preferentially oxidized by microsomal aldehyde dehydrogenases. Disulfiram significantly inhibited mitochondrial (45 per cent) and cytosolic (93 per cent) NAD +-dependent aldehyde dehydrogenase, but it did not cause significant inhibition of microsomal NAD +-dependent activity. Disulfiram inhibited the NADP +-dependent aldehyde dehydrogenase activity (>71 per cent) in all subcellular fractions. Diethylstilbestrol activated both NAD +- and NADP +-dependent aldehyde dehydrogenases in mitochondria and cytosol. Microsomal aldehyde dehydrogenases were not affected by diethylstilbestrol.

NAD +-dependent acetaldehyde oxidation in Drosophila

1. 1. NAD +-dependent acetaldehyde oxidation was measured spectrophotometrically in homogenates of flies Drosophila melanogaster. 2. 2. The reaction was specifically triggered by addition of acetaldehyde and proceeded linearily over five minutes. 3. 3. At low acetaldehyde concentrations the reaction rate was rapidly maximal whereas higher acetaldehyde concentrations proved to be inhibitary. 4. 4. Enzyme activity in regard to NAD + concentration followed classical Michaelis kinetics. The apparent Km for NAD + was 0.05 mM. 5. 5. These data provide evidence for the presence of a NAD +-dependent aldehyde dehydrogenase. It is suggested that this enzyme is involved in the oxidation of acetaldehyde in Drosophila. 6. 6. The biochemical and biological significance of this enzyme in regard to ethanol metabolism and ethanol tolerance is discussed.

Species and strain - dependent aldehyde dehydrogenase: F. S. Messiha and H. F. Sproat, Departments of Pathology and Psychiatry, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, Texas 79430

Species and strain - dependent aldehyde dehydrogenase: F. S. Messiha and H. F. Sproat, Departments of Pathology and Psychiatry, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, Texas 79430

Genetic control of responsiveness of rat liver supernatant aldehyde dehydrogenase to phenobarbital and 3-methylcholanthrene.

The responsiveness of the hepatic supernatant NAD+-dependent aldehyde dehydrogenase with a high Km value (high Km-AldDH) to phenobarbital (PB) and 3-methylcholanthrene (3-MC) treatment was studied in male rats of three strains; Wistar, Long-Evans, and Sprague-Dawley. A remarkable strain difference in the response of the enzyme to PB or 3-MC was observed. In rats of the Wistar strain the enzyme activity remained unchanged ("non-responsive") in all rats after treatment with PB while it increased ("responsive") 5- to 19-fo-d in all rats after treatment with 3-MC. The enzyme activity increased 8- to 20-fold and 2- to 8-fold respectively after treatment with PB and 3-MC in all rats of the Long-Evans strain. In rats of the Sprague-Dawley strain the enzyme activity remained unchanged in half of all the rats treated with PB or 3-MC and increased 2- to 7-fold over the basal level in half of the treated rats. The non-responsive rats to PB were all responsive to 3-MC treatment while the responsive rats to PB were responsive in 65% and non-responsive in 35% to 3-MC treatment.

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.