The effect of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The effect of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. 2. Disulfiram causes an immediate inhibition of the enzyme reaction. The effect on the cytoplasmic enzyme is much greater than on the mitochondrial enzyme. 3. In both cases, the initial partial inhibition is followed by a gradual irreversible loss of activity. 4. The pH-rate profile of the inactivation of the mitochondrial enzyme by disulfiram and the pH-dependence of the maximum velocity of the enzyme-catalysed reaction are both consistent with the involvement of a thiol group. 5. Excess of 2-mercaptoethanol or GSH abolishes the effect of disulfiram. However, equimolar amounts of either of these reagents and disulfiram cause an effect greater than does disulfiram alone. It was shown that the mixed disulphide, Et2N-CS-SS-CH2-CH2OH, strongly inhibits aldehyde dehydrogenase. 6. The inhibitory effect of diethyldithiocarbamate in vitro is due mainly to contamination by disulfiram.     

Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase.

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.     

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin, resveratrol and diethylstilbestrol

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD(+)-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD(+) concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD(+), by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.     

Bioactivation of nitroglycerin by purified mitochondrial and cytosolic aldehyde dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC(50) values of 42 +/- 2.9 and 3.1 +/- 0.4 microm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.     

Some properties of aldehyde dehydrogenase from sheep liver mitochondria.

Aldehyde dehydrogenase from sheep liver mitochondria was purified to homogeneity as judged by electrophoresis on polyacrylamide gels, and by sedimentation-equilibrium experiments in the analytical ultracentrifuge. The enzyme has a molecular weight of 198000 and a subunit size of 48000, indicating that the molecule is a tetramer. Fluorescence and spectrophotometric titrations indicate that each subunit can bind 1 molecule of NADH. Enzymic activity is completely blocked by reaction of 4mol of 5,5'-dithiobis-(2-nitrobenzoate)/mol of enzyme. Excess of disulfiram or iodoacetamide decreases activity to only 50% of the control value, and only two thiol groups per molecule are apparently modified by these reagents.     

The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The liver microsomal metabolism of [4-14C]cholesterol, endogenous cholesterol, 7 alpha-hydroxy-4-[6 beta-3H]cholesten-3-one, 5-beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol and [3H]lithocholic acid was studdied in control and clofibrate (ethyl p-chlorophenoxyisobutyrate)-treated rats. 2. The extent of 7 alpha-hydroxylation of exogenous [414C]cholesterol and endogenous cholesterol, the latter determined with a mass fragmentographic technique, was the same in the two groups of rats. The extent of 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one and 5 beta-cholestane-3 alpha, 7 alpha-diol was increased by about 60 and 120% respectively by clofibrate treatment. The 26-hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol was not significantly affected by clofibrate. The 6 beta-hydroxylation of lithocholic acid was about 80% higher in the clofibrate-treated animals than in the controls. 3. The results are discussed in the context of present knowledge about the liver microsomal hydroxylating system and bile acid formation in patients with hypercholesterolaemia, treated with clofibrate.     

Rat liver constitutive and phenobarbital-inducible cytosolic aldehyde dehydrogenases are highly homologous proteins that function as distinct isozymes

Rat liver contains two class 1 aldehyde dehydrogenases (ALDHs): a constitutive isozyme (ALDH1) and a phenobarbital-inducible isozyme (ALDH-PB). Defining characteristics of mammalian class 1 ALDHs include a homotetrameric structure, high expression in liver, sensitivity to the inhibitor disulfiram, and high activity for the oxidation of retinal. It is often presumed that ALDH-PB is the rat ortholog of mammalian ALDH1, and the identity of rat ALDH-PB is commonly interchanged with ALDH1. In this study, we characterized recombinant rat liver cytosolic ALDH1 and ALDH-PB. Previous reports indicate that ALDH-PB is a homodimer; however, we found by mass spectrometry and gel electrophoresis that it is a homotetramer. ALDH1 mRNA was highly expressed in untreated rat liver, while ALDH-PB had very weak expression, in contrast to a previous report that ALDH-PB mRNA is expressed in untreated rat liver. Rat liver ALDH1 had a high affinity for retinal (K(m) = 0.6 microM), while no oxidation by ALDH-PB could be detected with 20 microM retinal. ALDH1 was more efficient at oxidizing acetaldehyde, propionaldehyde, and benzaldehyde and was more sensitive to disulfiram inhibition. We conclude that rat liver ALDH1 is the ortholog of mammalian liver ALDH1. Furthermore, despite a high level of sequence identity and classification as a class 1 ALDH, ALDH-PB does not function like ALDH1. ALDH-PB is not merely an inducible ALDH1 isozyme; it is a distinct ALDH isozyme.     

Isolation and characterization of rat liver cytosolic aldehyde dehydrogenases induced by phenanthrene or benzo[a]pyrene

The cytosolic aldehyde dehydrogenase was isolated from the liver of Wistar rats treated with phenanthrene (non-carcinogenic) or benzo[a]pyrene (carcinogenic polycyclic aromatic hydrocarbon). The benzo[a]pyrene-induced enzyme has higher Km values for small aliphatic aldehydes and a lower molecular weight than the phenanthrene-induced enzyme. It is more resistant to changes of pH and to inhibition by disulfiram, but more sensitive to heat denaturation than the phenanthrene-induced enzyme. The phenanthrene-induced aldehyde dehydrogenase is very similar to the normal uninduced aldehyde dehydrogenase, whereas the benzo[a]pyrene-induced aldehyde dehydrogenase has common properties with the TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced enzyme and the hepatoma-specific enzyme.     

Hydride transfer stereospecificity of rat liver aldehyde dehydrogenases

The stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.     

Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver.

In the presence of acetic anhydride or butyric anhydride, liver aldehyde dehydrogenases catalyse the oxidation of NADH at pH 7.0 and 25 degrees C. The maximum velocities and Michaelis constants for NADH at saturating anhydride concentrations are independent of which anhydride is used, the values being V'max. = 12 min-1 and Km for NADH = 9 micrometer for the mitochondrial enzyme and V'max = 25 min-1 and Km for NADH = 20 micrometer for the cytoplasmic enzyme. Substitution of [4A-2H]NADH for NADH resulted in 2-fold and 4-fold decreases in rate for the mitochondrial and cytoplasmic enzymes respectively.     

The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.     

Some properties of pig kidney-cortex aldehyde reductase.

Aldehyde reductase was purified from pig kidney cortex to homogeneity by a new procedure. The molecular weight of the enzyme was estimated by sedimentation equilibrium to be 43 700 and by gel electrophoresis in the presence of sodium dodecyl sulphate to be 41 700. The enzyme is clearly a monomer. The enzyme preparation contained no significant quantities of zinc, manganese or copper and had no essential histidine or thiol groups. Changes in the absorption and fluorescence spectra of NADPH were observed on formation of the enzyme-NADPH complexes. Large changes in the fluorescence spectra were also observed in the presence of sodium barbitone or Warfarin. These changes were used as the basis of active-site titrations, which showed that the enzyme had one active site per molecule. The dissociation constants of NADPH and NADP+ from binary complexes with the enzyme were estimated in spectrophotometric titrations.     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

Structures of human alcohol and aldehyde dehydrogenases

Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized.     

Neurodegeneration and motor dysfunction in mice lacking cytosolic and mitochondrial aldehyde dehydrogenases: implications for Parkinson's disease

Previous studies have reported elevated levels of biogenic aldehydes in the brains of patients with Parkinson's disease (PD). In the brain, aldehydes are primarily detoxified by aldehyde dehydrogenases (ALDH). Reduced ALDH1 expression in surviving midbrain dopamine neurons has been reported in brains of patients who died with PD. In addition, impaired complex I activity, which is well documented in PD, reduces the availability of the NAD(+) co-factor required by multiple ALDH isoforms to catalyze the removal of biogenic aldehydes. We hypothesized that chronically decreased function of multiple aldehyde dehydrogenases consequent to exposure to environmental toxins and/or reduced ALDH expression, plays an important role in the pathophysiology of PD. To address this hypothesis, we generated mice null for Aldh1a1 and Aldh2, the two isoforms known to be expressed in substantia nigra dopamine neurons. Aldh1a1(-/-)×Aldh2(-/-) mice exhibited age-dependent deficits in motor performance assessed by gait analysis and by performance on an accelerating rotarod. Intraperitoneal administration of L-DOPA plus benserazide alleviated the deficits in motor performance. We observed a significant loss of neurons immunoreactive for tyrosine hydroxylase (TH) in the substantia nigra and a reduction of dopamine and metabolites in the striatum of Aldh1a1(-/-)×Aldh2(-/-) mice. We also observed significant increases in biogenic aldehydes reported to be neurotoxic, including 4-hydroxynonenal (4-HNE) and the aldehyde intermediate of dopamine metabolism, 3,4-dihydroxyphenylacetaldehyde (DOPAL). These results support the hypothesis that impaired detoxification of biogenic aldehydes may be important in the pathophysiology of PD and suggest that Aldh1a1(-/-)×Aldh2(-/-) mice may be a useful animal model of PD.     

Inactivation of cytosolic aldehyde dehydrogenase via S-nitrosylation in ethanol-exposed rat liver

Aldehyde dehydrogenase (ALDH) isozymes are critically important in the metabolism of acetaldehyde, thus preventing its accumulation after ethanol-exposure. We previously reported that mitochondrial ALDH2 could be inactivated via S-nitrosylation in ethanol-exposed rats. This study was aimed at investigating whether cytosolic ALDH1, with a relatively-low-Km value (11-18 microM) for acetaldehyde, could be also inhibited in ethanol-exposed rats. Chronic or binge ethanol-exposure significantly decreased ALDH1 activity, which was restored by addition of dithiothreitol. Immunoblot analysis with the anti-S-nitroso-Cys antibody showed one immunoreactive band in the immunoprecipitated ALDH1 only from ethanol-exposed rats, but not from pair-fed controls, suggesting S-nitrosylation of ALDH1. Therefore inactivation of ALDH1 via S-nitrosylation can result in accumulation of acetaldehyde upon ethanol-exposure.     

Inducibility of liver cytosolic aldehyde dehydrogenase activity in various animal species

1. The inducibility of hepatic cytosolic aldehyde dehydrogenase activity was studied in rat, mouse, guinea pig, chicken, frog, salamander and rainbow trout, by using two different types of inducers of drug metabolism. 2. Phenobarbital (a type I inducer of drug metabolizing enzymes) increased total liver cytosolic aldehyde dehydrogenase activity (up to 20-fold) in a genetically defined substrain of responsive rats (RR) and only slightly, if at all, in a non-responsive substrain (rr). On the contrary, both types of rats showed a highly induced aldehyde dehydrogenase activity after treatment with methylcholanthrene (a type II inducer). Phenobarbital is affecting mainly an isozyme of aldehyde dehydrogenase which is best measured with propionaldehyde as the substrate and NAD as the coenzyme (P/NAD). 3. Administration of phenobarbital to mice produced only a slight increase (2-fold) in the P/NAD aldehyde dehydrogenase activity. 4. Methylcholanthrene treatment caused a 2-fold increase of the hepatic P/NAD aldehyde dehydrogenase activity in the chicken. 5. In the guinea pig, phenobarbital produced an approximate 3-fold increase of the P/NAD activity. Methylcholanthrene had a similar effect, although to a lesser extent. 6. In the salamander, a 4-fold increase was detected in the enzyme activity measured with benzaldehyde as the substrate and NADP as the coenzyme (B/NADP), after treatment with either phenobarbital or methylcholanthrene. 7. The hepatic aldehyde dehydrogenase activities were found unchanged in the rainbow trout, after treatment with phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin. 8. The rat model remains the only one examined that shares with human hepatocytes strong inducibility of the B/NADP aldehyde dehydrogenase isozyme upon treatment with polycyclic aromatic hydrocarbons.     

Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD(+) and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD(+) and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD(+), estimates of 2x10(5) litre.mol(-1).s(-1) and 2s(-1) were obtained for k(+1) and k(-1) respectively. Although k(+3) could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s(-1), k(+2) and k(-2) could not be determined uniquely, but could be related by the equation: (k(-2)+k(+3))/k(+2) =50x10(-6)mol.litre(-1). No significant isotope effect was observed when [1-(2)H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k(-2)+k(+3))/k(+2)=2.3x10 (-3)mol.litre(-1) and k(+3) is 23s(-1). When [1,2,2,2-(2)H]acetaldehyde was used, no isotope effect was observed on k(+3), but there was a significant effect on k(+2) and k(-2). A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.