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.     

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.     

Effects of diethylstilbestrol, 2,2'-dithiodipyridine, and chloral hydrate on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase

The binding of diethylstilbestrol (DES) to aldehyde dehydrogenase (ALDH) has a very similar effect on the dehydrogenase activity of the enzyme as has modification of the enzyme by 2,2'-dithiodipyridine [Kitson, T.M. (1982) Biochem. J. 207, 81-89]. The latter modification may occur at the site of the esterase activity of the enzyme [Kitson, T.M. (1985) Biochem. J. 228, 765-767]. This suggests that DES might be a competitive inhibitor of the esterase reaction. However, in the absence of oxidized nicotinamide adenine dinucleotide (NAD+) or reduced nicotinamide adenine dinucleotide (NADH), and at low concentrations of substrate (4-nitrophenyl acetate, PNPA), DES is a potent partial noncompetitive inhibitor. It is concluded therefore that DES binds at a site different from the esterase active site and that the enzyme-DES complex retains some ability to act as an esterase. High concentrations of PNPA appear to displace DES from its binding site. In the presence of NAD+, DES is a weaker inhibitor, and in the presence of NADH, DES has very little effect. Esterase activity is enhanced by NADH when PNPA concentrations are high but is inhibited when they are low. The rate of reaction of ALDH with 2,2'-dithiodipyridine is only slightly reduced by DES, suggesting that the site at which thiol modifiers react and the DES binding site are different. When ALDH is modified by 2,2'-dithiodipyridine, it has reduced esterase activity, which declines further as the modified enzyme loses its 2-thiopyridyl label. In the presence of NAD+, chloral hydrate is a simple competitive inhibitor of the esterase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some comparisons of pig and sheep liver cytosolic aldehyde dehydrogenases

1. The pig enzyme was purified to homogeneity and was found to be a tetramer of apparently identical subunits. 2. The pig enzyme was found to contain 1 mol NADH/mol enzyme which is tightly bound, which is not directly involved in catalysis and which so far has not been removed from the enzyme so as to produce an active apoenzyme. 3. The pig enzyme seems to contain only one functioning active site/tetramer. 4. The pig and sheep enzymes are compared in respect of NADH binding, substrate specificity, immunological response and surface charge.     

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.     

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.     

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.     

Genetic basis for the polymorphism of rat liver cytosolic aldehyde dehydrogenase

Liver aldehyde dehydrogenase (ALDH), the enzyme involved in the oxidation of aldehydes such as acetaldehyde derived from ethanol, exists in multiple forms in most mammals. Up to five separable forms have been identified from the cytosolic fraction of Wistar rat liver. We investigated the genetic basis of a particular set of three enzyme forms by selective breeding and analysis of electrophoretic patterns of liver ALDH by isoelectric focusing. The forms of liver ALDH investigated were at pI 5.8 or 6.2, or a triple form with enzymes at pI 5.8, 6.0, and 6.2. There are two alleles found at the ALDH locus which encode in homozygotes for one of two electrophoretically separable ALDH forms. A rat heterozygous at the locus forms both ALDH types plus a hybrid. The alleles are expressed codominantly, found at an autosomal locus, and remain constant postpartum. The activities associated with the triplet enzyme form were statistically indistinguishable from a 1:2:1 ratio. This suggests that the enzymes hybridize to form a set of dimers or tetramers of the form A₂, AB, B₂ or A₄, A₂B₂, B₄, respectively.     

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.     

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.     

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.     

Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents.

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2'-dithiodipyridine and 4,4'-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4'-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2'-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5'-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.     

Mechanism of inactivation of sheep liver cytoplasmic aldehyde dehydrogenase by disulfiram.

Stoicheiometric amounts of [14C]disulfiram react rapidly with sheep liver cytoplasmic aldehyde dehydrogenase to give loss of catalytic activity and incorporation of the expected amount of radioactivity. In a subsequent slower reaction the label is lost from the enzyme without re-emergence of enzymic activity. The results imply that in vivo disulfiram may act as an oxidation-reduction catalyst for the inactivation of aldehyde dehydrogenase.     

Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase.

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2'-Dithiodipyridine is an activator of the enzyme.     

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD(+). Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD(+) site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.     

Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme--NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K_i=47 μM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane–carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.     

Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase.

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.     

Interaction of human aldehyde dehydrogenase with aromatic substrates and ligands

The substrate benzaldehyde (but not propionaldehyde) could elute aldehyde dehydrogenase from a p-hydroxyacetophenone-affinity column, and inhibit the esterase activity (K(i)=47 microM), indicating that this simple aromatic aldehyde binds to the free enzyme and possibly in the substrate-binding site. Thus, the kinetic mechanism for aldehyde dehydrogenase might be dependent upon which aldehyde is used in the reaction. Chloramphenicol which also elutes the enzyme from the affinity column, shows a discriminatory effect by inhibiting the ALDH1 oxidation of benzaldehyde and activating that of propionaldehyde while showing no effect when assayed with hexanal or cyclohexane-carboxaldehyde. Chloramphenicol is an uncompetitive inhibitor against NAD when benzaldehyde is the substrate. We propose that this drug might interact with both the benzaldehyde and NAD binding sites.