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.     

Kinetics of p-nitrophenyl pivalate hydrolysis catalysed by cytoplasmic aldehyde dehydrogenase.

The effects of modifiers (NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol and p-nitrobenzaldehyde) on the hydrolysis of p-nitrophenyl (PNP) pivalate (PNP trimethylacetate) catalysed by cytoplasmic aldehyde dehydrogenase are reported. In each case a different inhibition pattern is obtained to that observed when the substrate is PNP acetate; for example, propionaldehyde and chloral hydrate competitively inhibit the hydrolysis of PNP acetate, but are mixed inhibitors with PNP pivalate. The kinetic results can be rationalized in terms of different rate-determining steps: acylation of the enzyme in the case of the pivalate but acyl-enzyme hydrolysis for the acetate. This is confirmed by stopped-flow studies, in which a burst of p-nitrophenoxide is observed when the substrate is PNP acetate, but not when it is the pivalate. PNP pivalate inhibits the dehydrogenase activity of the enzyme competitively with the aldehyde substrate; this is most simply explained if the esterase and dehydrogenase reactions occur at a common enzymic site.     

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.     

Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids.

A soluble aldehyde dehydrogenase (EC 1.2.1.3) was partially purified from Rhizobium japonicum bacteroids and from free-living R. japonicum 61A76. The enzyme was activated by NAD+, NADH, and dithiothreitol, and it reduced NAD(P)+. Acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and succinic semialdehyde were substrates. The Km for straight-chain aldehydes decreased with increasing carbon chain length. The aldehyde dehydrogenase was inhibited by 6-cyanopurine, but not by metronidazole. These compounds inhibited acetylene reduction, but not respiration, by isolated bacteroids.     

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.     

Human prostatic aldehyde dehydrogenase of healthy controls and diseased prostates.

Aldehyde dehydrogenase (ALDH, EC 1.2.1.3) of the human prostate was the subject of investigation in this study. The possible physiological role of aldehyde dehydrogenase in the human prostate might be to detoxify aldehydes arising from the oxidation of the polyamines via monoamine or diamine oxidases. The specific activity of the enzyme with 1 mM propionaldehyde as substrate and 0.5 mM NAD at pH 7.4 in the control normal prostates and prostates afflicted with the disease, benign prostatic hyperplasia (BPH), was 26.06 +/- 2.96 and 5.17 +/- 0.48 nmol/g prostate per min, respectively. When 100 microM gamma-aminobutyraldehyde was used as a substrate, the specific activity in the normal controls and prostates with benign prostatic hyperplasia was 19.80 +/- 1.33 and 2.95 +/- 2.46 nmol/g prostate per min, respectively. Upon isoelectric focusing of the extracts of the control prostates when the gels were developed for aldehyde dehydrogenase activity, there were three aldehyde dehydrogenase activity bands visible, pI 4.9 (mitochondrial), 5.4 (cytosolic) and about 6.0-6.5, on the IEF gels developed with gamma-aminobutyraldehyde as a substrate. With the extracts of prostates with benign prostatic hyperplasia the pI 4.9 band was significantly reduced, the pI 5.4 band enhanced and the approx. pI 6.0 band was not detectable on the IEF gels with propionaldehyde as a substrate. There was no detectable aldehyde dehydrogenase activity in the extract of the prostate with cancer on IEF gels nor in the activity assays with propionaldehyde or gamma-aminobutyraldehyde as substrates.     

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.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.     

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.     

Enhancement of aldehyde dehydrogenase activity in human and rat hepatocyte cultures by 3-methylcholanthrene

Aldehyde dehydrogenase was measured in primary cultures of hepatocytes obtained with a two-step collagenase perfusion either from human hepatic tissue or from livers of Fisher rats. Basal enzyme activity declines gradually as a function of time in culture, but remains at all times higher when measured with propionaldehyde and NAD (P/NAD) than with benzaldehyde and NADP (B/NADP). Treatment of the cultures with 2 M of 3-methylcholanthrene for four days significantly increased the B-NADP activity of human and rat hepatocytes (tenfold and eightfold respectively). In human hepatocytes 3-methylcholanthrene increases also the P/NAD activity, but to a lesser extent (twofold), compared to the B/NADP activity. Due to the significant enhancement of B/NADP activity in cultures of human and rat hepatocytes after application of 3-methylcholanthrene, the initial difference in the basal activity levels between the P/NAD and B/NADP forms diminishes or, in the case of human hepatocytes, is even inverted. These results show for the first time that aldehyde dehydrogenase activity is increased in cultured human hepatocytes. This biochemical property is preserved in human and rat hepatocyte cultures, despite the rather quick loss of the basal aldehyde dehydrogenase activity.Abbreviations ALDH aldehyde dehydrogenase - B benzaldehyde - p-p-DDT 1,1,1,-trichlo-2,2,bis(p-chlorophenyl)ethane - DMSO dimethylsulfoxide - 3-MC 3-methylcholanthrene - MEM Minimal Essential Medium - P proprionaldehyde - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells 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.     

Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme.

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 X 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

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.     

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Bromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase

Human liver aldehyde dehydrogenase isozymes E1 and E2 (EC 1.2.1.3) are both completely and irreversibly inactivated by bromoacetophenone (2-bromo-1-phenylethanone). Steady-state kinetics with both acetophenone and chloroacetophenone indicated interaction with the same enzyme form as the aldehyde substrate. Saturation kinetics with chloroacetophenone and bromoacetophenone indicated interaction at a specific site on the enzyme surface and gave a dissociation constant similar to that from steady-state kinetics, suggesting that the same processes were being observed by both methods and that the active site may be involved. Protection against inactivation was afforded by chloral and NAD together. Stoichiometry of inactivation showed the first 2 equiv per tetramer to abolish the majority of catalytic activity; 4 equiv inactivated both isozymes with complete loss of esterase, NAD-stimulated esterase, and dehydrogenase activities. Peptide mapping of enzyme modified with [carbonyl-14C]bromoacetophenone of CNBr digests (E1) and tryptic digests (E1 and E2) showed one peptide to be preferentially labeled. The above results together with the similarity of bromoacetophenone to the substrate benzaldehyde suggest bromoacetophenone may react with a residue in the active site of aldehyde dehydrogenase. Amino acid analysis of the labeled E1 tryptic fragment indicated reaction with a different peptide from that with which iodoacetamide reacts.     

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.     

Purification of liver aldehyde dehydrogenase by p-hydroxyacetophenone-sepharose affinity matrix and the coelution of chloramphenicol acetyl transferase from the same matrix with recombinantly expressed aldehyde dehydrogenase.

p-Hydroxyacetophenone was coupled to epoxy-activated Sepharose 6B to generate an affinity chromatographic matrix to purify aldehyde dehydrogenase. Purified beef liver mitochondrial aldehyde dehydrogenase specifically bound to the support and could be eluted with p-hydroxyacetophenone. A post-ammonium sulfate (30-55%) fraction of bovine liver was applied to the affinity gel column and aldehyde dehydrogenase was effectively purified, although not to complete homogeneity, indicating the potential selectivity of the matrix. Both beef liver cytosolic and mitochondrial aldehyde dehydrogenase bound to the column. A post-Cibacron blue Sepharose Cl-6B affinity-fractionated liver mitochondrial aldehyde dehydrogenase was purified to complete homogeneity by p-hydroxyacetophenone-Sepharose, thus eliminating the need for the isoelectric focusing step often employed. p-Hydroxyacetophenone was found to be a competitive inhibitor against propionaldehyde and noncompetitive against NAD. Escherichia coli lysates of recombinantly expressed aldehyde dehydrogenase were purified from E. coli lysates with one major 25-kDa protein contaminant also binding to the column, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 25-kDa contaminant was found to be chloramphenicol acetyl transferase from sequence analysis and binding studies.