Showing papers on "Aldehyde dehydrogenase published in 1982"

Human aldehyde dehydrogenase: mechanism of inhibition of disulfiram

Abstract: Disulfiram labeled with carbon-14 reacts specifically with human liver aldehyde dehydrogenase E1 with loss of catalytic activity and no incorporation of label. Carbon-14-labeled diethyldithiocarbamate is formed and the number of enzyme sulfhydryl groups decreases from 34 to 30 during this process. Activity is recovered by-mercaptoethanol but not by glutathione, the physiological reducing agent.

Structural mutation in a major human aldehyde dehydrogenase gene results in loss of enzyme activity.

Abstract: Most Caucasians have two major liver aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, while approximately 50% of Orientals have only ALDH1 isozyme, missing the ALDH2 isozyme. A remarkably higher frequency of acute alcohol intoxication among Orientals than among Caucasians could be related to the absence of the ALDH2 isozyme, which has a low apparent Km for acetaldehyde. Examination of liver extracts by two-dimensional crossed immunoelectrophoresis revealed that an atypical Japanese liver, which had no ALDH2 isozyme, contained an enzymatically inactive but immunologically cross-reactive material corresponding to ALDH2, beside the active ALDH1 isozyme. Therefore, the absence of ALDH2 isozyme in atypical Orientals is not due to regulatory mutation, gene deletion, or nonsense mutation, but must be due to a structural mutation in a gene for the ALDH2 locus, resulting in synthesis of enzymatically inactive abnormal protein.

[76] Alcohol dehydrogenase from acetic acid bacteria, membrane-bound

Abstract: Publisher Summary This chapter describes the assay method, purification, and properties of alcohol dehydrogenase isolated from acetic acid bacteria. Alcohol dehydrogenase of acetic acid bacteria acts on a wide range of primary alcohols, except methanol. The enzyme acts as a vinegar producer by coupling with aldehyde dehydrogenase. The enzyme is localized on the outer surface of the cytoplasmic membrane and the oxidation of substrate is linked to its respiratory chain. The enzyme differs from alcohol dehydrogenase of methanol utilizing bacteria, which without exception requires ammonia for full activity. The reaction rate is estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phenazine methosulfate; (b) by colorimetry in the presence of potassium ferricyanide; (c) by polarography with an oxygen electrode; or (d) by manometry in a conventional Warburg apparatus. The assay method with potassium ferricyanide is employed because of its simplicity for routine assay. The steps involved in the purification of alcohol dehydrogenase from G. suboxydans and A. aceti are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) diethylaminoethyl (DEAE)-Sephadex column chromatography I and II, and (4) hydroxyapatite column chromatography.

Aldehyde Dehydrogenases in Rat Brain. Subcellular Distribution and Properties

Abstract: Kinetic studies suggested the presence of several forms of NAD-dependent aldehyde dehydrogenase (ALDH) in rat brain. A subcellular distribution study showed that low- and high-Km activities with acetaldehyde as well as the substrate-specific enzyme succinate semialdehyde dehydrogenase were located mainly in the mitochondrial compartment. The low-Km activity was also present in the cytosol (less than 20%). The low-Km activity in the homogenate was only 10-15% of the total activity with acetaldehyde as the substrate. Two Km values were obtained with both acetaldehyde (0.2 and 2000 microM) and 3,4-dihydroxyphenylacetaldehyde (DOPAL) (0.3 and 31 microM), and one Km value with succinate semialdehyde (5 microM). The main part of the aldehyde dehydrogenase activities with acetaldehyde, DOPAL, and succinate semialdehyde, but only little activity of the marker enzyme for the outer membrane (monoamine oxidase, MAO), was released from a purified mitochondrial fraction subjected to sonication. Only small amounts of the ALDH activities were released from mitochondria subjected to swelling in a hypotonic buffer, whereas the main part of the marker enzyme for the intermembrane space (adenylate kinase) was released. These results indicate that the ALDH activities with acetaldehyde, DOPAL and succinate semialdehyde are located in the matrix compartment. The low-Km activity with acetaldehyde and DOPAL, but not the high-Km activities and succinate semialdehyde dehydrogenase, was markedly stimulated by Mg2+ and Ca2+ in phosphate buffer. The low- and high-Km activities with acetaldehyde showed different pH optima in pyrophosphate buffer.

Identification of a segment containing a reactive cysteine residue in human liver cytoplasmic aldehyde dehydrogenase (isoenzyme E1).

Abstract: A single cysteine residue is selectively alkylated by iodoacetamide in cytoplasmic human liver aldehyde dehydrogenase (isoenzyme E1). The amino acid sequence of a 35-residue fragment containing this residue is determined, showing two additional cysteine residues and also three histidine residues. The alkylation is selective for Cys-30 of this fragment, with only little alkylation even at an adjacent residue, Cys-29. The region examined is likely to be of significance in the reaction of this isoenzyme with disulfiram since disulfiram blocks the selective alkylation.

[82] Aldehyde dehydrogenase from acetic acid bacteria, membrane-bound

Abstract: Publisher Summary This chapter describes the assay method, purification, and properties of aldehyde dehydrogenase. Aldehyde dehydrogenase of acetic acid bacteria acts on a wide range of aliphatic aldehydes, except formaldehyde. The enzyme is localized on the outer surface of the cytoplasmic membrane of an organism and the oxidation of aldehyde is linked to its respiratory chain. This enzyme acts as a vinegar producer in acetic acid bacteria by coupling with alcohol dehydrogenase. The rate of aldehyde oxidation is estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phenazine methosulfate; (b) by colorimetry in the presence of potassium ferricyanide; (c) by polarography with an oxygen electrode; or (d) by manometry in a conventional Warburg apparatus. The steps involved in the preparation of Gluconobacter suboxydans and Acetobacter aceti are also discussed in the chapter. All operations are carried out at 0-5°C, unless otherwise stated. Sodium acetate buffer, pH 5.3, is used for enzyme purification from G. suboxydans. Potassium phosphate buffer is used for the purification from A. aceti.

Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

Abstract: Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

The D(V/K) isotope effect of the cytochrome P-450-mediated oxidation of ethanol and its biological applications.

Abstract: Purified preparations of rat liver microsomes consist of flat discoid structures, presumably vesicles. They catalyze oxidation of ethanol to acetaldehyde in the absence of alcohol dehydrogenase and catalase with a specific activity of 1.4-53 nmol acetaldehyde formed min−1 (nmol cytochrome P-450)−1. The D(V/ K) isotope effect of this microsomal ethanol-oxidizing system was 1.15. This value is clearly different from those of alcohol dehydrogenase (3.0) and catalase (1.9). Particulate aldehyde dehydrogenases present in the preparation are active at unphysiologically high concentrations of aldehyde. Coincidently, the activity of the microsomal ethanol- oxidizing system, was well as the D(V/K) isotope effect associated with it, was increased significantly. This observation confirms the enzymic nature of the reaction studied. In microsomes washed once in KCI, ethanol oxidation was catalyzed mainly by catalase, but also by the microsomal ethanol-oxidizing system, as judged from the D(V / K) isotope effect upon the net reaction. In the presence of sodium azide alcohol dehydrogenase activity could be demonstrated. In hepatocytes from rat or pig, 4-methyl pyrazole at high concentrations reduced the rate of ethanol oxidation to 3-10%. The D(V/K) isotope effect upon the residual ethanol oxidation reflected activities of both catalase and the microsomal ethanol- oxidizing system. The activity of this latter system was inhibited by 4-methyl pyrazole competitively with ethanol, Ki= 5.7 mM. When this inhibition is taken into account, the uninhibited membrane system activity in these cells amount to maximally 10% of the rate of ethanol oxidation in the absence of inhibitor.

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

Abstract: 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.

Inhibition of aldehyde dehydrogenases in rat brain and liver by disulfiram and coprine.

Abstract: Rats were treated with either coprine or disulfiram and the inhibition of aldehyde dehydrogenase (ALDH) in liver and brain mitochondria was measured with acetaldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and succinate semialdehyde at different concentrations. The inhibition pattern was similar for both inhibitors, but the degree of inhibition was lower with disulfiram. The ALDH activity both in the liver and the brain was inhibited at low concentrations of acetaldehyde and DOPAL, but not with succinate semialdehyde. The high-Km enzyme activities with acetaldehyde were not inhibited in liver and brain. The activity at high concentration of DOPAL was inhibited in the liver, but only slightly affected in the brain, suggesting the presence of a brain enzyme with an intermediate Km value for DOPAL. In contrast with the results observed in viva, it was found that the high-Km activities with acetaldehyde and DOPAL in brain mitochondrial preparations were more sensitive to the inhibitors in vitro than the low-Km activities. Kinetic studies on ALDH preparations from brain and liver mitochondria suggested that acetaldehyde and DOPAL are metabolized by the same low-Km ALDH.

Further studies of the action of disulfiram and 2,2'-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase.

Abstract: 1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2'-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2'-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.

Proton release during the pre-steady-state oxidation of aldehydes by aldehyde dehydrogenase. Evidence for a rate-limiting conformational change

Abstract: A transient release of protons with an amplitude corresponding to one proton per active site has been observed for the oxidation of propionaldehyde, acetaldehyde, and benzaldehyde by sheep liver cytoplasmic aldehyde dehydrogenase at pH 7.6 with phenol red as indicator. At saturating substrate levels, the rate constants for the proton burst are in each case the same, and for acetaldehyde and propionaldehyde show the same dependence on the concentrations of the substrates, as the rate constants for the transient production of NADH reported previously [MacGibbon, A.K.H., Blackwell, L.F., & Buckley, P.D. (1977) Biochem. J. 167, 469-477]. Although, with propionaldehyde as a substrate, a full proton burst is also observed at pH 6.0, no proton burst is observed at pH 9.0. For 4-nitrobenzaldehyde, there is no burst in NADH production, but a burst in proton release is observed, showing that proton release precedes hydride transfer. No protons were released during the binding of the substrate analogues acetone and chloral hydrate nor on reaction of the enzyme with the inhibitor tetraethylthiuram disulfide (disulfiram). A model is proposed in which the rate-limiting step in the pre-steady-state phase of the reaction is a conformational change which occurs after the binding of aldehydes to the enzyme. As a result of the conformational change, the environment of a functional group on the enzyme, which initially has a pKa of about 8.5, is perturbed to give a final pKa value for the group of less than 5. Computer simulations were used to show that the model accurately reproduces all of the experimental data. The lack of observation of a second transient proton release, as required by the overall stoichiometry, argues that its release occurs in a slow step prior to NADH dissociation.

Mechanism of alcohol sensitivity and disulfiram-ethanol reaction.

Abstract: Human aldehyde dehydrogenase (ALDH) consists of two main isozymes with low and high Km for aldehyde. ALDH isozymes in hair sheats were tested from 40 Japanese using isoelectric focusing and blood acetaldehyde determination with gas chromatography. About 43% of Japanese, who lacked the low Km enzyme (ALDH I) showed an elevated acetaldehyde concentration due to their inability to metabolize acetaldehyde quickly and effectively. Studies regarding the inhibitory reaction of disulfiram and its metabolites have been performed. Among the metabolites, diethylamine inhibited the low Km enzyme strongly. It is presumed that vasomotor symptoms and high acetaldehyde concentration in blood after alcohol intake in patients who are treated with disulfiram might be mainly due to a decrease in activity of the low Km enzyme caused by diethylamine which is produced in vivo as one of the metabolites from disulfiram, rather than to an inhibitory reaction of disulfiram only. Thus, alcohol sensitivity in Mongoloids and disulfiram-ethanol reaction may have a common mechanism.

Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids.

Abstract: 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.

[81] Aldehyde dehydrogenases from Pseudomonas aeruginosa

Abstract: Publisher Summary 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 + .

Chlorpropamide-alcohol flushing, aldehyde dehydrogenase activity, and diabetic complications.

Abstract: Many diabetics who take chlorpropamide (a sulphonylurea compound) experience facial flushing after drinking even small amounts of alcohol. These flushers have a noticeably lower prevalence of late complications of diabetes (microangiopathy, macroangiopathy, and neuropathy) than non-flushers. This flush reaction is accompanied by increased blood acetaldehyde concentrations, suggesting an inhibition of aldehyde dehydrogenase activity. In the present study the activity of this enzyme in erythrocytes was assessed in the absence of chlorpropamide. Erythrocyte homogenates obtained from flushers and non-flushers were incubated with acetaldehyde and the rate of metabolism studies. Flushers eliminated acetaldehyde more slowly at a low range of concentrations (0--30 mumol/l), suggesting a difference in aldehyde dehydrogenase activity. Further studies are needed to clarify the role of this enzyme in the pathogenesis of diabetic complications.

Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase.

Abstract: Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.

Aldehyde dehydrogenase from bakers' yeast.

Abstract: Publisher Summary This chapter describes the assay method, purification, and properties of aldehyde dehydrogenase isolated from bakers' yeast. Aldehyde dehydrogenase activity is assayed at 25°C by measuring the rate of formation of nicotinamide adenine dinucleotide dehydrogenase (NADH) at 340 nm. The proteolytic products from the native enzyme are found in the process of the purification of aldehyde dehydrogenase from yeast. The preparation method in the presence of phenylmethylsulfonyl fluoride (PMSF) as a serine esterase inhibitor diminishes the amount of proteolytic degradation products. Fresh bakers' yeast is washed twice with three volumes of cold distilled water and stored at -25°C before use. The steps involved in the purification of aldehyde dehydrogenase are (1) the preparation of crude extract, (2) protamine sulfate precipitation, (3) first and second ammonium sulfate fractionation, (4) diethylaminoethyl (DEAE)-sepharose chromatography, (5) gel filtration, and (6) blue sepharose chromatography. All steps are performed at about 4°C. The purified enzyme migrates as a single component when subjected to polyacrylamide electrophoresis in the absence and in the presence of sodium dodecyl sulfate (SDS) and does not show any sign of proteolytically degraded forms.

Mammalian enzymes of trimethyllysine conversion to trimethylaminobutyrate

Abstract: The biosynthesis of carnitine proceeds from trimethyllysine (TML) by beta-hydroxylation by a liver or kidney mitochondrial enzyme, which requires oxygen, alpha-ketoglutarate, ferrous iron, and ascorbate. This dioxygenase is rapidly inactivated by preincubation with Fe2+, but not Fe3+. The evidence suggests that superoxide anion is involved in the hydroxylation. beta-Hydroxytrimethyllysine undergoes aldol cleavage to glycine and trimethylaminobutyraldehyde under the influence of serine hydroxymethyltransferase and possibly a specific aldolase. The next step, the aldehyde oxidation, is catalyzed by a specific NAD-dependent aldehyde dehydrogenase from liver cytosol. The product, trimethylaminobutyrate, is then hydroxylated by a cytosolic dioxygenase to carnitine. This enzyme, which has the same cofactor requirements as TML hydroxylase, is found in the liver of all species examined, but is absent from the kidney of some species.

4-Aminobutyraldehyde dehydrogenase activity in rat brain.

Abstract: 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.

Subcellular Distribution of Human Brain Aldehyde Dehydrogenase

Abstract: Two human brain surgery biopsies and one autopsy sample were subjected to subcellular fractionation. With either 0.12 or 6 mM-acetaldehyde as substrate, about half of the total aldehyde dehydrogenase activity was found in the mitochondrial (+ synaptosomal) fraction and less activity in the cytosolic, nuclear, and microsomal fractions. High-affinity activity was found only in the mitochondrial fraction. The enzyme in all fractions had a higher affinity for indole-3-acetaldehyde than for acetaldehyde. The kinetic data indicate the presence of several distinct aldehyde dehydrogenase isozymes that have ample capacity to oxidize both aliphatic and aromatic aldehydes in human brain.

The activation of aldehyde dehydrogenase by diethylstilboestrol and 2,2'-dithiodipyridine.

Abstract: 1 The activation of sheep liver cytoplasmic aldehyde dehydrogenase by diethylstilboestrol and by 2,2'-dithiodipyridine is described The effects of the two modifiers are very similar with respect to variation with acetaldehyde concentration, pH and temperature Thus the degree of activation is maximal when the enzyme is assayed at approx 1 mM-acetaldehyde, is greater at 25 degrees C than at 37 degrees C, and is greater at pH 74 than at pH 975 With low concentrations of acetaldehyde both modifiers decrease the enzyme activity 2 Diethylstilboestrol affects the sheep liver cytoplasmic enzyme in a very similar way to that previously described for a rabbit liver cytoplasmic enzyme Preliminary experiments show that the same is true for a preparation of human liver aldehyde dehydrogenase It is proposed that sensitivity to diethylstilboestrol (and steroids) is a common property of all mammalian cytoplasmic aldehyde dehydrogenases