A chemical and enzymological account of the multiple forms of human liver aldehyde dehydrogenase. Implications for ethnic differences in alcohol metabolism

Three major low-pI zones of aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) may be visualized with specific histochemical staining after starch gel electrophoresis at pH 7.4 of Caucasian human liver extracts, whereas about 50% of Chinese human liver extracts show only two such zones. The three zones of activity were purified to apparent homogeneity from Caucasian liver. The substrate specificity of each form was investigated by double reciprocal plots using 13 aldehydes of various chemistries. The acetaldehyde-preferring isozyme I lacking in 50% of Chinese livers had a slightly lower native and subunit molecular weight than the "universal' isozymes IIa and IIb. All forms were highly sensitive to disulfiram inhibition. This inhibition could be protected against, or reversed, by dithiothreitol. 2,2'-Dithiodipyridine was a slower inhibitor of isoenzyme I. All three purified forms of the enzyme, as well as crude extracts of normal and isozyme I-deficient Chinese livers, showed positive immunoreactivity to antibodies prepared in rabbits against type I enzyme. Tryptic peptide maps of forms IIa and IIb were almost identical, whereas that of form I, although showing some similarities, was clearly different. These results provide a consistent explanation for the acetaldehyde-mediated extreme sensitivity to moderate alcohol ingestion shown normally by about 50% of oriental subjects and during disulfiram (Antabuse) therapy by all subjects.     

Two aldehyde dehydrogenases from human liver. Isolation via affinity chromatography and characterization of the isozymes.

Human liver extracts show two major bands with aldehyde dehydrogenase (Aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) activity via starch gel electrophoresis at pH 7.0. Both bands have been purified to apparent homogeneity via classical chromatography combined with affinity chromatography on 5'-AMP-Sepharose 4B. The slower migrating band, enzyme 1, when assayed at pH 9.5 has a low Km for NAD (8 micrometer) and a high Km for acetaldehyde (approx. 0.1 mM). It is very strongly inhibited by disulfiram at pH 7.0 with a Ki of 0.2 micrometer. The faster migrating band, enzyme 2, has a low Km for acetaldehyde, (2--3 micrometer at pH 9.5), a higher Km for NAD (70 micrometer at pH 9.5), and is not inhibited by disulfiram at pH 7.0. The two enzymes are very similar to the F1 and F2 isozymes of horse liver purified by Eckfeldt et al. (Eckfeldt, J., Mope, L., Takio, K. and Yonetani, T. (1976) J. Biol, Chem. 251, 236-240) in molecular weight, subunit composition, amino acid composition and extinction coefficient. Preliminary kinetic characterizations of the enzyme are presented.     

Horse liver aldehyde dehydrogenase. Purification and characterization of two isozymes.

Two isozymes of horse liver aldehyde dehydrogenase (aldehyde, NAD oxidoreductase (EC 1.2.1.3)), F1 and F2, have been purified to homogeneity using salt fractionation followed by ion exchange and gel filtration chromatography. The specific activities of the two isozymes in a pH 9.0 system with propionaldehyde as substrate were approximately 0.35 and 1.0 mumol of NADH/min/mg of protein for the F1 and F2 isozymes, respectively. The multiporosity polyacrylamide gel electrophoresis molecular weights of the F1 and F2 isozymes were approximately 230,000 and 240,000 respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave subunit molecular weight estimates of 52,000 and 53,000 for the F1 and F2 isozymes, respectively. The amino acid compositions of the two isozymes were found to be similar; the ionizable amino acid contents being consistent with the electrophoretic and chromatographic behavior of the two isozymes. Both isozymes exhibited a broad aldehyde specificity, oxidizing a wide variety of aliphatic and aromatic aldehydes and utilized NAD as coenzyme, but at approximately 300-fold higher coenzyme concentration could use NADP. The F1 isozyme exhibited a very low Km for NAD (3 muM) and a higher Km for acetaldehyde (70 muM), while the F2 isozyme was found to have a higher Km for NAD (30 muM) and a low Km for acetaldehyde (0.2 muM). The two isozymes showed similar chloral hydrate and p-chloromercuribenzoate inhibition characteristics, but the F1 isozyme was found to be several orders of magnittude more sensitive to disulfiram, a physiological inhibitor of acetaldehyde oxidation. Based on its disulfiram inhibition characteristics, it has been suggested that the F1 isozyme may be the primary enzyme for oxidizing the acetyldehyde produced during ethanol oxidation in vivo.     

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

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

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for gamma-aminobutyraldehyde.

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.     

Purification of aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation from rabbit intestinal microsomes

This work presents the purification and further characterization of the aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation, from rabbit intestinal microsomes. Microsomal aldehyde dehydrogenase was solubilized with cholate and purified by using chromatography on 6-amino-n-hexyl-Sepharose and 5'-AMP-Sepharose. The purified enzyme migrated as a single polypeptide band with molecular weight of 60,000 on SDS-polyacrylamide gel. By gel filtration in the presence of detergent, its apparent molecular weight was estimated to be 370,000. In the detergent-free solution, in contrast, it had a much higher molecular weight, indicating its association in forming large aggregates. The pH optimum was 9.0 when pyrophosphate buffer was used. The enzyme was active toward various aliphatic aldehydes with more than three carbons. The Km value for substrate seemed to decrease with increase in the chain length. The microsomal aldehyde dehydrogenase was not affected by disulfiram and MgCl2, which were, in contrast, highly inhibitory towards the activity of the cytosolic aldehyde dehydrogenase separated from intestinal mucosa.     

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.     

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

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.     

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.     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

Subcellular localization of acetaldehyde dehydrogenase in human liver

The subcellular distribution of aldehyde dehydrogenase activity was determined in human liver biopsies by analytical sucrose density-gradient centrifugation. There was bimodal distribution of activity corresponding to mitochondrial and cytosolic localizations. At pH 9.6 cytosolic aldehyde dehydrogenase had a lower apparent Kappm for NAD (0.03 mmol l-1), than the mitochondrial enzyme (Kappm NAD = 1.1 mmol l-1). Also, the pH optimum for cytosolic aldehyde dehydrogenase activity (pH 7.5) was lower than that for the mitochondrial enzyme activity (pH 9.0), and the cytosolic enzyme activity was more sensitive to inhibition by disulfiram in vitro. Disulfiram (40 mumol l-1) caused a 70% reduction in cytosolic aldehyde dehydrogenase activity, but only a 30% reduction in mitochondrial enzyme activity after 10 min incubation. The liver cytosol may therefore be the major site of acetaldehyde oxidation in vivo in man.     

Involvement of lactaldehyde dehydrogenase in several metabolic pathways of Escherichia coli K12

Lactaldehyde dehydrogenase (E.C. 1.2.1.22) of Escherichia coli has been purified to homogeneity. It has four apparently equal subunits (molecular weight 55,000 each) and four NAD binding sites per molecule of native enzyme. The enzyme is inducible, only under aerobic conditions, by at least three different types of molecules, the sugars fucose and rhamnose, the diol ethylene glycol and the amino acid glutamate. The enzyme catalyzes the irreversible oxidation of several aldehydes with a Km in the micromolar range for alpha-hydroxyaldehydes (lactaldehyde, glyceraldehyde, or glycolaldehyde) and a higher Km, in the millimolar range, for the alpha-ketoaldehyde methylglyoxal. It displays substrate inhibition with all these substrates. NAD is the preferential cofactor. The functional and structural features of the enzyme indicate that it is not an isozyme of other E. coli aldehyde dehydrogenases such as glyceraldehyde phosphate dehydrogenase, glycolaldehyde dehydrogenase, or acetaldehyde dehydrogenase. The enzyme, previously described as specific for lactaldehyde, is thus identified as a dehydrogenase with a fairly general role in aldehyde oxidation, and it is probably involved in several metabolic pathways.     

Purification and characterization of aldehyde dehydrogenase from rat liver mitochondria

Nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent dehydrogenase activities from rat liver mitochondria have been copurified to homogeneity using combined DEAE, Sepharose, and affinity chromatographic procedures. The enzyme has a native molecular weight of 240,000 and subunit molecular weight of 60,000. The enzyme is tetrameric consisting of four identical subunits as revealed by electrophoresis and terminal analyses. A partial summary of physical properties is provided. The amino acid composition by acid hydrolysis is reported. Specific activities for various NAD(P)+ analogs and alkanal substrates were compared. The action of the effectors chloral hydrate, disulfiram, diethylstilbestrol, and Mg2+ and K+ ions were also investigated.     

2,2''-Dithiodipyridine activates aldehyde dehydrogenase and protects the enzyme against inactivation by disulfiram.

Small concentrations of 2,2'-dithiodipyridine cause a rapid activation of sheep liver cytoplasmic aldehyde dehydrogenase in the presence of NAD+. Enzyme pre-modified by 2,2'-dithiodipyridine is largely protected against the potent inactivatory effect of disulfiram. 2,2'-Dithiobis-(5-nitropyridine) inactivates the enzyme. The implications of these results are discussed with reference to various possible classes of thiol group in aldehyde dehydrogenase.     

Kinetic and structural evidence for the identification of 11-hydroxythromboxane B2 dehydrogenase as cytosolic aldehyde dehydrogenase

We have recently purified 11-hydroxythromboxane B₂ dehydrogenase from porcine kidney and identified it as cytosolic aldehyde dehydrogenase (EC 1.2.1.3) based on amino acid analysis and other protein characteristics. In the present paper we have studied the catalytic interaction of thromboxane B₂ (TXB₂) with different aldehyde substrates and a potent aldehyde dehydrogenase inhibitor, disulfiram. TXB₂ was a competitive inhibitor of the aldehyde dehydrogenase reaction in assays with 3,4-dihydroxyphenylacetaldehyde, a high affinity substrate. The conversion of TXB₂ to 11-dehydro-TXB₂ was also inhibited by propanal and disulfiram.

The protein characteristics of the enzyme have also been further studied. The native enzyme is a tetramer and has an isoelectric point of 7.0 which is comparable with that of cytosolic aldehyde dehydrogenases from other species. Taken together the present data further indicate that 11-hydroxythromboxane B₂ dehydrogenase is identical with cytosolic aldehyde dehydrogenase and that substrates and inhibitors of aldehyde dehydrogenase interact with thromboxane metabolism in vitro.     

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.     

The use of pH-gradient ion-exchange chromatography to separate sheep liver cytoplasmic aldehyde dehydrogenase from mitochondrial enzyme contamination, and observations on the interaction between the pure cytoplasmic enzyme and disulfiram.

1. Sheep liver cytoplasmic aldehyde dehydrogenase can be purified from contamination with the mitochondrial form of the enzyme by pH-gradient ion-exchange chromatography. The method is simple, reproducible and efficient. 2. The purified cytoplasmic enzyme retains about 2% of its original activity in the presence of a large excess of disulfiram. This suggests that the disulfiram-reactive thiol groups are not essential for covalent interaction with the aldehyde substrate during catalysis, as has sometimes been suggested. 3. Between 1.5 and 2.0 molecules of disulfiram per tetrameric enzyme molecule account for the observed loss of activity, suggesting that the enzyme may have only two functional active sites. 4. Experiments show that disulfiram-modified enzyme retains the ability to bind NAD+ and NADH.     

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.     

Effect of disulfiram and its reduced metabolite, diethyl-dithiocarbamate on aldehyde dehydrogenase of human erythrocytes

Inhibition of human erythrocyte aldehyde dehydrogenase (ALDH) activity was studied using disulfiram and its reduced metabolite, diethyldithiocarbamate (DDC). The enzyme was rapidly inactivated by disulfiram and the inhibition was protected by reduced glutathione (GSH), in a concentration dependent manner when the enzyme premixed with GSH was reacted with disulfiram. Higher reactivity of the thiol group of the enzyme than that of GSH to disulfiram was suggested from the observation that half of the enzyme activity was inhibited when the ratio of disulfiram to GSH was 1:10. Although DDC alone showed no inhibitory effect on the enzyme, inactivation was mediated by a low concentration of heme-containing peroxidases, but not by methemoglobin. Under this condition, the inhibition potential was not protected, even with a high concentration of GSH. The constant reoxidation system of DDC is probably directly related to the enzyme inactivation.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)