Liver Aldehyde Dehydrogenase Research Articles

Involvement of serine 74 in the enzyme-coenzyme interaction of rat liver mitochondrial aldehyde dehydrogenase.

It has been suggested that the active site nucleophile in sheep liver aldehyde dehydrogenase was not a cysteine residue but was a serine located at position 74 [Loomes, K. M., Midwinter, G. G., Blackwell, L. F., & Buckley, P. D. (1990) Biochemistry 29, 2070-2080]. This enzyme form has not yet been cloned and expressed, but since the rat liver mitochondrial enzyme has been and shares 70% sequence homology with other cytosolic aldehyde dehydrogenases, the residue in the rat enzyme was converted into an alanine to test for the necessity of a hydroxyl group at that position. The recombinantly expressed mutant enzyme possessed 10% catalytic activity, but the Km for NAD increased from 10 to 1900 microM while the Kms for various aldehydes were unchanged. Kinetic analysis revealed that the dissociation constant for NAD also increased in the mutant as did k1, the on velocity for NAD binding. The mutant enzyme bound poorly to an AMP-Sepharose column and did not interact as well with NADH, as determined by fluorescence enhancement binding studies, or with ADP-ribose, a competitive inhibitor. Pulse-chase analysis showed that the mutant was as stable as was the recombinantly expressed native enzyme. It was less stable to heat denaturation at 50 degrees C (half-life of 1 min compared to 4). Converting the alanine to a cysteine or a threonine did not restore native-like properties of the enzyme. These mutants had kinetic properties very similar to those of the alanine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase.

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.

Human liver aldehyde dehydrogenases: new method of purification of the major mitochondrial and cytosolic enzymes and re-evaluation of their kinetic properties

A new purification procedure, based on dye-adsorption and affinity chromatography, has been developed for the isolation of the two major ALDH isozymes from human liver: ALDH-1 (cytosolic, pI 5.2) and ALDH-2 (mitochondrial, pI 4.9). The procedure affords milligram quantities of ALDH-1 and -2 at 850- and 275-fold purifications, respectively, from 50 g of liver in two days. Kinetic parameters for acetaldehyde oxidation were determined with these purified enzymes, because there is a wide discrepancy in the absolute magnitude of these parameters in the biochemical literature. The Michaelis constants for ALDH-1 and -2, determined from initial velocities (for ALDH-1) and single reaction progress curves (for ALDH-2), are 180 ±10 μ M and 0.20 ± 0.02 μ M , respectively (pH 7.5 and 9.5, saturating NAD + in both cases). This three orders of magnitude difference in K m values is much greater than that reported previously in all but one study.

In Vivo Pharmacodynamic Studies of the Disulfiram Metabolite S‐Methyl N,N‐Diethylthiolcarbamate Sulfoxide:Inhibition of Liver Aldehyde Dehydrogenase

S-methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) is proposed to be the metabolite of disulfiram responsible for the in vivo inhibition of liver low Km aldehyde dehydrogenase (ALDH) in the rat. Studies were conducted in male Sprague-Dawley rats and also in vitro using both rat liver mitochondrial and purified bovine mitochondrial low Km ALDH to investigate further the pharmacodynamic and pharmacokinetic characteristics of DETC-MeSO. Administration of DETC-MeSO to rats produced a rapid and maximal inhibition of liver mitochondrial low Km ALDH within 2 hr, which was still inhibited 30% after 168 hr. After DETC-MeSO treatment, the maximum plasma concentration of DETC-MeSO was reached within 0.5 hr, with DETC-MeSO being undetectable 2 hr after DETC-MeSO dosing. Although a trace amount of DETC-Me was detected in the plasma 0.5 hr after DETC-MeSO administration to rats, this disappeared within 1 hr. When rats were treated with disulfiram, the maximal plasma concentration of DETC-MeSO was found within 2 hr, with only a very small quantity of DETC-MeSO still detectable after 8 hr. Rats also were given the disulfiram metabolites diethyldithiocarbamate (DDTC), diethyldithiocarbamate-methyl ester (DDTC-Me), and S-methyl N,N-diethylthiolcarbamate (DETC-Me), and plasma analyzed for DETC-MeSO 2 hr after the administration of these metabolites. DETC-MeSO was detected in plasma, further illustrating that DETC-MeSO can be found in plasma after the administration of either disulfiram, or the subsequent in vivo metabolites DDTC, DDTC-Me, or DETC-Me.(ABSTRACT TRUNCATED AT 250 WORDS)

Human aldehyde dehydrogenase: chromosomal assignment of the gene for the isozyme that metabolizes ?-aminobutyraldehyde

A cDNA clone of the E3 isozyme of human liver aldehyde dehydrogenase consisting of a 1320-base pair (bp) coding region and a 180-bp non-coding region at the 3' end was used for chromosomal localization of the E3 gene. Using a panel of human/hamster somatic cell hybrids we have localized, the gene coding for the E3 isozyme to human chromosome 1.

Aldehyde dehydrogenase of mice inhibited by thiocarbamate herbicides

The herbicide S -ethyl N,N -dipropylthiocarbamate (EPTC) and three of its candidate metabolites (the sulfoxide, N -depropyl and S -methyl derivatives) inhibit mitochondrial low-K m aldehyde dehydrogenase (ALDH) in liver by 56 to 82% 2 hr after these thiocarbamates are administered intraperitoneally (ip) to mice at 8 mg/kg. They also greatly elevate the acetaldehyde level (determined as the O -benzyloxime ether) in blood (up to 500 μM) and brain (up to 3 ppm) 30 min after two ip treatments, the first with the thiocarbamate at 40 mg/kg and 2 hr later with ethanol at 1000 mg/kg. EPTC at 4 mg/kg inhibits liver ALDH activity by 50% and at 8 and 18 mg/kg gives half of the maximum ethanol-dependent elevation of acetaldehyde levels in blood and brain, respectively. The in vivo effects of other thiocarbamate herbicides at 8 mg/kg on ALDH activity and 40 mg/kg on acetaldehyde levels decrease in the order of thiobencarb, pebulate, vernolate and molinate > butylate and triallate >> cycloate. The percentage inhibition of liver ALDH activity generally correlates with the elevation in blood and brain acetaldehyde under these treatment protocols. B.W. Hart and M.D. Faiman ( Biochem. Pharmacol. 43 403–406, 1992) have shown that the alcohol-aversion drug disulfiram is metabolized to S -methyl N,N -diethylthiocarbamate and its sulfoxide as the penultimate and ultimate metabolites inhibiting ALDH. Thus, the thiocarbamate herbicides and their metabolites are similar to the disulfiram metabolites not only in homologous structure but also in their potency range as ALDH inhibitors in vivo . On this basis some of the thiocarbamate herbicides may sensitize agricultural workers to ethanol intoxication.

O-52 - Effect of nifedipine on the activities of subcellular aldehyde dehydrogenases in rat liver.

O-52 - Effect of nifedipine on the activities of subcellular aldehyde dehydrogenases in rat liver.

Influence of cyclopropylethyl-containing amines and amides of the isoenzyme forms of rat liver aldehyde dehydrogenase

Influence of cyclopropylethyl-containing amines and amides of the isoenzyme forms of rat liver aldehyde dehydrogenase

Human aldehyde dehydrogenase. cDNA cloning and primary structure of the enzyme that catalyzes dehydrogenation of 4-aminobutyraldehyde.

Human liver aldehyde dehydrogenase (E3 isozyme), with wide substrate specificity and low Km for 4-aminobutyraldehyde, was only recently characterized [Kurys, G., Ambroziak, W. & Pietruszko, R. (1989) J. Biol. Chem. 264, 4715-4721] and in this study we report on its primary structure. Polyclonal antibodies, specific for the E3 isozyme and three oligonucleotide probes derived from amino acid sequence of the E3 protein, were used for isolation of the first cDNA clone encoding the human enzyme (1503 bp; coding for 440 amino acid residues). Additional clones were obtained by using the first isolated clone as a probe. The largest clone of 1635 bp coded for 462 amino acid residues; it was longer at the 3'end of the cDNA non-coding region. The identity of the clone was established by DNA sequencing and by comparison with peptide sequences derived from the E3 protein, which constituted approximately 29% of the total primary structure of the E3 isozyme. The start codon was never encountered despite a variety of different approaches (500 amino acid residues were expected on the basis of SDS-gel molecular-mass determination of the E3 isozyme subunit). Despite the great catalytic similarity between the E3 and E1 isozymes [Ambroziak, W. & Pietruszko, R. (1991) J. Biol. Chem. 266, 13011-13018], the primary structure of the E3 isozyme has only approximately 40.6% of positional identity with that of the E1 isozyme. Sequence comparison with GenBank and Protein Identification Resource database sequences indicated no primary structure of aldehyde dehydrogenase more closely resembling the E3 isozyme than that of Escherichia coli betaine aldehyde dehydrogenase (52.7% positional identity), a prokaryotic enzyme specific for betaine aldehyde.

Bioactivation of S-methyl N, N-Diethylthiolcarbamate to S-methyl N, N-diethylthiolcarbamate sulfoxide : Implications for the role of cytochrome P450

Diethyldithiocarbamate (DDTC), diethyldithiocarbamate methyl ester (DDTC-Me), S-methyl N, N-diethylthiolcarbamate (DETC-Me) and S-methyl N, N-diethylthiolcarbamate sulfoxide (DETC-MeSO) are all metabolites of disulfiram. All inhibit rat liver low K m aldehyde dehydrogenase (ALDH) in vivo, with the order of potency being DETC-MeSO > DETC-Me > DDTC-Me > DDTC. Studies were carried out both in vivo and in vitro to further investigate the role of bioactivation as a requirement for the action of disulfiram as a liver ALDH inhibitor. The cytochrome P450 inhibitor 1-benzylimidazole (NBI) was employed as a pharmacological tool to study the metabolism of DETC-Me to DETC-MeSO. Administration of NBI to rats prior to DETC-Me.treatment blocked the inhibition of liver mitochondrial low K m ALDH by DETC-Me. This was accompanied by an increase in plasma DETC-ME and a decrease in plasma DETC-MeSO. Pretreatment of rats with NBI prior to DETC-MeSO administration did not block the inhibition of liver mitochondrial low K m ALDH by DETC-MeSO. In in vitro studies, the inclusion of NBI in the incubation containing rat liver microsomes, mitochondria and an NADPH-generating system blocked the formation of DETC-MeSO and inhibition of liver mitochondrial low K m ALDH by DETC-Me. DETC-MeSO was found to be a potent inhibitor of rat liver mitochondrial low K m ALDH both in vivo and in vitro. The data suggest that the metabolism of DETC-Me to DETC-MeSO is mediated by cytochrome P450, and that inhibition of cytochrome P450 by inhibitors such as NBI block the inhibition of low K m ALDH by DETC-Me.

In vivo effects of disulfiram and cyanamide on canine liver aldehyde dehydrogenase isoenzymes as detected by high-performance (pressure) liquid chromatography.

Methods for analysis of aldehyde dehydrogenase isoenzymes using high-performance (pressure) liquid chromatography (HPLC) were used to determine in vivo effects of disulfiram and cyanamide on canine liver aldehyde dehydrogenase (ALDH) isoenzymes. Liver ALDH isoenzymes from control and disulfiram- or cyanamide-treated dogs were separated by ion-exchange HPLC, and enzyme activity was detected using a postcolumn reactor. Two major peaks of ALDH activity (peaks I and II) were detected. Varying the composition of the reaction column reagents resulted in alterations in the elution profiles consistent with the kinetic properties of individual isoenzymes (i.e., ALDH IB in peak I and ALDH IIB in peak II), including estimates of the Km for acetaldehyde and the effects of magnesium ions on ALDH activity. Disulfiram treatment decreased both peaks depending on disulfiram dose and length of treatment, with peak I being more sensitive to inactivation than peak II. Reagents containing MgCl2 (1 mM) decreased peak I and increased peak II compared with EDTA (1 mM) for samples from both control and disulfiram-treated animals. These data are consistent with the assignment of the disulfiram-sensitive isoenzyme (ALDH IB) to peak I and the isoenzyme stimulated by magnesium ions (ALDH IIB) to peak II. In vivo cyanamide treatment produced similar decreases in both peaks to a maximum decrease of approximately 30% of control depending on cyanamide dose. Peak I, however, was more sensitive than peak II to in vitro inactivation by cyanamide, which suggests that cytosolic ALDH in the dog (in contrast to other mammals) is more sensitive to inactivation than mitochondrial ALDH.

Import, processing, and two-dimensional NMR structure of a linker-deleted signal peptide of rat liver mitochondrial aldehyde dehydrogenase.

Previous NMR studies (Karslake, C., Piotto, M. E., Pak, Y. M., Weiner, H., and Gorenstein, D. G. (1990) Biochemistry 29, 9872-9878) had shown that a 22-amino acid signal peptide of rat liver aldehyde dehydrogenase (ALDH) when bound to a micelle had two amphiphilic alpha-helices, one located at the N terminus and the other at the C terminus. It was shown that deletion of either helix caused the precursor protein not to be imported (Wang, Y., and Weiner, H., (1993) J. Biol. Chem. 268, 4759-4765). The two helices are separated by a Arg-Gly-Pro flexible "linker" region, and to test the role of this linker region in the import and processing of the precursor protein, we deleted it from the ALDH signal peptide and precursor protein. The 19-amino acid signal peptide of ALDH, to which has been added 3 residues at the C terminus and from which has been deleted the 3-residue flexible linker region, has been studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment the peptide contains a single alpha-helical segment that extends almost the entire length of the peptide. NH exchange experiments show residues on the hydrophobic face of the peptide to exchange much more slowly than those of the hydrophilic face. Combined with the previous study, these results suggest that precursor protein import simply requires a sufficiently long amphiphilic helix (or helices) to bind stably to the membrane. The N and C helices of native ALDH are only about 6-8 residues long; this represents only about two turns of a helix, and either helix on its own does not provide enough stabilization to ensure folding and binding to the membrane. The linker-deleted ALDH peptide contains a single helix of 12-14 residues that is long enough to provide a hydrophobic surface that can stably interact with the hydrophobic interior of the membrane. The function of the C helix in the native signal peptide is therefore to enhance the stability and binding of the N-terminal signal to the membrane. Significantly, unlike native ALDH precursor protein, the linker-deleted signal peptide precursor protein could no longer be processed after import into mitochondria. As explained by modeling of the alpha-helix and the NH exchange rate data, the precursor protein requires that the first several residues of the mature protein be part of the hydrophobic membrane associated face of the helix.(ABSTRACT TRUNCATED AT 400 WORDS)

Substrate specificity of rat liver aldehyde dehydrogenase with chloroacetaldehydes.

Chlorinated acetaldehydes have been the focus of research due to their role as reactive intermediates and their possible occurrence in chlorinated drinking water. This study investigated the in vitro substrate specificity of cytosolic and mitochondrial rat liver aldehyde dehydrogenase toward these compounds. Monochloroacetaldehyde was found to be extensively metabolized by these enzymes, to an even greater extent than the standard substrate propionaldehyde. Dichloroacetaldehyde was metabolized to a much lesser extent, and chloral hydrate is not metabolized by this enzyme family. The Km (mM) and Vmax (Vmax for propionaldehyde set to 100) values with the low Km cytosolic enzyme were monochloroacetaldehyde 0.046 and 582, and dichloroacetaldehyde 0.13 and 54.9, and those with the high Km cytosolic enzyme were dichloroacetaldehyde 0.35 and 23.4. The values with the low Km mitochondrial enzyme were monochloroacetaldehyde 0.057 and 462 and dichloroacetaldehyde 0.038 and 12.9, and those with the high Km mitochondrial enzyme were monocloroacetaldehyde 0.024 and 55.5 and dichloroacetaldehyde 0.29 and 3.44. These data suggest that aldehyde dehydrogenase plays a significant role in the metabolism of monochloroacetaldehyde and, to some extent, dichloroacetaldehyde. Some evidence also suggested that alcohol dehydrogenase plays a significant role in the metabolism of dichloroacetaldehyde and chloral hydrate.

The presequence of rat liver aldehyde dehydrogenase requires the presence of an alpha-helix at its N-terminal region which is stabilized by the helix at its C termini.

Previous nuclear magnetic resonance data showed that the conformation of the signal peptide of rat liver mitochondrial aldehyde dehydrogenase in a micelle environment contained a N-helix and a C-helix which were separated by a flexible hinge region (Karslake, C., Piotto, M.E., Pak, Y.K., Weiner, H. and Gorenstein, D. (1990) Biochemistry 29, 9872-9878). Deletion of either helix from the presequence caused complete loss of the precursor import. Switching the positions of the two helices had little effect on import but decreased processing efficiency. Import was not affected by replacing the C-helix with the N-terminal helix of the presequence of cytochrome c oxidase subunit IV, however, it was decreased by replacing the C-helix with the C-terminal random coil of the oxidase's presequence. Circular dichroism studies on the synthesized signal peptides indicated that a helix in the C-segment of aldehyde dehydrogenase signal peptide was needed to stabilize the N-helix. It is concluded that a stable helix in the N-terminal region is necessary for a functional mitochondrial presequence. This helix could be obtained from its own sequence, or from the interaction with other portions of the presequence.

Microsomal aldehyde dehydrogenase or its cross-reacting protein exists in outer mitochondrial membranes and peroxisomal membranes in rat liver.

We investigated the subcellular distribution of microsomal aldehyde dehydrogenase (msALDH) in rat liver and revealed by the immunoblotting method that msALDH or a cross-reacting 54-kDa protein(s) exists in the outer mitochondrial membranes and peroxisomes. Anti-msALDH antibody markedly inhibited the decanal aldehyde dehydrogenase activity of the outer mitochondrial membranes as well as that of the microsomes. Immunogold electron microscopic observations showed that gold particles are localized over the ER, outer mitochondrial membranes and peroxisomal membranes. These results suggest that msALDH or its cross-reacting related protein is distributed not only in the ER membranes but also in the mitochondrial outer membranes and peroxisomal membranes.

Maternally-mediated developmental lithium toxicity in the mouse

1. 1. Breast fed maternally-mediated developmental LiCl toxicity was determined in mice offspring as a function of offspring's gender and duration of maternal intake of LiCl (1 mEq). 2. 2. The female offspring were more sensitive than the males to major organ weight changes by maternal exposure to LiCl. 3. 3. Maternal intake of LiCl from preconception until weaning of the nurslings induced offspring hepatic alcohol dehydrogenase and heart lactate dehydrogenase in both sexes which was isoenzyme specific for the latter. 4. 4. The offspring also showed induction of liver aldehyde dehydrogenase but only as consequence of postnatal exposure to LiCl. 5. 5. The results indicate offspring developmental toxicity as a consequence of maternal exposure to Li salts and breast feeding.

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.

Identification of human liver aldehyde dehydrogenases that catalyze the oxidation of aldophosphamide and retinaldehyde

Biotransformation of the biologically and pharmacologically important aldehydes, retinaldehyde and aldophosphamide, is mediated, in part, by NAD(P)-dependent aldehyde dehydrogenases catalyze the oxidation of the aldehydes to their respective acids, retinoic acid and carboxyphosphamide. Not known at the onset of this investigation was which of the several known human aldehyde dehydrogenases (ALDHs) catalyze these reactions. Thus, human liver aldehyde dehydrogenases were chromatographically resolved and the ability of each to catalyze the oxidation of retinaldehyde and aldophosphamide was assessed. Only one, namely ALDH-1, catalyzed the oxidation of retinaldehyde; the K + m value was 0.3 μM. Three, namely ALDH-1, ALDH-2 and succinic Semialdehyde dehydrogenase, catalyzed the oxidation of aldophosphamide; K m values were 52, 1193, and 560 μM, respectively. ALDH-4, ALDH-5 and betaine aldehyde dehydrogenase did not catalyze the oxidation of either aldophosphamide or retinaldehyde. ALDH-1 and succinic Semialdehyde dehydrogenase accounted for 64 and 30%, respectively, of the total hepatic aldehyde dehydrogenase-catalyzed aldophosphamide (160 μM) oxidation. ALDH-1-catalyzed oxidation of aldophosphamide was noncompetitively inhibited by chloral hydrate; the K i value was 13μM. ALDH-2- and succinic semialdehyde dehydrogenase-catalyzed oxidation of aldophosphamide was relatively insensitive to inhibition by chloral hydrate. These observations strongly suggest an important in vivo role for ALDH-1 in the catalysis of retinaldehyde and aldophosphamide biotransformation. Succinic semialdehyde dehydrogenase-catalyzed biotransformation of aldophosphamide may also be of some in vivo importance.

Modification of aldehyde dehydrogenase with dicyclohexylcarbodiimide: separation of dehydrogenase from esterase activity.

Dehydrogenase activity of the cytoplasmic (E1) isozyme of human liver aldehyde dehydrogenase (EC 1.2.1.3) was almost totally abolished (3% activity remaining) by preincubation with dicyclohexylcarbodiimide (DCC), while esterase activity with p-nitrophenyl acetate as substrate remained intact. The esterase reaction of the modified enzyme exhibited a hysteretic burst prior to achieving steady-state velocity; addition of NAD+ abolished the burst. The Km for p-nitrophenyl acetate was increased, but physicochemical properties remained unchanged. The selective inactivation of dehydrogenase activity was the result of covalent bond formation. Protection by NAD+ and chloral, saturation kinetics, and the stoichiometry and specificity of interaction indicated that the reaction of DCC occurred at the active site of the E1 isozyme. The results suggested the some amino acid other than aspartate or glutamate, possibly a cysteine residue, located on a large tryptic peptide of the E1 enzyme, may have reacted with DCC.

In vitro and in vivo inhibition of rat liver aldehyde dehydrogenase by s-methyl N, N-diethylthiolcarbamate sulfoxide, a new metabolite of disulfiram

In summary, these data provide the first evidence that DETC-MeSO is a natural metabolite of disulfiram, and a potent inhibitor of rat liver mitochondrial low Km ALDH both in vitro and in vivo. It is therefore proposed that, based upon evidence to date, DETC-MeSO appears to be the chemical species to which disulfiram must be bioactivated, and is the metabolite most likely responsible for disulfiram's inhibition of rat liver mitochondrial low Km ALDH in vivo. Characterization of the properties of DETC-MeSO as the metabolite responsible for disulfiram's action as an ALDH inhibitor is presently in the process of being completed.