Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase.

The free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1-oxy-2,2,5,5-tetramethyl-3-imidazoline-4-yl)disulfide, via a thiol-disulfide exchange reaction and with 2[14C]iodoacetic acid, via S-alkylation. The respective reactions were monitored by electron paramagenetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S-carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o-phenanthroline. This cysteine residue, which could serve as a putative ligand to the active-site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of < or 10 A between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 degrees C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Structures of human alcohol and aldehyde dehydrogenases

Human alcohol dehydrogenase is a dimeric zinc metalloenzyme for which forms of three classes, I, II and III, have been distinguished. Subunits hybridize within but not between classes. There are three types of subunit, alpha, beta, and gamma, in class I. The primary structures of all three forms have been established, as well as the overall properties and the effects of the amino acid substitutions between the various forms. Each subunit has 374 residues, of which 35 exhibit differences among the alpha, beta and gamma chains. Corresponding cDNA structures are also known, as are the genetic organization and details of the gene structures. Allelic variants occur at the beta and gamma loci. Corresponding amino acid substitutions have been characterized, and enzymatic differences between the allelic forms are explained by defined residue exchanges. The results also illustrate recent and repeated isozyme evolution, a subject where alcohol dehydrogenases exceptionally well offer detailed examples. Human aldehyde dehydrogenase occurs of two types, a mitochondrial and a cytosolic form. The enzymes are tetramers, do not contain functional metals, and have subunits which do not form inter-type hybrids. The primary structures have been determined, revealing a positional identity of 68% (in 500 residues) between the mitochondrial and cytosolic forms. The N-terminus is heterogeneous and is not blocked in the subunit of the mitochondrial enzyme, in contrast to that of the cytosolic enzyme or those of all the alcohol dehydrogenases (also cytosolic). A reactive cysteine residue at position 302 has been ascribed functional importance at or close to the active site, is conserved in the two aldehyde dehydrogenases, and is associated with the action of disulfiram on the enzyme. In Oriental populations, a mutant allelic variant of the mitochondrial protein with impaired enzyme function has also been characterized.     

The involvement of alcohol dehydrogenase and aldehyde dehydrogenase in alcohol/aldehyde metabolism in Drosophila melanogaster

In this study we have examined the roles of alcohol dehydrogenase, aldehyde oxidase, and aldehyde dehydrogenase in the adaptation of Drosophila melanogaster to alcohol environments. Fifteen strains were characterized for genetic variation at the above loci by protein electrophoresis. Levels of in vitro enzyme activity were also determined. The strains examined showed considerable variation in enzyme activity for all three gene-enzyme systems. Each enzyme was also characterized for coenzyme requirements, effect of inhibitors, subcellular location, and tissue specific expression. A subset of the strains was chosen to assess the physiological role of each gene-enzyme system in alcohol and aldehyde metabolism. These strains were characterized for both the ability to utilize alcohols and aldehydes as carbon sources as well as the capacity to detoxify such substrates. The results of the above analyses demonstrate the importance of both alcohol dehydrogenase and aldehyde dehydrogenase in the in vivo metabolism of alcohols and aldehydes.     

Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzaldehyde

Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.     

Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases

The amino acid sequence of sheep liver sorbitol dehydrogenase has been fitted to the high-resolution model of the homologous horse liver alcohol dehydrogenase by computer graphics. This has allowed construction of a model of sorbitol dehydrogenase that provides explanations why sorbitol is not a substrate for alcohol dehydrogenase, why ethanol is not a substrate for sorbitol dehydrogenase, and what determines its specificity for polyols. An important feature of the model is that one of the ligands to the active site zinc atom is a glutamic acid residue instead of a cysteine residue, which is the corresponding ligand in the homologous alcohol dehydrogenases. This is one component of the structural change that can be related to the different substrate specificities, showing how altered enzymic activity might be brought about by structural changes of the kind that it is now possible to introduce by site-directed mutagenesis and recombinant DNA techniques.     

Effect of chronic ethanol or acetaldehyde on hepatic alcohol and aldehyde dehydrogenases, aminotransferases and glutamate dehydrogenase

Ethanol or acetaldehyde orally administered (15% and 2% respectively in drinking water) to male Wistar rats for three months induced alterations in the main liver enzymes responsible for ethanol metabolism, aspartate and alanine aminotransferases and NAD glutamate dehydrogenase. Ethanol produced a significant decrease in the activity of soluble alcohol dehydrogenase, while acetaldehyde induced alterations both in soluble and mitochondrial aldehyde dehydrogenases: soluble activity was significantly higher than in the control and ethanol-treated groups, and mitochondrial activity was significantly diminished. Both soluble aspartate and alanine aminotransferases showed pronounced increases by the chronic effect of acetaldehyde, while mitochondrial activities were practically unchanged by the effect of ethanol or acetaldehyde. Mitochondrial NAD glutamate dehydrogenase showed a rise in its activity both by the effect of chronic ethanol and acetaldehyde consumption. The level of metabolites assayed in liver extracts showed marked differences between ethanol and acetaldehyde treatment which indicates that ethanol produced a remarkable increase in glutamate, aspartate and free ammonia together with marked decrease in pyruvate and 2-oxoglutarate concentrations. Acetaldehyde consumption induced a significant decrease in 2-oxoglutarate and pyruvate concentrations. These observations suggest that ethanol has an important effect on the urea cycle enzymes, while the effect of acetaldehyde contributes to the impairment of the citric acid cycle.     

Multifunctionality of liver alcohol dehydrogenase. Studies of aldehyde dehydrogenase activity

Kinetic studies of the liver alcohol dehydrogenase catalyzed dehydrogenation of aldehydes were carried out over a wide range of octanal concentrations. The effect of specific inhibitors of liver alcohol dehydrogenase on aldehyde dehydrogenase activity was examined. The results were consistent with a steady-state random mechanism with the formation of the ternary E · NADH octanal complex at low temperatures. This ternary complex becomes inconspicuous at high temperatures. The aldehyde dehydrogenase activity was found to associate with all ethanol-active isozymes. The dual dehydrogenase reactions are catalyzed by the same molecule, presumably in the region of the same domain. However, the two activities respond differently to structural changes.     

Genotypes of alcohol dehydrogenase and aldehyde dehydrogenase loci in Japanese alcohol flushers and nonflushers

Summary A much higher incidence of alcohol flushing among Orientals in comparison to Caucasians, i.e., >50% vs 5%–10%, has been attributed to racial differences in alcohol-metabolizing enzymes. A large majority of Orientals are atypical in alcohol dehydrogenase-2 locus (ADH ₂ ), and their livers exhibit significantly higher ADH activity than the livers of most Caucasians. Approximately 50% of Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH₂) activity, and elimination of acetaldehyde might be disturbed. We determined by means of hybridization of genomic DNA samples with allele specific oligonucleotide probes, genotypes of the ADH ₂ and ALDH ₂ loci in Japanese alcohol flushers and nonflushers. We found that all individuals with homozygous atypical ALDH ₂ ² /ALDH ₂ ² and most of those with heterozygous atypical ALDH ₁ ² /ALDH ₂ ² were alcohol flushers, while all subjects with homozygous usual ALDH ₁ ² /ALDH ₁ ² were nonflushers. Frequency of the atypical ADH ₂ ² was found to be higher in alcohol flushers than in nonflushers, but the statistical significance was not established in the sample size examined.     

Polymorphism of aldehyde dehydrogenase and alcohol sensitivity

The metabolism of acetaldehyde has received considerable attention in the past years owing to its acute and chronic toxic effects in humans. Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of acetaldehyde in liver and other organs. Two major isozymes of hepatic ALDH (ALDH I or E2 and ALDH II or E1), which differ in their structural and functional properties, have been characterized in humans. The ALDH I with a low Km for acetaldehyde is predominantly of mitochondrial origin and ALDH II which has a relatively higher Km is of cytosolic origin. An inherited deficiency of ALDH I isozyme has been found among Japanese and Chinese which is primarily responsible for producing acute alcohol sensitivity symptoms (flushing response) after drinking mild doses of alcohol. Biochemical, immunochemical and molecular genetics data indicate a structural mutation in the ALDH I isozyme gene responsible for the loss in catalytic activity. Population genetic studies indicate a wide prevalence of this ALDH polymorphism among individuals of Mongoloid race. Flushing response to alcohol shows familial resemblances and preliminary family data from Japan, China and Korea hint to an autosomal codominant inheritance for ALDH I isozyme deficiency. The ALDH polymorphism is apparently responsible for the low incidence of alcoholism in Japanese, Chinese and Koreans. Alcohol-induced sensitivity due to ALDH isozyme deficiency may act as an inhibitory factor against excessive alcohol drinking thereby imparting a protection against alcoholism.     

Differences between alcohol dehydrogenases. Structural properties and evolutionary aspects.

Comparisons of the primary structures of yeast and horse liver alcohol dehydrogenases reveal that the enzymes are homologous but distantly related. The overall positional identity is 25% between common regions, and several deletions/insertions occur in either enzyme, the longest apparently corresponding to 21 residues, showing that the different subunit sizes are largely explained by internal differences. Variabilities in the structural similarities can be coupled with functional requirements but not directly with whole domains in the previously known tertiary structure of the horse protein. The two most similar regions of the enzymes affect active-site segments and the two most dissimilar regions seem to affect a loop structure without known function, and a segment participating in subunit interactions. The dissimilarities may probably be correlated with changes in zinc-binding properties and quaternary structures. The extra region corresponding to the large internal chain-length difference shows an apparent coincidence in sequence to a following segment of the horse enzyme, and additional elements of internal coincidences, or superficial similarities with other dehydrogenases, are noticed. These characteristics are not fully distinguishable from chance distributions but in view of the extensive species variations in alcohol dehydrogenases some evolutionary considerations may not be excluded, in which case a model relating all regions of these and associated enzymes to a common ancestor is shown to be compatible with all known observations.     

Liver alcohol dehydrogenase and aldehyde dehydrogenase in the Japanese: isozyme variation and its possible role in alcohol intoxication.

Forty autopsy livers from Japanese individuals were studied concerning alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes using electrophoretic and enzyme assay methods. A remarkably high frequency (85%) was found for the atypical ADH phenotype. The gene frequencies of ADH22 and ADH32 were .625 and .05, respectively. The usual ALDH phenotype showed two major isozyme bands, a faster migrating (low Km for acetaldehyde) and a slower migrating isozyme (high Km for acetaldehyde). Fifty-two percent of the specimens had an unusual phenotype of ALDH, which showed only the slower migrating isozyme. The usual phenotype was inhibited about 20%--30% by disulfiram and the unusual type up to 90%. Such a high incidence in the Japanese of the unusual phenotype, which lacks in the low Km isozyme, suggests that the initial intoxicating symptoms after alcohol drinking in these subjects might be due to delayed oxidation of acetaldehyde rather than its higher-than-normal production by typical or atypical ADH.     

The intramucosal distribution of gastric alcohol dehydrogenase and aldehyde dehydrogenase activity in rats.

Using qualitative and microquantitative histo-chemical techniques, alcohol dehydrogenase and aldehyde dehydrogenase activity was studied in the gastric mucosa of male and female rats. Alcohol dehydrogenase was demonstrated by staining reactions with maximum activity in surface and neck cells and with clearly weaker activity also in parietal cells. Aldehyde dehydrogenase could be detected in surface and neck cells, and also to a comparable degree in the parietal cells. Quantitative analyses of microdissected samples yielded high values for alcohol dehydrogenase activity exclusively in the superficial part of the gastric mucosa, whereas low-Km aldehyde dehydrogenase activity showed a decreasing gradient from the surface to the deeper parts of the mucosa. Sex differences could not be confirmed.     

Localization of alcohol and aldehyde dehydrogenases in the human spinal cord and brain

Location of aldehyde dehydrogenase (AldDG) and alcohol dehydrogenase (ADG) has been studied in 38 nuclei of the human brain. Neurons with a high AldDG activity predominate in the nucleus of the descending root of the trigeminal nerve, motor nuclei of the craniocerebral nerves (trigeminal, facial, abducent, blocking, sublingual, supraspinal), motor nuclei of the anterior horns of the spinal cord, lateral vestibular nucleus, posterior nucleus of the vagus nerve, pedunculopontine nucleus, superior salivary nucleus, and in the nucleus of Westphal-Edinger-Jacobovich. Neurons with a moderate AldDG activity predominate in the superior olivary complex, nucleus of the lateral loop, parabrachial (pigmented) mesencephalic nucleus and reticular lateral nucleus. A low enzymatic activity is specific for neurons of the pons proper, inferior vestibular nucleus, trapezoid body of the inferior olivary complex, dentate nucleus of the cerebellum, reticular nucleus of the tegmen of Bekhterev's pons and posterior nucleus of Gudden's suture. A high ADG activity is revealed in piriform neurons of the cerebellar cortex. Functional importance of ADG and AldDG activity in the brain is discussed.     

Intralobular distribution of rat liver aldehyde dehydrogenase and alcohol dehydrogenase

1. The activity of liver microsomal high Km-ALDH and mitochondrial low Km-ALDH, which may be primarily responsible for the oxidation of acetaldehyde after ethanol administration was found to be predominantly distributed in the centrilobular area. 2. The activities of other ALDH isozymes in mitochondrial and soluble fractions were evenly distributed in periportal and perivenous regions. 3. The activity of ADH which is involved in production of acetaldehyde was predominantly located in the periportal area. 4. From these results it seems unlikely that a concentration of acetaldehyde after ethanol ingestion is higher in perivenous hepatocytes than in periportal ones. Additional data would be needed to understand fully the mechanism by which ethanol induces predominantly centrilobular liver injury.     

No effect of chronic ethanol administration on the activity of alcohol dehydrogenase and aldehyde dehydrogenases in the near-term pregnant guinea pig

The objective of this study was to determine the effect of chronic maternal administration of moderate-dose ethanol on alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the guinea pig at near-term pregnancy. The activity of each enzyme in the maternal liver, fetal liver, and placenta of the guinea pig at 59 days of gestation (term, 66 days) was determined spectrophotometrically following chronic daily oral administration of two doses of 1 g ethanol/kg maternal body weight or isocaloric sucrose solution. There was no experimental evidence of ethanol-induced malnutrition in the mother or growth retardation in the fetus. There was a statistically significant increase (65%) in the microsomal cytochrome P-450 content of the maternal liver for the ethanol treatment compared with the sucrose treatment. The alcohol dehydrogenase, low Km aldehyde dehydrogenase, and high Km aldehyde dehydrogenase activities in the maternal liver, fetal liver, and placenta were not statistically different for the ethanol-treated compared with the sucrose-treated animals. This also was the case for the maternal blood and fetal blood ethanol and acetaldehyde concentrations, determined at 2h after maternal administration of 1 g ethanol/kg maternal body weight. These data demonstrate that the ethanol- and acetaldehyde-oxidizing enzyme activities in the maternal-placental-fetal unit of the guinea pig at near-term pregnancy were not changed by chronic administration of moderate-dose ethanol.     

Hepatic aldehyde dehydrogenase activity in Peromyscus genetically deficient in alcohol dehydrogenase

1. Hepatic aldehyde dehydrogenase (ALDH) activity was measured in two strains of deer-mouse, Peromyscus maniculatus. 2. There is no difference in the subcellular distribution of ALDH activity in the two strains. Animals of AdhN/AdhN genotype, lacking liver alcohol dehydrogenase (ADH), had 90% of total ALDH activity in the mitochondrial fraction compared to 94% for the AdhF/AdhF animals with normal ADH activity. Almost all of the remaining ALDH activity was in the hepatic cytosol with less than 1% in the microsomal fraction. 3. By contrast, in mice (Mus musculus) 43% of total hepatic ALDH activity was found in the cytosolic fraction and 55% in the mitochondrial. 4. It was concluded that the subcellular distribution of hepatic ALDH activity in Peromyscus does not vary with the presence or absence of ADH and that this ALDH distribution is not similar to that reported for other rodents.     

Isoenzyme polymorphism of the sorbitol dehydrogenase and the NADP-dependent isocitrate dehydrogenases in the fish family Cyprinidae

Among members of the fish family Cyprinidae , the existence of a diploid-tetraploid relationship is well established. The analysis of individual gene loci, using isoenzyme polymorphism as genetic markers, does not always confirm the expected gene duplication in the tetraploids. Of the markers used in this study, only the M-form of the NADP-dependent isocitrate dehydrogenase follows this expectation; the data suggest the existence of a single gene locus for the enzyme in diploids, while the observations on tetraploids were consistent with control by two distinct loci. For two other enzymes, the S-form of the NADP-dependent isocitrate dehydrogenase and sorbitol dehydrogenase, no difference seems to exist in the number of gene loci between diploids and tetraploids. A comparison between Cyprinid fish (order Ostariophysi ) and members of the order Isospondyli in which another diploid-tetraploid relationship was established, reveals that gene duplications are more frequently demonstrable within tetraploid Isospondyli than in tetraploid Cyprinidae. From this, it is concluded that polyploidization occurred earlier in evolution of Cyprinidae than of Isospondyli.     

The equilibrium between vitamin A alcohol and aldehyde in the presence of alcohol dehydrogenase

Vitamin A is reversibly dehydrogenated to vitamin A aldehyde (retinene) in isolated retinal rods and in liver extracts containing alcohol dehydrogenase and coenzyme 1.The reaction is probably involved in the utilization of vitamin A for the regeneration of bleached visual purple.The equilibrium constant k_H of the dehydrogenation is 3.3 × 10^?9, about 300 times more favorable to the aldehyde than k_H for ethyl alcohol.