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X Parés

Publications and source records attributed to X Parés.

At least 19 recordsLinked to original sources
PubMed2003

S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localisation.

S-nitrosoglutathione (GSNO) formation represents a mechanism for storage and transport of nitric oxide. Analysis of human liver and Saccharomyces cerevisiae extracts has revealed the presence of only one enzyme able to significantly reduce GSNO, identified as glutathione-dependent formaldehyde dehydrogenase (FALDH). GSNO is the best substrate known for the human and yeast enzymes (kcat/Km = 444,400 and 350,000 mM(-1) min(-1), respectively). Although NADH is the preferred cofactor, some activity with NADPH (Km = 460 microM) can be predicted in vivo. The subcellular localization demonstrates a cytosolic and nuclear distribution of FALDH in living yeast cells. This agrees with previous results in rat, and suggests a role in the regulation of GSNO levels in the cytoplasmic and nuclear compartments of the eukaryotic cell.

Aldehyde Oxidoreductases↗
PubMed2001

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase.

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.

Acetoin↗
PubMed2001

Kinetic effects of a single-amino acid mutation in a highly variable loop (residues 114-120) of class IV ADH.

Class IV alcohol dehydrogenase shows a deletion at position 117 with respect to class I enzymes, which typically have a Gly residue. In class I structures, Gly117 is part of a loop (residues 114-120) that is highly variable within the alcohol dehydrogenase family. A mutant human class IV enzyme was engineered in which a Gly residue was inserted at position 117 (G117ins). Its kinetic properties, regarding ethanol and primary aliphatic alcohols, secondary alcohols and pH profiles, were determined and compared with the results obtained in previous studies in which the size of the 114-120 loop was modified. For the enzymes considered, a smaller loop was associated with a lower catalytic efficiency towards short-chain alcohols (ethanol and propanol) and secondary alcohols, as well as with a higher K(m) for ethanol at pH 7.5 than at pH 10.0. The effect can be rationalized in terms of a more open, solvent-accessible active site in class IV alcohol dehydrogenase, which disfavors productive binding of ethanol and short-chain alcohols, specially at physiological pH.

Alcohol Dehydrogenase↗
PubMed2001

N-terminal acetylation in a third protein family of vertebrate alcohol dehydrogenase/retinal reductase found through a 'proteomics' approach in enzyme characterization.

A recent finding of a novel class of retinol-active alcohol dehydrogenase (ADH) in frog prompted analysis of this activity in other vertebrate forms. Surprisingly, yet another and still more unrelated ADH was identified in chicken tissues. It was found to be a member of the aldo-keto reductase (AKR) enzyme family, not previously known as an ADH in vertebrates. Its terminal blocking group and the N-terminal segment, not assigned by protein and cDNA structure analysis, were determined by electrospray tandem mass spectrometry after protein isolation by two-dimensional gel electrophoresis. The N terminus is Acetyl-Ala- and the N-terminal segment contains two consecutive Asn residues. The results establish the new ADH enzyme of the AKR family and show the usefulness of combined gel separation and mass spectrometry in enzyme-characterization.

Acetylation↗
PubMed2001

Distribution of alcohol dehydrogenase mRNA in the rat central nervous system. Consequences for brain ethanol and retinoid metabolism.

The localization of alcohol dehydrogenase (ADH) in brain regions would demonstrate active ethanol metabolism in brain during alcohol consumption, which would be a new basis to explain the effects of ethanol in the central nervous system. Tissue sections from several regions of adult rat brain were examined by in situ hybridization to detect the expression of genes encoding ADH1 and ADH4, enzymes highly active with ethanol and retinol. ADH1 mRNA was found in the granular and Purkinje cell layers of cerebellum, in the pyramidal and granule cells of the hippocampal formation and in some cell types of cerebral cortex. ADH4 expression was detected in the Purkinje cells, in the pyramidal and granule cells of the hippocampal formation and in the pyramidal cells of cerebral cortex. High levels of ADH1 and ADH4 mRNAs were detected in the CNS epithelial and vascular tissues: leptomeninges, choroid plexus, ependymocytes of ventricle walls, and endothelium of brain vessels. Histochemical methods detected ADH activity in rodent cerebellar slices, while Western-blot analysis showed ADH4 protein in homogenates from several brain regions. In consequence, small but significant levels of ethanol metabolism can take place in distinct areas of the CNS following alcohol consumption, which could be related to brain damage caused by a local accumulation of acetaldehyde. Moreover, the involvement of ADH in the synthesis of retinoic acid suggests a role for the enzyme in the regulation of adult brain functions. The impairment of retinol oxidation by competitive inhibition of ADH in the presence of ethanol may be an additional origin of CNS abnormalities caused by ethanol.

Alcohol Dehydrogenase↗
PubMed2000

Characterization of a (2R,3R)-2,3-butanediol dehydrogenase as the Saccharomyces cerevisiae YAL060W gene product. Disruption and induction of the gene.

The completion of the Saccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these, YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD(+), but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2, 3-butanediol (K(m) = 14 mm, k(cat) = 78,000 min(-)(1)) and meso-butanediol (K(m) = 65 mm, k(cat) = 46,000 min(-)(1)) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K(m) = 4.5 mm, k(cat) = 98,000 min(-)(1)) to (2R,3R)-2,3-butanediol and meso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH). S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3. 5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R, 3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family.

Acetoin↗
PubMed2000

Molecular basis for differential substrate specificity in class IV alcohol dehydrogenases: a conserved function in retinoid metabolism but not in ethanol oxidation.

Mammalian class IV alcohol dehydrogenase enzymes are characteristic of epithelial tissues, exhibit moderate to high K(m) values for ethanol, and are very active in retinol oxidation. The human enzyme shows a K(m) value for ethanol which is 2 orders of magnitude lower than that of rat class IV. The uniquely significant difference in the substrate-binding pocket between the two enzymes appears to be at position 294, Val in the human enzyme and Ala in the rat enzyme. Moreover, a deletion at position 117 (Gly in class I) has been pointed out as probably responsible for class IV specificity toward retinoids. With the aim of establishing the role of these residues, we have studied the kinetics of the recombinant human and rat wild-type enzymes, the human G117ins and V294A mutants, and the rat A294V mutant toward aliphatic alcohols and retinoids. 9-cis-Retinol was the best retinoid substrate for both human and rat class IV, strongly supporting a role of class IV in the generation of 9-cis-retinoic acid. In contrast, 13-cis retinoids were not substrates. The G117ins mutant showed a decreased catalytic efficiency toward retinoids and toward three-carbon and longer primary aliphatic alcohols, a behavior that resembles that of the human class I enzyme, which has Gly(117). The K(m) values for ethanol dramatically changed in the 294 mutants, where the human V294A mutant showed a 280-fold increase, and the rat A294V mutant a 50-fold decrease, compared with those of the respective wild-type enzymes. This demonstrates that the Val/Ala exchange at position 294 is mostly responsible for the kinetic differences with ethanol between the human and rat class IV. In contrast, the kinetics toward retinoids was only slightly affected by the mutations at position 294, compatible with a more conserved function of mammalian class IV alcohol dehydrogenase in retinoid metabolism.

Alanine↗
PubMed2000

Genetic polymorphism of alcohol dehydrogenase in europeans: the ADH2*2 allele decreases the risk for alcoholism and is associated with ADH3*1.

Polymorphism at the ADH2 and ADH3 loci of alcohol dehydrogenase (ADH) has been shown to have an effect on the predisposition to alcoholism in Asian individuals. However, the results are not conclusive for white individuals. We have analyzed the ADH genotype of 876 white individuals from Spain (n = 251), France (n = 160), Germany (n = 184), Sweden (n = 88), and Poland (n = 193). Peripheral blood samples from healthy controls and groups of patients with viral cirrhosis and alcohol-induced cirrhosis, as well as alcoholics with no liver disease, were collected on filter paper. Genotyping of the ADH2 and ADH3 loci was performed using polymerase chain reaction-restriction fragment length polymorphism methods on white cell DNA. In healthy controls, ADH2*2 frequencies ranged from 0% (France) to 5.4% (Spain), whereas ADH3*1 frequencies ranged from 47. 6% (Germany) to 62.5% (Sweden). Statistically significant differences were not found, however, between controls from different countries, nor between patients with alcoholism and/or liver disease. When all individuals were grouped in nonalcoholics (n = 451) and alcoholics (n = 425), ADH2*2 frequency was higher in nonalcoholics (3.8%) than in alcoholics (1.3%) (P =.0016), whereas the ADH3 alleles did not show differences. Linkage disequilibrium was found between ADH2 and ADH3, resulting in an association of the alleles ADH2*2 and ADH3*1, both coding for the most active enzymatic forms. In conclusion, the ADH2*2 allele decreases the risk for alcoholism, whereas the ADH2*2 and ADH3*1 alleles are found to be associated in the European population.

Adult↗
PubMed2000

Pharmacogenetics of the alcohol dehydrogenase system.

Alcohol dehydrogenase (ADH) constitutes a complex enzyme system with different forms and extensive multiplicity. A combination of constant and variable properties regarding function, multiplicity and structure of ADH is highlighted for the human system and extended to ADH forms in general. Future perspectives suggest continued studies in specific directions for distinction of metabolic, regulatory and pharmacogenetic roles of ADH.

Alcohol Dehydrogenase↗
PubMed1999

A double residue substitution in the coenzyme-binding site accounts for the different kinetic properties between yeast and human formaldehyde dehydrogenases.

Glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzymatic system for formaldehyde detoxification in all eukaryotic and many prokaryotic organisms. The enzyme of yeasts and some bacteria exhibits about 10-fold higher k(cat) and K(m) values than those of the enzyme from animals and plants. Typically Thr-269 and Glu-267 are found in the coenzyme-binding site of yeast FALDH, but Ile-269 and Asp-267 are present in the FALDH of animals. By site-directed mutagenesis we have prepared the T269I and the D267E mutants and the D267E/T269I double mutant of Saccharomyces cerevisiae FALDH with the aim of investigating the role of these residues in the kinetics. The T269I and the D267E mutants have identical kinetic properties as compared with the wild-type enzyme, although T269I is highly unstable. In contrast, the D267E/T269I double mutant is stable and shows low K(m) (2.5 microM) and low k(cat) (285 min(-1)) values with S-hydroxymethylglutathione, similar to those of the human enzyme. Therefore, the simultaneous exchange at both residues is the structural basis of the two distinct FALDH kinetic types. The local structural perturbations imposed by the substitutions are suggested by molecular modeling studies. Finally, we have studied the effect of FALDH deletion and overexpression on the growth of S. cerevisiae. It is concluded that the FALDH gene is not essential but enhances the resistance against formaldehyde (0.3-1 mM). Moreover, the wild-type enzyme (with high k(cat) and K(m)) provides more resistance than the double mutant (with low k(cat) and K(m)).

Aldehyde Oxidoreductases↗
PubMed1999

Structural and enzymatic properties of a gastric NADP(H)- dependent and retinal-active alcohol dehydrogenase.

A class IV-type, gastric alcohol dehydrogenase (ADH) has been purified from frog (Rana perezi) tissues, meaning detection of this enzyme type also in nonmammalian vertebrates. However, the protein is unique among vertebrate ADHs thus far characterized in having preference for NADP(+) rather than NAD(+). Similarly, it deviates structurally from other class IV ADHs and has a phylogenetic tree position outside that of the conventional class IV cluster. The NADP(+) preference is structurally correlated with a replacement of Asp-223 of all other vertebrate ADHs with Gly-223, largely directing the coenzyme specificity. This residue replacement is expected metabolically to correlate with a change of the reaction direction catalyzed, from preferential alcohol oxidation to preferential aldehyde reduction. This is of importance in cellular growth regulation through retinoic acid formed from retinol/retinal precursors because the enzyme is highly efficient in retinal reduction (k(cat)/K(m) = 3.4.10(4) mM(-1) min(-1)). Remaining enzymatic details are also particular but resemble those of the human class I/class IV enzymes. However, overall structural relationships are distant (58-60% residue identity), and residues at substrate binding and coenzyme binding positions are fairly deviant, reflecting the formation of the new activity. The results are concluded to represent early events in the duplicatory origin of the class IV line or of a separate, class IV-type line. In both cases, the novel enzyme illustrates enzymogenesis of classes in the ADH system. The early origin (with tetrapods), the activity (with retinoids), and the specific location of this enzyme (gastric, like the gastric and epithelial location of the human class IV enzyme) suggest important functions of the class IV ADH type in vertebrates.

Alcohol Dehydrogenase↗
PubMed1999

Recommended nomenclature for the vertebrate alcohol dehydrogenase gene family.

The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies.

Alcohol Dehydrogenase↗
PubMed1998

Retinoids, omega-hydroxyfatty acids and cytotoxic aldehydes as physiological substrates, and H2-receptor antagonists as pharmacological inhibitors, of human class IV alcohol dehydrogenase.

Kinetic constants of human class IV alcohol dehydrogenase (sigmasigma-ADH) support a role of the enzyme in retinoid metabolism, fatty acid omega-oxidation, and elimination of cytotoxic aldehydes produced by lipid peroxidation. Class IV is the human ADH form most efficient in the reduction of 4-hydroxynonenal (k(cat)/Km: 39,500 mM(-1) min(-1)). Class IV shows high activity with all-trans-retinol and 9-cis-retinol, while 13-cis-retinol is not a substrate but an inhibitor. Both all-trans-retinoic and 13-cis-retinoic acids are potent competitive inhibitors of retinol oxidation (Ki: 3-10 microM) which can be a basis for the regulation of the retinoic acid generation and of the pharmacological actions of the 13-cis-isomer. The inhibition of class IV retinol oxidation by ethanol (Ki: 6-10 mM) may be the origin of toxic and teratogenic effects of ethanol. H2-receptor antagonists are poor inhibitors of human and rat classes I and IV (Ki > 0.3 mM) suggesting a small interference in ethanol metabolism at the pharmacological doses of these common drugs.

Alcohol Dehydrogenase↗
PubMed1998

First pass metabolism of ethanol is strikingly influenced by the speed of gastric emptying.

BACKGROUND: Ethanol undergoes a first pass metabolism (FPM) in the stomach and liver. Gastric FPM of ethanol primarily depends on the activity of gastric alcohol dehydrogenase (ADH). In addition, the speed of gastric emptying (GE) may modulate both gastric and hepatic FPM of ethanol. AIMS: To study the effect of modulation of GE on FPM of ethanol in the stomach and liver. METHODS: Sixteen volunteers (eight men and eight women) received ethanol (0.225 g/kg body weight) orally and intravenously, and the areas under the ethanol concentration time curves were determined to calculate FPM of ethanol. In seven of these subjects, FPM of ethanol was measured after the intravenous administration of 10 mg metoclopramide (MCP) and 20 mg N-butylscopolamine (NBS) in separate experiments to either accelerate or delay GE. GE was monitored sonographically by integration of the antral area of the stomach every five minutes for 90 minutes after oral ethanol intake. In addition, gastric biopsy specimens were taken to determine ADH activity and phenotype, as well as to evaluate gastric histology. Blood was also drawn for ADH genotyping. RESULTS: GE time was significantly delayed by the administration of NBS as compared with controls (p<0.0001) and as compared with the administration of MCP (p<0.0001). This was associated with a significantly enhanced FPM of ethanol with NBS compared with MCP (p = 0.0004). A significant correlation was noted between GE time and FPM of ethanol (r = 0.43, p = 0.0407). Gastric ADH activity did not significantly correlate with FPM of ethanol. CONCLUSION: FPM of ethanol is strikingly modulated by the speed of GE. Delayed GE increases the time of exposure of ethanol to gastric ADH and may therefore increase gastric FPM of ethanol. In addition, hepatic FPM of ethanol may also be enhanced as the result of slower absorption of ethanol from the small intestine. Thus a knowledge of GE time is a major prerequisite for studying FPM of ethanol in humans.

Administration, Oral↗
PubMed1997

Alcohol dehydrogenase of human and rat blood vessels. Role in ethanol metabolism.

Alcohol dehydrogenase (ADH) activity has been detected in all arteries and veins examined from humans and rat. In distinct human autopsy vessels, activity values range from 0.9 +/- 0.2 to 9.9 +/- 7.7 mU/mg. Distribution of the activity in human aorta was: intima (23.5%), media (74%) and adventia (2.5%). In most of the samples the beta1 beta1 isozyme of class I ADH was the only form responsible for the ADH activity. Class IV ADH (sigma sigma-ADH) was present in three of the 28 individuals examined. The rat blood vessels showed class IV, but not class I, ADH localized in endothelium and media. The physiological role of vascular ADH is probably related to retinoid metabolism and elimination of lipid peroxidation aldehydes. A contribution to human ethanol metabolism is supported by the significant amount of low-Km activity and the extension of the vascular system.

Adult↗
PubMed1996

Arabidopsis formaldehyde dehydrogenase. Molecular properties of plant class III alcohol dehydrogenase provide further insights into the origins, structure and function of plant class p and liver class I alcohol dehydrogenases.

A glutathione-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) has been characterized from Arabidopsis thaliana. This plant enzyme exhibits kinetic and molecular properties in common with the class III forms from mammals, with a K(m) for S-hydroxymethylglutathione of 1.4 microM, an anodic electrophoretic mobility (pI: 5.3-5.6) and a cross-reaction with anti-(rat class III alcohol dehydrogenase) antibodies. The enzyme structure, deduced from the cDNA sequence, fits into the complex system of alcohol dehydrogenases and shows that all life forms share the class III protein type. The corresponding mRNA is 1.4 kb and present in all plant organs; a single copy of the gene is found in the genome. The class III structural variability is different from that of the ethanol-active enzyme types in both vertebrates (class I) and plants (class P), although class P conserves more of the class III properties than class I does. Also the enzymatic properties differ between the two ethanol-active classes. Active-site variability and exchanges at essential residues (Leu/Gly57, Asp/Arg115) may explain the distinct kinetics. These patterns are consistent with two different metabolic roles for the ethanol-active enzymes, a more constant function, reduction of acetaldehyde during hypoxia, for class P, and a more variable function, the detoxication of alcohols and participation in metabolic conversions, for class I. A sequence motif, Pro-Xaa-Ile/Val-Xaa-Gly-His-Glu-Xaa-Xaa-Gly, common to all medium-chain alcohol dehydrogenases is defined.

Alcohol Dehydrogenase↗