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Western blot analysis of ADH-F in chicken liver homogenate . Lane 1, 100 ng of recombinant ADH-F. Lanes 2 and 3, 1 and 10 g of liver homogenate, respectively. Numbers on the right indicate isoelectric points of the isoelectic focusing standard proteins.
Source publication
This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues, Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDN...
Contexts in source publication
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... protein corresponding to the wild-type ADH-F was de- tected in the chicken liver homogenate with the rabbit anti- serum raised against recombinant ADH-F (Fig. 3). This anti- serum cross-reacted with 100 ng of human class I, II, and IV but not class III ADH proteins (not shown), all of which have similar subunit molecular weights and cannot be separated by SDS-polyacrylamide gel electrophoresis. Thus, isoelectric fo- cusing was employed to separate the 80-kDa dimers of chick ADH isozymes ...
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... have similar subunit molecular weights and cannot be separated by SDS-polyacrylamide gel electrophoresis. Thus, isoelectric fo- cusing was employed to separate the 80-kDa dimers of chick ADH isozymes according to their isoelectric points. Recombi- nant ADH-F appeared as a smear of multiple protein bands with pI values ranging from 7.1 to 8.0 (Fig. 3, lane 1). All bands exhibited activity with 100 mM ethanol, 5 mM trans-2-hexen-1- ol, and 100 M 3-hydroxysteroid alcohols. Protein bands bind- ing anti-ADH-F antibodies also appeared in the chicken liver homogenate separated by isoelectric focusing in the same range of pI values as the recombinant ADH-F (Fig. 3, lanes 2 and ...
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... pI values ranging from 7.1 to 8.0 (Fig. 3, lane 1). All bands exhibited activity with 100 mM ethanol, 5 mM trans-2-hexen-1- ol, and 100 M 3-hydroxysteroid alcohols. Protein bands bind- ing anti-ADH-F antibodies also appeared in the chicken liver homogenate separated by isoelectric focusing in the same range of pI values as the recombinant ADH-F (Fig. 3, lanes 2 and ...
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... new ADH gene encoded an active enzyme when pro- duced as a recombinant protein in E. coli. Antiserum against this recombinant ADH-F recognized protein bands in the chicken liver homogenate with the same range of isoelectric points as the multiple ADH-F forms (Fig. 3). The slightly more basic pI of the recombinant protein is consistent with the lack of N-acetylation in E. coli-expressed proteins ...
Citations
... These two classes, identified at DNA level, are the most divergent within mammalian ADHs. On the other hand, ADH7, previously named ADH-F due to its fetal expression, is a steroid/retinoid dehydrogenase that was first described in chicken [14]. Finally, ADH8 is a unique NADP + -dependent ADH isolated from the stomach of the frog Rana perezi and its proposed function is the reduction of retinaldehyde to retinol [15]. ...
... ADH1C has unique features among substrate-binding residues. At position 93, the lack of an aromatic ring expands the substrate cleft and permits the accommodation of large substrates, as in human ADH1A and chicken ADH7 [14,65,66]. In contrast, an unusual Phe116 would narrow the entrance, although still may allow productive binding of retinoids as occurs in X. laevis ADH8B [67] . ...
... The novel reptilian genomic sequences supported the class assignment of X. tropicalis ADH7, sharing identity percentages of 71.2% and 67.4% with turtle ADH7 and anole ADH7B, respectively. As listed inTable 7, all ADH7 enzymes show Thr48, involved in the stereospecificity for secondary alcohols; a small residue such as Cys at position 93 (Pro in chicken), indicative of high K m values for ethanol and correct positioning of steroid sub- strates [14]; Phe140 and Leu141 (in most sequences); and similar coenzyme-binding residues. Positions 112 to 126 are almost identical, and His115 and Trp142 are common for all ADH7 enzymes. ...
The alcohol dehydrogenase (ADH) gene family uniquely illustrates the concept of enzymogenesis. In vertebrates, tandem duplications gave rise to a multiplicity of forms that have been classified in eight enzyme classes, according to primary structure and function. Some of these classes appear to be exclusive of particular organisms, such as the frog ADH8, a unique NADP+-dependent ADH enzyme. This work describes the ADH system of Xenopus, as a model organism, and explores the first amphibian and reptilian genomes released in order to contribute towards a better knowledge of the vertebrate ADH gene family.
Xenopus cDNA and genomic sequences along with expressed sequence tags (ESTs) were used in phylogenetic analyses and structure-function correlations of amphibian ADHs. Novel ADH sequences identified in the genomes of Anolis carolinensis (anole lizard) and Pelodiscus sinensis (turtle) were also included in these studies. Tissue and stage-specific libraries provided expression data, which has been supported by mRNA detection in Xenopus laevis tissues and regulatory elements in promoter regions. Exon-intron boundaries, position and orientation of ADH genes were deduced from the amphibian and reptilian genome assemblies, thus revealing syntenic regions and gene rearrangements with respect to the human genome. Our results reveal the high complexity of the ADH system in amphibians, with eleven genes, coding for seven enzyme classes in Xenopus tropicalis. Frogs possess the amphibian-specific ADH8 and the novel ADH1-derived forms ADH9 and ADH10. In addition, they exhibit ADH1, ADH2, ADH3 and ADH7, also present in reptiles and birds. Class-specific signatures have been assigned to ADH7, and ancestral ADH2 is predicted to be a mixed-class as the ostrich enzyme, structurally close to mammalian ADH2 but with class-I kinetic properties. Remarkably, many ADH1 and ADH7 forms are observed in the lizard, probably due to lineage-specific duplications. ADH4 is not present in amphibians and reptiles.
The study of the ancient forms of ADH2 and ADH7 sheds new light on the evolution of the vertebrate ADH system, whereas the special features showed by the novel forms point to the acquisition of new functions following the ADH gene family expansion which occurred in amphibians.
... ADH7: Class VII forms were initially described in birds (Kedishvili et al, 1997). In chicken, ADH7 (or ADH-F) is flanked by two closely related sequences, annotated as 'similar to ADH-F'. ...
Multiple members of the MDR-ADH (MDR: Medium-chain dehydrogenases/reductases; ADH: alcohol dehydrogenase) family are found in vertebrates, although the enzymes that belong to this family have also been isolated from bacteria, yeast, plant and animal sources. Initial understanding of the physiological roles and evolution of the family relied on biochemical studies, protein alignments and protein structure comparisons. Subsequently, studies at the genetic level yielded new information: the expression pattern, exon-intron distribution, in silico-derived protein sequences and murine knockout phenotypes. More recently, genomic and EST databases have revealed new family members and the chromosomal location and position in the cluster of both the first and new forms. The data now available provide a comprehensive scenario, from which a reliable picture of the evolutionary history of this family can be made.
... 6, a high K m for ethanol (about 30 mM) and moderate sensitivity to pyrazole inhibition (Cheng & Yoshida, 1991; Yasunami et al. 1991). An additional class of ADH (tentatively designated class VI) was reported in the liver of deer mice (Peromyscus maniculatus; Zheng et al. 1993) and rats (Hoog & Brandt, 1995) and class VII ADH was cloned from chicken (Kedishvili et al. 1997); to date the human homologues have not been found. There is a new nomenclature for ADH, shown inTable 1 , but the remainder of the present review will use the older system. ...
Alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH2) are responsible for metabolizing the bulk of ethanol consumed as part of the diet and their activities contribute to the rate of ethanol elimination from the blood. They are expressed at highest levels in liver, but at lower levels in many tissues. This pathway probably evolved as a detoxification mechanism for environmental alcohols. However, with the consumption of large amounts of ethanol, the oxidation of ethanol can become a major energy source and, particularly in the liver, interferes with the metabolism of other nutrients. Polymorphic variants of the genes for these enzymes encode enzymes with altered kinetic properties. The pathophysiological effects of these variants may be mediated by accumulation of acetaldehyde; high-activity ADH variants are predicted to increase the rate of acetaldehyde generation, while the low-activity ALDH2 variant is associated with an inability to metabolize this compound. The effects of acetaldehyde may be expressed either in the cells generating it, or by delivery of acetaldehyde to various tissues by the bloodstream or even saliva. Inheritance of the high-activity ADH beta2, encoded by the ADH2*2 gene, and the inactive ALDH2*2 gene product have been conclusively associated with reduced risk of alcoholism. This association is influenced by gene-environment interactions, such as religion and national origin. The variants have also been studied for association with alcoholic liver disease, cancer, fetal alcohol syndrome, CVD, gout, asthma and clearance of xenobiotics. The strongest correlations found to date have been those between the ALDH2*2 allele and cancers of the oro-pharynx and oesophagus. It will be important to replicate other interesting associations between these variants and other cancers and heart disease, and to determine the biochemical mechanisms underlying the associations.
... As compared to the Km values for retinol of three ADHs expressed in the human gastric mucosa, i.e. Class I (γ), Class III, and Class IV ADHs, the finding is reasonable (3,11,14). The association between the Class IV ADH activity and the ATRA formation level was further confirmed even after adjusting subjects' age and status of H. pylori infection in another multiple regression analysis. ...
Aldehyde and alcohol dehydrogenases (ALDHs and ADHs) are both highly multiple enzymes with many different forms in metabolically linked functional pathways. However, they are derived from separate protein families with distinct properties. Those of the vertebrate ADHs have been well characterized since long, and their different classes belong to the MDR protein family of known structures from several sources. However, the properties and multiplicity of the ALDHs have only recently been partly characterized. Presently, just three of the at least twelve different ALDH classes (Yoshida et al., 1998) are kn in three-dimensional structure (Liu et al., 1997; Steinmetz et al., 1997; Johansson et al.998), several have not been purified and characterized enzymatically, and little is known about their species or class variabilities.
The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.
Mammalian alcohol dehydrogenases ADH1 (class I ADH) and ADH4 (class IV ADH) function as retinol dehydrogenases contributing to the synthesis of retinoic acid, the active form of vitamin A involved in growth and development. Xenopus laevis ADH1 and ADH4 genes were isolated using polymerase chain reaction primers corresponding to conserved motifs of vertebrate ADHs. The predicted amino acid sequence of Xenopus ADH1 was clearly found to be an ortholog of ADH1 from the related amphibian Rana perezi. Phylogenetic tree analysis of the Xenopus ADH4 sequence suggested this enzyme is likely to be an ADH4 ortholog, and this classification was more confidently made when based also on the unique expression patterns of Xenopus ADH1 and ADH4 in several retinoid-responsive epithelial tissues. Northern blot analysis of Xenopus adult tissues indicated nonoverlapping patterns of ADH expression, with ADH1 mRNA found in small intestine, large intestine, liver, and mesonephros and ADH4 mRNA found in esophagus, stomach, and skin. These nonoverlapping tissue-specific patterns are identical to those previously observed for mouse ADH1 and ADH4, thus providing further evidence that Xenopus ADH1 and ADH4 are orthologs of mouse ADH1 and ADH4, respectively. During Xenopus embryonic development ADH1 mRNA was first detectable by Northern blot analysis at stage 35, whereas ADH4 mRNA was undetectable through stage 47. Whole-mount in situ hybridization indicated that ADH1 expression was first localized in the pronephros during Xenopus embryogenesis, thus conserved with mouse embryonic ADH1 which is first expressed in the mesonephros. ADH4 expression was not detected in Xenopus embryos by whole-mount in situ hybridization but was localized to the gastric mucosa of the adult stomach, a property shared by mouse ADH4. Conserved expression of ADH1 and ADH4 in retinoid-responsive epithelial tissues of amphibians and mammals argue that these enzymes may perform essential retinoid signaling functions during development of the pronephros, mesonephros, liver, and lower digestive tract in the case of ADH1 and in the skin and upper digestive tract in the case of ADH4.
Isozymes of alcohol and other dehydrogenases convert ethanol and retinol to their corresponding aldehydes in vitro. In addition, new pathways of retinol metabolism have been described in hepatic microsomes that involve, in part, cytochrome P450s, which can also metabolize various drugs. In view of these overlapping metabolic pathways, it is not surprising that multiple interactions between retinol, ethanol, and other drugs occur. Accordingly, prolonged use of alcohol, drugs, or both, results not only in decreased dietary intake of retinoids and carotenoids, but also accelerates the breakdown of retinol through cross-induction of degradative enzymes. There is also competition between ethanol and retinoic acid precursors. Depletion ensues, with associated hepatic and extrahepatic pathology, including carcinogenesis and contribution to fetal defects. Correction of deficiency through vitamin A supplementation has been advocated. It is, however, complicated by the intrinsic hepatotoxicity of retinol, which is potentiated by concomitant alcohol consumption. By contrast, beta-carotene, a precursor of vitamin A, was considered innocuous until recently, when it was found to also interact with ethanol, which interferes with its conversion to retinol. Furthermore, the combination of beta-carotene with ethanol results in hepatotoxicity. Moreover, in smokers who also consume alcohol, beta-carotene supplementation promotes pulmonary cancer and, possibly, cardiovascular complications. Experimentally, beta-carotene toxicity was exacerbated when administered as part of beadlets. Thus ethanol, while promoting a deficiency of vitamin A also enhances its toxicity as well as that of beta-carotene. This narrowing of the therapeutic window for retinol and beta-carotene must be taken into account when formulating treatments aimed at correcting vitamin A deficiency, especially in drinking populations.
