Multiplication of the class I alcohol dehydrogenase locus in mammalian evolution

Chromosomal DNA samples derived from various primates and other mammals (horse, sheep, rabbit, and mouse) were digested with restriction endonuclease and hybridized with a probe of the sixth exon of the human ADH gene, which is highly conserved in the class I alcohol dehydrogenase of these mammalian species. The copy number of the class I ADH gene in each species was estimated from the number of hybridized bands. Primate DNA samples showed three distinct bands in the blots of PstI digest and DraI digest. Moreover, most of the bands from primate DNA showed a similarity in size so as to allow us to assign the ADH1, ADH2, and ADH3 homologues in each species. In contrast, mouse has only one gene, and rabbit, sheep, and horse seem to have only two genes, for the class I ADH, which showed divergent hybridization bands. These results are consistent with the view that the human class I ADH gene cluster has been generated through gene multiplication events which occurred before the Catarrhini branch point in the course of primate evolution.     

Molecular evolution of the zinc-containing long-chain alcohol dehydrogenase genes

Phylogenetic relationships and rates of nucleotide substitution were studied for alcohol dehydrogenase (ADH) genes by using DNA sequences from mammals and plants. Mammalian ADH sequences include the three class I genes and a class II gene from humans and one gene each from baboon, rat, and mouse. Plant sequences include two ADH genes each from maize and rice, three genes from barley, and one gene each from wheat and two dicots, Arabidopsis and pea. Phylogenetic trees show that relationships among ADH genes are generally consistent with taxonomic relationships: mammalian and plant ADH genes are classified into two distinct groups; primate class I genes are clustered; and two dicot sequences are clustered separately from monocot sequences. Accelerated evolution has been detected among the duplicated ADH genes in plants, in which synonymous substitutions occurred more often within the coenzyme-binding domain than within the catalytic domains.     

Structural relationships among class I isozymes of human liver alcohol dehydrogenase

The alpha subunit of human liver alcohol dehydrogenase has been submitted to structural analysis. Together with earlier work on the beta and gamma subunits, the results allow conclusions on the relationship of all known forms of the class I type of the enzyme. Two segments of the alpha subunit were determined; one was also reinvestigated in the beta and gamma subunits. The results establish 11 residue replacements among class I subunits in the segments analyzed and show that the alpha, beta, and gamma protein chains each are structurally distinct in the active site regions, where replacements affect positions influencing coenzyme binding (position 47; Gly in alpha, Arg in beta and gamma) and substrate specificity (position 48; Thr in alpha and beta, Ser in gamma). Residue 128, previously not detected in beta and gamma subunits, corresponds to a position of another isozyme difference (Arg in beta and gamma, Ser in alpha). The many amino acid replacements in alcohol dehydrogenases even at their active sites illustrate that in judgements of enzyme functions absolute importance of single residues should not be overemphasized. Available data suggest that alpha and gamma are the more dissimilar forms within the family of the three class I subunits that have resulted from two gene duplications. The class distinction of alcohol dehydrogenases previously suggested from enzymatic, electrophoretic, and immunological properties therefore also holds true in relation to their structures.     

Rabbit liver alcohol dehydrogenase: isolation and characterization of class I isozymes

Livers of rabbits contain three classes of alcohol dehydrogenase (ADH) isozymes which are highly analogous to the human classes. Class I ADHs migrate toward cathode on starch gel and are very sensitive to 4-methylpyrazole (4-MePz) inhibition. Class II ADH migrates slowly toward anode and is less sensitive to 4-MePz. Class III ADH migrates rapidly toward anode and is insensitive to 4-MePz. There are one class II, one class III and at least three class I ADH isozymes present in the rabbit liver. The three class I isozymes purified to homogeneity are all dimers with subunit molecular weight of 41700. Two are heterodimers composed of A-, C-chains and B-, C-chains, respectively. The third one is a homodimer, contains only the C-chain. These results indicate that among all the mammals examined, rabbit ADH bears the greatest resemblance to the human enzyme.     

In vitro dissociation and reassociation of human alcohol dehydrogenase class I isozymes

Freezing (-78 degrees C) and thawing (25 degrees C) a heterodimeric human alcohol dehydrogenase class I isozyme in the presence of 0.1 M sodium phosphate/0.1 mM DTT, pH 7.0, and the subsequent separation of the scrambled isozymes by HPLC are used to prepare homodimers from heterodimers, with recovery of enzyme activity ranging from 80 to 95%. The ratio of the three isozymes obtained from a heterodimer follows the binomial distribution of 1:2:1, indicating random reassociation of the two subunits. The physical and enzymatic properties of the reassociated isozymes are the same as those obtained directly from human liver preparations. The nature of subunit-subunit interactions of human ADH class I isozymes is examined by optimizing the conditions required for the formation of the new dimers "in vitro". The effect of a number of reagents previously used in the reversible dissociation of dehydrogenases is investigated. The coenzyme NAD+ is a potent inhibitor of the dissociation of dimers during the freeze/thaw procedure. The presence of sodium phosphate in the enzyme solution is essential during the freezing and thawing experiment. No appreciable dissociation/reassociation occurs in TES, HEPES, or even potassium phosphate. The reversible dissociation is due primarily to the decrease in pH because of the low solubility of Na2HPO4 at low temperatures. The reassociation occurs after thawing in a temperature-dependent process. There is no reactivation if the enzyme is incubated at 0 degrees C after thawing, while at 25 degrees C high recovery in activity is achieved in a time period ranging from 15 to 90 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase

The class III human liver alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, separates electrophoretically into a major anodic form (₁) of known structure, and at least one minor, also anodic but a slightly faster migrating form (₂). The primary structure of the minor form isolated by ion-exchange chromatography has now been determined. Results reveal an amino acid sequence identical to that of the major form, suggesting that the two derive from the same translation product, with the minor form modified chemically in a manner not detectable by sequence analysis. This pattern resembles that for the classical alcohol dehydrogenase (class I). Hence, the ₁/₂ multiplicity does not add further primary forms to the complex alcohol dehydrogenase system but shows the presence of modified forms also in class III.     

Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics

Ethanol is teratogenic to many vertebrates. We are utilizing zebrafish as a model system to determine whether there is an association between ethanol metabolism and ethanol-mediated developmental toxicity. Here we report the isolation and characterization of two cDNAs encoding zebrafish alcohol dehydrogenases (ADHs). Phylogenetic analysis of these zebrafish ADHs indicates that they share a common ancestor with mammalian class I, II, IV, and V ADHs. The genes encoding these zebrafish ADHs have been named Adh8a and Adh8b by the nomenclature committee. Both genes were genetically mapped to chromosome 13. The 1450-bp Adh8a is 82, 73, 72, and 72% similar at the amino acid level to the Baltic cod ADH8 (previously named ADH1), the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. Also, the 1484-bp Adh8b is 77, 68, 67, and 66% similar at the amino acid level to the Baltic cod ADH8, the human ADH1B2, the mouse ADH1, and the rat ADH1, respectively. ADH8A and ADH8B share 86% amino acid similarity. To characterize the functional properties of ADH8A and ADH8B, recombinant proteins were purified from SF-9 insect cells. Kinetic studies demonstrate that ADH8A metabolizes ethanol, with a V(max) of 13.4 nmol/min/mg protein, whereas ADH8B does not metabolize ethanol. The ADH8A K(m) for ethanol as a substrate is 0.7 mm. 4-Methyl pyrazole, a classical competitive inhibitor of class I ADH, failed to inhibit ADH8A. ADH8B has the capacity to efficiently biotransform longer chain primary alcohols (>/=5 carbons) and S-hydroxymethlyglutathione, whereas ADH8A does not efficiently metabolize these substrates. Finally, mRNA expression studies indicate that both ADH8A and ADH8B mRNA are expressed during early development and in the adult brain, fin, gill, heart, kidney, muscle, and liver. Together these results indicate that class I-like ADH is conserved in zebrafish, albeit with mixed functional properties.     

Molecular evolution of mammalian ribonucleases 1

There have been many studies on the chemistry of mammalian pancreatic ribonucleases (ribonucleases 1), but the functional biology of this family of homologous proteins is still largely unknown. Many studies have been performed on the molecular evolution and properties of this enzyme from species belonging to a large number of mammalian taxa, including paralogous gene products resulting from recent gene duplications. Novel ribonuclease 1 sequences were determined for three rodent species (gundi, brush-tailed porcupine, and squirrel), rabbit, a fruit bat, elephant, and aardvark, and the new sequences were used for deriving most parsimonious networks of ribonucleases from different mammalian orders, including earlier determined nucleotide sequences and also a larger set of protein sequences. Weak support for interordinal relationships were obtained, except for an Afrotheria clade containing elephant and aardvark. Results of current analyses and also those obtained 20 years ago on amino acid sequences confirm conclusions derived recently from larger data sets of other molecules. Several examples of recent gene duplications in ribonucleases 1 are discussed, with respect to illustrate the concepts of orthology and paralogy. Previously evidence was presented for extensive parallelism between sequence regions with attached carbohydrate (about one quarter of the molecule) of unrelated species with cecal digestion (pig and guinea pig). These features are also present in the sequences of elephant and fruit bat, species with cecal digestion, but with a very low ribonuclease content in their pancreas.     

cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.     

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.     

Molecular evolution of the mammalian prion protein

Prion protein (PrP) sequences are until now available for only six of the 18 orders of placental mammals. A broader comparison of mammalian prions might help to understand the enigmatic functional and pathogenic properties of this protein. We therefore determined PrP coding sequences in 26 mammalian species to include all placental orders and major subordinal groups. Glycosylation sites, cysteines forming a disulfide bridge, and a hydrophobic transmembrane region are perfectly conserved. Also, the sequences responsible for secondary structure elements, for N- and C-terminal processing of the precursor protein, and for attachment of the glycosyl-phosphatidylinositol membrane anchor are well conserved. The N-terminal region of PrP generally contains five or six repeats of the sequence P(Q/H)GGG(G/-)WGQ, but alleles with two, four, and seven repeats were observed in some species. This suggests, together with the pattern of amino acid replacements in these repeats, the regular occurrence of repeat expansion and contraction. Histidines implicated in copper ion binding and a proline involved in 4-hydroxylation are lacking in some species, which questions their importance for normal functioning of cellular PrP. The finding in certain species of two or seven repeats, and of amino acid substitutions that have been related to human prion diseases, challenges the relevance of such mutations for prion pathology. The gene tree deduced from the PrP sequences largely agrees with the species tree, indicating that no major deviations occurred in the evolution of the prion gene in different placental lineages. In one species, the anteater, a prion pseudogene was present in addition to the active gene.     

The major piscine liver alcohol dehydrogenase has class-mixed properties in relation to mammalian alcohol dehydrogenases of classes I and III.

The major alcohol dehydrogenase of cod liver has been purified, enzymatically characterized, and structurally analyzed in order to establish original functions and relationships among the deviating classes of the enzyme in mammalian tissues. Interestingly, the cod enzyme exhibits mixed properties--many positional identities with a class III protein, but functionally a class I enzyme--blurring the distinction among the classes of alcohol dehydrogenase. The two domain interfaces, affected by movements upon coenzyme binding, both exhibit substitutions in a manner thus far unique to the cod enzyme. In contrast, coenzyme-binding residues are highly conserved. At the active site, inner and outer parts of the substrate pocket show different extents of amino acid replacement. In total, no less than 7-10 residues of 11 in the substrate binding pocket differ from those of all the mammalian classes, explaining the substrate specificities. However, the inner part of the substrate pocket is very similar to that of the class I enzymes, which is compatible with the observed characteristics of the cod enzyme: ethanol is an excellent substrate (Km = 1.2 mM) and 4-methylpyrazole is a strong inhibitor (Ki = 0.1 microM). These values are about as low as those typical for the ethanol-active class I mammalian enzyme and do not at all resemble those for class III, for which ethanol is hardly a substrate and pyrazole is hardly an inhibitor. Further out in the substrate pocket, several residues differ from the mammalian classes, affecting large substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genotyping of human class I alcohol dehydrogenase. Analysis of enzymatically amplified DNA with allele-specific oligonucleotides

Large inter-individual differences are noted in the susceptibility to alcohol-related problems. Part of this variation may be due to the different isoenzyme patterns of the alcohol-metabolizing enzymes and, consequently, different pharmacokinetics of alcohol degradation. We have used the polymerase chain reaction and oligonucleotide hybridization to amplify and analyze class I alcohol dehydrogenase isoenzyme-specific genomic DNA. The method unambiguously distinguishes between different allelic variants and thus provides a new means of elucidating the alcohol dehydrogenase isoenzyme pattern of humans.