Purification and molecular properties of mouse alcohol dehydrogenase isozymes

Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues have been purified to homogeneity using triazine-dye affinity chromatography. The enzymes are dimers with similar but distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: A, 43000; B, 39000, and C, 47000. Zinc analyses and 1,10-phenanthroline inhibition studies indicated that the A and C subunits each contained two atoms of zinc, with at least one being involved catalytically, whereas the B subunit probably contained a single non-catalytic zinc atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length; C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. In contrast, B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at very high concentrations (greater than 0.5 M). The isozyme exhibited low Km and high Vmax values, however, with medium-chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirm genetic analyses [Holmes, Albanese, Whitehead and Duley (1981) J. Exp. Zool. 215, 151-157] which are consistent with at least three structural genes encoding alcohol dehydrogenase in the mouse and confirm the role of the major liver isozyme (A2) in ethanol metabolism.     

A novel ancestral protein ofDrosophila alcohol dehydrogenase inStreptomyces?

Polyclonal antibodies raised against purifiedDrosophila alcohol dehydrogenase (ADH) were used in Western blot analyses to search for structurally and/or immunologically related proteins in prokaryotes and eukaryotes. No immunological-reactive protein was detected in a flesh fly, a locust, and butterflies. Immunological similarity with the 50-kDa PQQ-glucose dehydrogenase (GluDH)-B enzyme ofAcinetobacter calcoaceticus was found, but the cross-reactivity apparently is dependent on the high hydrophilic character of this protein. Antibodies against PQQ-GluDH did not recognizeDrosophila ADH. In five of seven species of the gram-positive soil bacteria actinomycetes tested, a protein approximately 28–30 kDa in subunit size was strongly recognized by α-DADH. It is probably not one of the two proteins with known homology toDrosophila ADH,viz., theactIII gene product and 20β-hydroxysteroid dehydrogenase. The protein is present in both the soluble and the pellet-membrane fraction of the cells. The protein has a late temporal expression in surface-grown cultures and, therefore, might be involved in secondary metabolism.     

The quinohaemoprotein alcohol dehydrogenase from Gluconacetobacter xylinus: molecular and catalytic properties

Gluconacetobacter xylinus possesses a constitutive membrane-bound oxidase system for the use of ethanol. Its alcohol dehydrogenase complex (ADH) was purified to homogeneity and characterized. It is a 119-kDa heterodimer (68 and 41 kDa subunits). The peroxidase reaction confirmed the presence of haem C in both subunits. Four cytochromes c per enzyme were determined by pyridine hemochrome spectroscopy. Redox titrations of the purified ADH revealed the presence of four haem c redox centers, with apparent mid-point potential values (Em₇) of −33, +55, +132 and +310 mV, respectively. The ADH complex contains one mol of pyrroloquinoline quinone as determined by HPLC. The enzyme was purified in full reduced state; oxidation was induced by potassium ferricyanide and substrate restores full reduction. Activity responses to pH were sharp, showing two distinct optimal pH values (i.e. pH 5.5 and 6.5) depending on the electron acceptor used. Purified ADH oxidizes primary alcohols (C2–C6) but not methanol. Noteworthy, aliphatic aldehydes (C1–C4) were also good substrates. Myxothiazol and antymicin A were powerful inhibitors of the purified ADH complex, most likely acting at the ubiquinone acceptor site in subunit II.     

Variation in the biochemical properties of the Drosophila alcohol dehydrogenase allozymes

Thirteen Drosophila Adh variants have been characterized with respect to gene expression, substrate preference, thermostability, and specific activity. The results suggest that the variants may be grouped into two biochemical classes, typified by the properties of the two most common enzyme forms, ADH-F and ADH-S. Membership of these classes cannot be predicted from electrophoretic mobility, nor is any simple classification possible with regard to the characteristics of level of gene expression (in terms of ADH activity or ADH protein) or thermostability of the gene product.     

The partial characterization of alcohol dehydrogenase null alleles from natural populations ofDrosophila melanogaster

Twenty-three alcohol dehydrogenase (ADH) putative null alleles extracted from four Tasmanian (Australia) populations of Drosophila melanogaster produce no ADH activity and are unable to form active heterodimers with either AdhF or AdhS. Twelve of these nulls were tested by enzyme-linked immunosorbent assay (ELISA) and did not produce any ADH cross-reacting material (CRM). The null homozygotes had similar, but slightly lower, mortalities on ethanol-supplemented media compared to an artificially induced null allele. Heterozygotes between the null alleles and standard AdhF and AdhS alleles had intermediate ADH activity and CRM levels.     

Recent origin for a thermostable alcohol dehydrogenase allele ofDrosophila melanogaster

Summary The nucleotide sequence of theFast-Chateau Douglas isolate of the thermostable alcohol dehydrogenase allele is compared with the sequences of theSlow andFast alleles ofDrosophila melanogaster. Conceptual translation of theFChD sequence indicates that the thermostable polypeptide has the diagnostic FAST amino acid replacement at residue 192 and an additional replacement of serine for proline at residue 214. This suggests aFast origin for the thermostableAdh allele. However, some of the biochemical properties of the FCHD protein resemble those of the SLOW rather than the FAST polypeptides. The serine for proline replacement confers upon the thermostable polypeptide substrate specificities and some kinetic parameters similar to the SLOW protein. The same replacement substitution within the third coding exon also appears to alter the ADH protein concentration to a level similar to the SLOW polypeptide and the probable effect is at the level of mRNA concentration. The low level of nucleotide sequence variation, other than that leading to the amino acid substitution, suggests a recent origin for the thermostable allele. The time since divergence of theFChD sequence fromFast is estimated to be approximately 260,000–470,000 years.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

Agarose-bound horse-liver alcohol dehydrogenase. Dependence of molecular properties and activity on coupling conditions.

1. Spectroscopic methods for protein and active-site determination with the same sample of immobilised horse liver alcohol dehydrogenase have been developed. 2. The influence of pH, active-site protection of the soluble enzyme and protein concentration on coupling of alcohol dehydrogenase with cyanogen-bromide-activated Sepharose has been investigated. In phosphate buffer (pH 8.0) products with over 90% active-site retention have been synthesized. The binary complex alcohol-dehydrogenase . NADH gives a preparation with the same active-site content but a lower apparent specific activity compared to the unprotected enzyme. Increase in protein concentration yields products with the same active-site content relative to bound protein but the apparent specific activity is decreased. 3. The great similarity in spectroscopic properties of soluble and immobilised enzyme, as well as of their ternary complexes, shows that no significant conformational change has taken place during immobilisation. 4. Exchange of the non-catalytic Zn2+ against Co2+ yields a hybrid Sepharose--Co2Zn2-alcohol-dehydrogenase with over 90% active-site retention during metal exchange. The absorption spectra of the soluble and immobilised hybrid are identical.     

Nonlatitudinal environmental correlations for alcohol dehydrogenase in Southern African populations ofDrosophila melanogaster

The relation between alcohol dehydrogenase (ADH) allozyme frequencies and several environmental variables, as well as latitude and longitude, was examined in 17 natural populations of Drosophila melanogaster in Namibia, South Africa, and Zimbabwe. Unlike several other studies, we found no latitudinal allele-frequency clines and no significant correlation with temperature. We found that the frequency of the most common allele (AdhS) was positively correlated with an increase in rainfall. We suggest that substrate-induced toxicity may explain the geographic variability in our results.     

Molecular consequences of two formaldehyde-induced mutations in the alcohol dehydrogenase gene ofDrosophila melanogaster

Adhfn23 and Adhfn24 are two formaldehyde-induced, homozygous-viable, alcohol dehydrogenase-null mutants that bear lesions in the gene that codes for the alcohol dehydrogenase (ADH; EC 1.1.1.1) of Drosophila melanogaster. Adhfn23 contains a 34-base pair deletion in the C-terminal coding region of the alcohol dehydrogenase structural gene. By immunological and molecular analysis, we show that the deletion shifts the translation reading frame and results in a prematurely truncated polypeptide product (10 amino acids shorter than wild type) that cross-reacts with antibody raised against ADH. The steady-state level of alcohol dehydrogenase mRNA present in this mutant is close (97%) to that in the wild type, but the steady-state level of alcohol dehydrogenase-like protein is 50% lower. Moreover, the rate of alcohol dehydrogenase synthesis in Adhfn23 flies is reduced to 60% of that found in the wild type. Hence both the rate of synthesis and the rate of degradation of alcohol dehydrogenase are affected. In contrast, Adhfn24 which contains an 11-base pair deletion in the N-terminal coding region of the ADH gene, synthesizes no immunodetectable protein, and the amount of alcohol dehydrogenase mRNA is less than half that of wild-type flies. As with Adhfn23, the deletion in Adhfn24 results in a change in the reading frame. Unlike Adhfn23, however, nucleic acid sequence data indicate that polypeptide chain elongation can proceed for a considerable distance (over 130 amino acids) beyond the deletion. Based upon antigenic binding-site predictions, the resultant aberrant protein (projected 195 amino acids in length) would share few antigenic sites with the alcohol dehydrogenase from the wild type, which may account for the lack of immunoprecipitable material in this mutant. The contrasting effects these two deletions have on the Drosophila ADH mRNA levels and ADH protein levels are discussed.     

Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria.     

The purification and biochemical properties of alcohol dehydrogenase—“Fast (chateau douglas)” from Drosophila melanogaster

Alcohol dehydrogenase has been purified from Drosophila melanogaster lines bearing the Adh ^F, Adh^S, and Adh ^FCh.D. alleles. Biochemical investigations show that the properties of the purified enzymes are very similar to those of crude enzyme extracts except that the pure enzymes are more heat stable. ADH-FCh.D. resembles ADH-S very closely in specific activity, substrate specificity, and a number of kinetic parameters including limiting values for K _m(app.) for ethanol. However, it is considerably more heat stable than either of the two common variants. ADH-F differs from ADH-S and ADH-FCh.D. particularly with regard to the rate of oxidation of secondary alcohols. Atomic absorbtion spectroscopy shows that all three allozymes lack zine or other divalent cations as active-site components. Peptide mapping experiments identify one very active cysteinyl residue; and amide residues in the NAD⁺ binding domain.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

Catalytic and molecular properties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha strain Bo

The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent k(cat)/K(m) and K(i) values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k(cat)/K(m) value of 788 x 10(4) M(-1) s(-1). The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.     

The effect of temperature and genetic background on the phenotypic expression of severalVestigial strains ofDrosophila melanogaster

The eightvestigial strains,vg, vg-co-iso,vg;se-co-iso,vg-ms-1-co-iso,vg-ms-1; 3-co-iso,vg-ms-co-iso,vg-ms;se andvg-ms;se-co-iso showed various temperature responses. The five strains of thevg-ms group showed a greater response to temperature than did the three strains of thevg group. This tendency became more pronounced the higher the temperature was. The difference in temperature response between thevg-ms-1; 3-co-iso andvg-ms-co-iso strains indicates that the phenotypic expression ofvestigial is influenced by a modifier or modifiers located on the second chromosome of the Oregon(iso) strain. It was found that the X chromosome of the Oregon(iso) strain showed a slight modifier action in females but not in males of thevg-ms mutant.These gene in thevg-ms;se strain seemed to enhance the sensitivity to heat, and to inhibit the emergence of adult flies from pupal cases at 30°C when combined with thevg-ms gene, but no interaction was seen between these gene and thevg gene. From the results of this experiment, it is assumed that thevg-ms mutant either has a new recessive allele of thevg gene, or a modifier gene(s) closely linked tovg.This work forms part of a thesis for the doctorate of Kyoto University.     

Effect of environmental alcohol on in Vivo properties of Drosophila alcohol dehydrogenase

The effects of environmental 2-propanol on the in vivo properties of Drosophila alcohol dehydrogenase (E.C. 1.1.1.1.) are presented. Exposed flies were found to exhibit a significant decrease in ADH specific activity with a concomitant increase in the enzyme's relative in vivo stability and concentration. The possible adaptive significance of the observed responses is discussed.This work was supported by NSF grant #DEB 7815466 to J.M. Journal Paper No. J-9979 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2272.     

Changes in native alcohol dehydrogenase activity ofDrosophila upon treatment with guanidine hydrochloride,urea and heat

Fifty-two isochromosomal lines ofDrosophila melanogaster were examined for the existence of additional genetic variations in ADH activity subsequent to treatment With guanidine hydrochloride, urea and heat. A wealth of hidden variation was discovered among and within the Mexican populations of the insect after treatment with the denaturants. Dedicated to the late Dr. Sarah B. Pipkin of Howard University.     

The effect of aqueous surfactant solutions on alcohol dehydrogenase (LADH)

Alcohol dehydrogenase (LADH) was studied in aqueous solutions of surfactants to determine its structural and catalytic characteristics. Fluorescence, circular dichroism (CD), and electron paramagnetic resonance (ERP) techniques were used to study structural changes to the enzyme. The activity of LADH in catalyzing the oxidation of ethanol was investigated. Short-chain alkyl sulfonates and sulfates did not deactivate LADH or alter its structure. Longer and branched alkyl sulfates and sulfonates, as well as a cationic surfactant (CTAB), affected both LADH activity and conformation. (c) 1993 John Wiley & Sons, Inc.     

The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol. The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM. Pyrazole notably inhibited the enzymatic activity. The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature.