Alcohol Dehydrogenase Mutants of Chinese Hamster Somatic Cells Resistant to Allyl Alcohol

Alcohol dehydrogenase (alcohol: NAD oxidoreductase, E.C. 1.1.1.1.) mutants of Chinese hamster somatic cells were isolated as resistant to allyl alcohol (ALL^R). The ALL^R phenotypes of the mutant clones were reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Several mutants, Adh-1, Adh-2, Adh-9 and Adh-13, resistant to allyl alcohol were characterized. They have between 15 and 40% of the alcohol dehydrogenase activity of the wild-type cell lines. Cell-cell hybridization experiments using Adh-1 and wild-type Chinese hamster cells indicate that resistance to allyl alcohol is recessive to the wild-type allele. This phenotype is therefore a useful marker to analyze gene segregation of somatic cell mutations and to study the expression of the genes involved in the metabolism of ethanol in mammalian cells.     

The Action of the Notch Locus in DROSOPHILA MELANOGASTER. II. Biochemical Effects of Recessive Lethals on Mitochondrial Enzymes

We show that six mapped recessive lethal point mutations of the Notch locus affect mitochondrial enzyme activities: NADH oxidase, NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. The mutant N^264-40, which has the same morphological and embryological effects as the Notch⁸ deletion, demonstrates the same biochemical effects and dosage relations as Notch⁸. The other five mapped recessive lethals also affect four enzymic activities. They show specific patterns of activity that depend in several cases on the wild-type chromosome in the heterozygous females. That effect occurs with mutants located in the extreme right part of the Notch locus where some mutations, according to other authors, show temperature-sensitive expression.     

Purification and partial characterization of alcohol dehydrogenase from wheat.

Further support for hypotheses proposed earlier for the genetic control and subunit composition of the alcohol dehydrogenase of Triticum has been obtained through the purification and partial characterization of the enzyme. The alcohol dehydrogenase of the wheat T. monococcum was purified 103-fold to a specific activity of 55,900 units/mg. Purification was achieved using streptomycin sulfate precipitation, gel filtration chromatography, DEAE-cellulose anion-exchange chromatography, and preparative isoelectric focusing. The native enzyme has a molecular weight of 116,000 and a dimeric subunit structure. The apparent Michaelis constants are 1.2 × 10^?2m for ethanol and 1 × 10^?4m for NAD. The substrate specificity of wheat alcohol dehydrogenase differs significantly from the substrate specificities of the enzymes of horse and yeast.     

Cholinesterase activity in some species of theAllium genus

In partly purified protein complexes obtained from 22 species of theAllium genus and 6 cultivars ofAllium cepa the activity of cholinesterases was detected and measured using the method of Ellman et al. The degree of its inhibition with 10^-4 M neostigmine was also tested. It was found that the activity of cholinesterase differed in individual species up to two hundred times, while the differences in the inhibitory activity of 10^-4 M neostigmine occurred only in a few cases. Individual sections and cultivars could not be characterized on the basis of the differences in the activities of the cholinesterases. Of all the sections that ofPhyllodolon shows the highest average activity. In the case of the tested cultivars distinctly the lowest activity was observed in cv. Kastická. The values of the enzymatic activity measured by Ellman’s method in this plant material include the activity of specific and unspecific cholinesterases and the part uninhibitable by neostigmine.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.     

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.     

Hepatic microsomal ethanol-oxidizing system: solubilization, isolation, and characterization

The solubilization and subsequent separation of the hepatic microsomal ethanol-oxidizing system from alcohol dehydrogenase and catalase activities by DEAE-cellulose column chromatography is described. Absence of alcohol dehydrogenase in the column eluates exhibiting microsomal ethanol-oxidizing system activity was demonstrated by the failure of NAD⁺ to promote ethanol oxidation at pH 9.6. Differentiation of the microsomal ethanol-oxidizing system from alcohol dehydrogenase was further shown by the apparent K_m for ethanol (7.2 mm, insensitivity of the microsomal ethanol-oxidizing system to the alcohol dehydrogenase inhibitor pyrazole (0.1 mm) and by the failure of added alcohol dehydrogenase to increase the ethanol oxidation. Absence of catalatic activity in these fractions was demonstrated by spectrophotometric and polarographic assay. Differentiation of the microsomal ethanol-oxidizing system from the peroxidatic activity of catalase was shown by the apparent K_m for oxygen (8.3 μm), insensitivity of the microsomal ethanol-oxidizing system to the catalase inhibitors azide and cyanide, and by the lack of a H₂O₂-generating system (glucose-glucose oxidase) to sustain ethanol oxidation in the eluates. The oxidation of ethanol to acetaldehyde by the alcohol dehydrogenase- and catalase-free fractions required NADPH and oxygen and was inhibited by CO. The column eluates showing microsomal ethanol-oxidizing system activity contained cytochrome P-450, NADPH-cytochrome c reductase, and phospholipids and also metabolized aminopyrine, benzphetamine, and aniline.     

Root and Nodule Enzymes of Ammonia Assimilation in Two Plant-Conditioned Symbiotically Ineffective Genotypes of Alfalfa (Medicago sativa L.)

Biochemical and physiological parameters associated with nitrogen metabolism were measured in nodules and roots of glasshouse-grown clones of two symbiotically ineffective alfalfa (Medicago sativa L.) genotypes supplied with either NO₃⁻ or NH₄⁺. Significant differences were observed between genotypes for nodule soluble protein concentrations and glutamine synthetase (GS) and glutamate synthase (GOGAT) specific activities, both in untreated controls and in response to applied N. Nodule soluble protein of both genotypes declined in response to applied N, while nodule GS, GOGAT, and glutamate dehydrogenase (GDH) specific activities either decreased or remained relatively constant. In contrast, no genotype differences were observed in roots for soluble protein concentrations and GS, GOGAT, and GDH specific activities, either in untreated controls or in response to applied N. Root soluble protein levels and GS and GOGAT specific activities of N-treated plants increased 2- to 4-fold within 4 days and then decreased between days 13 and 24. Root GDH specific activity of NH₄⁺-treated plants increased steadily throughout the experiment and was 50 times greater than root GS or GOGAT specific activities by day 24.     

Alcohol Dehydrogenase Activities of Wine Yeasts in Relation to Higher Alcohol Formation

Alcohol dehydrogenase activities were examined in cell-free extracts of 10 representative wine yeast strains having various productivities of higher alcohols (fusel oil). The amount of fusel alcohols (n-propanol, isobutanol, active pentanol, and isopentanol) produced by the different yeasts and the specific alcohol dehydrogenase activities with the corresponding alcohols as substrates were found to be significantly related. No such relationship was found for ethanol. The amounts of higher alcohols formed during vinification could be predicted from the specific activities of the alcohol dehydrogenases with high accuracy. The results suggest a close relationship between the control of the activities of alcohol dehydrogenase and the formation of fusel oil alcohols. Also, new procedures for the prediction of higher alcohol formation during alcoholic beverage fermentation are suggested.     

Orientation of chromatophores and spheroplast-derived membrane vesicles of Rhodopseudomonas sphaeroides: analysis by localization of enzyme activities.

Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown cells of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and l-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg²⁺-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg²⁺-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the l-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.     

Rapid estimation of the molecular weights of protein polypeptide chains using high-pressure liquid chromatography in 6 M guanidine hydrochloride.

Minute activities of alcohol dehydrogenase in blood serum can be determined from the fading of the yellow color of p-nitrosodimethylaniline in a recycling reaction. This aldehyde-like substrate is reduced enzymatically to a colorless compound in the presence of NADH which is generated by alcohol dehydrogenase from NAD and n-butanol. Using this method alcohol dehydrogenase activities can also be measured in other biological materials.     

Studies on soybean nodule senescence

Soybean Glycine max. L. Merr. nodule senescence was studied using the loss of acetylene reduction by intact tap root nodules as its indication. Tap root nodules from two varieties (Calland and Beeson) of field-grown soybeans were used. The specific activities of nitrogenase (micromoles/minute gram fresh weight of nodules) as measured by the acetylene reduction assay decreased abruptly between 58 to 65 and 68 to 75 days after planting the Beeson and Calland soybeans, respectively. Major changes were not detected in dry weight, total nitrogen, and leghemoglobin levels during the period when in vivo nitrogenase activity declined. Ammonium levels in the cytosol of nodules and poly-β-hydroxybutyrate increased moderately just prior to or coincidental with the loss of nitrogenase activity. Neither enzymes that have been postulated to be involved in ammonium assimilation nor NADP⁺-specific isocitrate dehydrogenase exhibited any large changes in specific activities during the initial period when nitrogenase activity declined.     

Purification and Characterization of an NAD+-Dependent XylB-Like Aryl Alcohol Dehydrogenase Identified in Acinetobacter baylyi ADP1

The gene xylB_ADP1 from Acinetobacter baylyi ADP1 (gene annotation number ACIAD1578), coding for a putative aryl alcohol dehydrogenase, was heterologously expressed in Escherichia coli BL21(DE3). The respective aryl alcohol dehydrogenase was purified by fast protein liquid chromatography to apparent electrophoretic homogeneity. The predicted molecular weight of 39,500 per subunit was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to the native M_w as determined by gel filtration, the enzyme forms dimers and therefore seems to be XylB related. The enzyme showed the highest activity at 40°C. For both the reduction and the oxidation reactions, the pH for optimum activity was 6.5. The enzyme was NADH dependent and able to reduce medium- to long-chain n-alkylaldehydes, methyl-branched aldehydes, and aromatic aldehydes, with benzaldehyde yielding the highest activity. The oxidation reaction with the corresponding alcohols showed only 2.2% of the reduction activity, with coniferyl alcohol yielding the highest activity. Maximum activities for the reduction and the oxidation reaction were 104.5 and 2.3 U mg⁻¹ of protein, respectively. The enzyme activity was affected by low concentrations of Ag⁺ and Hg²⁺ and high concentrations of Cu²⁺, Zn²⁺, and Fe²⁺. The gene xylB_ADP1 seems to be expressed constitutively and an involvement in coniferyl alcohol degradation is suggested. However, the enzyme is most probably not involved in the degradation of benzyl alcohol, anisalcohol, salicyl alcohol, vanillyl alcohol, cinnamyl alcohol, or aliphatic and isoprenoid alcohols.     

Allyl Alcohol Selection for Lower Alcohol Dehydrogenase Activity in Nicotiana plumbaginifolia Cultured Cells

One cell strain with stable tolerance to allyl alcohol (AA^r) was selected from 6 × 10⁸ suspension cultured Nicotiana plumbaginifolia Viviani cells. The selected strain contained one-half the alcohol dehydrogenase (ADH) activity of the wild type (NP) due to the loss of two of three bands of ADH activity seen on starch gels following electrophoresis of wild-type cell extracts. Anaerobic conditions, simulated by not shaking the suspension cultures, increased the ADH specific activity to more than 3-fold the initial level in both strains but did not change the number of activity bands or the relative levels of activity. The cell strain with decreased ADH activity lost viability more rapidly than the wild type under the anaerobic conditions. The AA^r cells were 10 times more tolerant to ethanol than the NP cells and were also somewhat more tolerant to acetaldehyde and antimycin A. The substrate specificities of the ADH enzymes from both strains were very similar. Further selection of AA^r cells with allyl alcohol produced strains with even lower ADH activity and selection under anaerobic conditions produced strains with increased ADH activity. Genetic studies indicate that the N. plumbaginifolia ADH activity bands arise from subunits produced by two nonallelic genes. This is the first example of the use of allyl alcohol to select for decreased ADH using cultured plant cells.     

Salmonella typhimurium Mutants with Altered Glutamate Dehydrogenase and Glutamate Synthase Activities

Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH₄)₂SO₄ as the nitrogen source (Asm⁻) had about one-fourth the normal glutamate synthase activity and one-half the glutamine synthetase activity. The asm mutations also prevented growth with alanine, arginine, or proline as nitrogen sources and conferred resistance to methionine sulfoximine. When a mutation (gdh-51) causing the loss of glutamate dehydrogenase activity was transferred into a strain with an asm-102 mutation, the resulting asm-102 gdh-51 mutant had a partial requirement for glutamate. A strain isolated as a complete glutamate auxotroph had a third mutation, in addition to the asm-102 gdh-51 lesions, that further decreased the glutamate synthase activities to 1/20 the normal level. Both the asm-102 and gdh-51 mutations were located on the S. typhimurium linkage map at sites distinct from those found for mutations causing similar phenotypes in Klebsiella aerogenes and Escherichia coli.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Correlation of Biological Activity and Reactor Performance in Biofiltration of Toluene with the Fungus Paecilomyces variotii CBS115145

A biofiltration system inoculated with the mold Paecilomyces variotii CBS115145 showed a toluene elimination capacity (EC) of around 250 g/m³ of biofilter/h, which was higher than the values usually reported for bacteria. P. variotii assimilated m- and p-cresols but not the o isomer. Initial toluene hydroxylation occurred both on the methyl group and through the p-cresol pathway. These results were corroborated by detecting benzyl alcohol, benzaldehyde, and p-cresol as volatile intermediates. In liquid cultures with toluene as a substrate, the activity of toluene oxygenase (TO) was 5.6 nmol of O₂/min/mg of biomass, and that of benzyl alcohol dehydrogenase was 16.2 nmol of NADH/min/mg of protein. Toluene biodegradation determined from the TO activity in the biofilter depended on the biomass distribution and the substrate concentration. The specific enzymatic activity decreased from 6.3 to 1.9 nmol of O₂/min/mg of biomass along the reactor. Good agreement was found between the EC calculated from the TO activity and the EC measured on the biofilter. The results were confirmed by short-time biofiltration experiments. Average EC measured in different biofiltration experiments and EC calculated from the TO activity showed a linear relation, suggesting that in the biofilters, EC was limited by biological reaction. As the enzymatic activities of P. variotii were similar to those reported for bacteria, the high performance of the fungal biofilters can possibly be explained by the increased transfer of the hydrophobic compounds, including oxygen, from the gas phase to the mycelia, overcoming the transfer problems associated with the flat bacterial biofilms.     

Structural and biochemical characterisation of a NAD+-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni²⁺-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni²⁺, activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD⁺/NADH, whereas no activity has been observed with NADP⁺/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn²⁺. The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni²⁺-dependent activity towards 1,3-propanediol.