Activity Of Horse Liver Alcohol Dehydrogenase Research Articles

Genetically Fused T4L Acts as a Shield in Covalent Enzyme Immobilisation Enhancing the Rescued Activity

Enzyme immobilisation is a common strategy to increase enzymes resistance and reusability in a variety of excellent ‘green’ applications. However, the interaction with the solid support often leads to diminished specific activity, especially when non-specific covalent binding to the carrier takes place which affects the delicate architecture of the enzyme. Here we developed a broadly applicable strategy where the T4-lysozyme (T4L) is genetically fused at the N-terminus of different enzymes and used as inert protein spacer which directly attaches to the carrier preventing shape distortion of the catalyst. Halomonas elongata aminotransferase (HEWT), Bacillus subtilis engineered esterase (BS2m), and horse liver alcohol dehydrogenase (HLADH) were used as model enzymes to elucidate the benefits of the spacer. While HEWT and HLADH activity and expression were diminished by the fused T4L, both enzymes retained almost quantitative activity after immobilisation. In the case of BS2m, the protective effect of the T4L effectively was important and led to up to 10-fold improvement in the rescued activity.

The Effect of Plant Compounds from Whisky Cask on Horse Liver Alcohol Dehydrogenase Activity

Aged whisky contains many plant derived compounds from oak wood cask, and the color of whisky gradually changes to deep amber. More than 500 kinds of compounds are known as minor components in aged whisky. The taste of whisky becomes smooth with aging, and we can sober up feeling good. However, detail effect of these plant derived components from whisky cask on alcohol metabolism has not been clarified. In this experiment, we investigated the relation between the structure of components and the rate of ethanol oxidation reaction by using 26 kinds of plant derived components from oak wood cask and its derivatives. All the compounds which we used have a benzene ring as a basic frame, and have a hydroxyl group, an aldehyde group, and a carboxyl group further. All 26 compounds from oak wood cask and its derivatives used in this experiment were inhibited the ethanol oxidation. Interestingly, the relative activity of compound with an aldehyde group (Benzaldehyde and its derivatives) inhibited most strongly to be 30~40%. However, the relative activity of Benzaldehyde with one hydroxyl group had about 70% of relative activity and it had only one hydroxyl group, it clarified that the inhibition effect becomes weaker. The inhibition effect of compounds with calboxyl group (Benzoic acid and its derivatives) was weak and showed 90% of re lative activity. However, in contrast to the case of Benzaldehyde, the inhabitation effect of Benzoic acid only with one hydroxyl group becomes strong. From these results, it is clarified that many compounds from oak wood cask inhibite the ethanol oxidation. But the mechanism of inhabitation is complex and the additivity of the functional group contribution is not applied simply.

Synthesis of fluorinated NAD as a soluble coenzyme for enzymatic chemistry in fluorous solvents and carbon dioxide

The synthesis of the coenzyme nicatinamide adenine dinucleotide (NAD) with an covalently attached fluorinated polymer is reported. The fluorinated NAD (FNAD) was rendered soluble in both fluorous solvents and liquid carbon dioxide due to the attachment of a perfluoropolyether. The enzyme horse liver alcohol dehydrogenase (HLADH) was active in catalyzing oxidation/reduction reactions using FNAD as a soluble coenzyme in a fluorous solvent, methoxynonafluorobutane (HFE), and liquid carbon dioxide. In both solvents, the activity of HLADH using FNAD was greater than the same molar amount of unmodified (insoluble) NAD, indicating that a soluble coenzyme results in more efficient reactions.

Prediction of the Remaining Activity of Horse Liver Alcohol Dehydrogenase after Exposure to Various Organic Solvents

Twenty solvents were selected from a principal component (PC) analysis of 103 organic solvents. The solvents were selected so as to obtain a set of test items that spanned a large range of molecular properties of the selected solvents. The activity of free and Celite-immobilized horse liver alcohol dehydrogenase (HLADH) obtained after 20 h exposure to these solvents were used to create a PLS model based on 14 solvent descriptors. A correlation coefficient of 0.997 between the predicted and the observed remaining activity was obtained.A slightly worse PLS model was obtained using only three solvent descriptors (log P, dielectric constant and melting point). A correlation coefficient of 0.957 between the predicted and the observed remaining activity was obtained. The latter model was used to predict the remaining activity of HLADH after its exposure to 93 organic solvents.

The role of lys-228 residue in horse liver alcohol dehydrogenase activity

Lys-228 in horse liver alcohol dehydrogenase isoenzyme E (HLADH-E) was mutated to glycineby site-directed mutagenesis. The specific activity of the mutant enzyme was increased about 4-fold and Michaelis constants for NAD+(Ka) and NADH (Kq) increased by about 350-and 50-fold, respectively. The wild-type enzyme and K228G mutant enzyme were treated with ethylacetimidate. Acetimidylation of the wild-type enzyme increased the activity about 10-fold, but the mutant enzyme was little affected. These results confirm that Lys-228 residue plays an important role in the activity of the enzyme through forming the hydrogen bond with adenosine ribose of NAD+.

Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex.

Long-chained 1-mercapto- n-alkanes as potent inhibitors toward liver alcohol dehydrogenase

Long-chained 1-mercapto- n-alkanes showed potent inhibitory effects on horse liver alcohol dehydrogenase (HLADH). The inhibitory effect of the thiols was enhanced by increasing the number of the alkyl carbon atoms up to 10–12 and steeply lowered by further increase in the carbon number. The HLADH activity was almost completely inhibited in competitive manner by an equivalent concentration of 1-mercapto- n-decane or - n-dodecane to that of the subunit of the dimeric zinc enzyme; inhibition constant Ki was 0.55 nM for the former. The present study strongly suggests that the thiols interact simultaneously with at least two sites of HLADH; the primary one could be the zinc atom in the active site of the enzyme, interacting with the sulfhydryl groups, and the other a hydrophobic binding site for their alkyl carbons.

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 > R-9 > R-4 > R-6 > R-10 > R-8 > R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD +, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 77–78, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.

Molecular associations

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 > R-9 > R-4 > R-6 > R-10 > R-8 > R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 77–78, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.