New human liver alcohol dehydrogenase forms with unique kinetic characteristics

All adult and infant human liver homogenates studied thus far show two previously unreported forms of alcohol dehydrogenase on starch gel electrophoresis. Under the conditions employed, these forms migrate toward the anode and readily stain for pentanol but virtually not for ethanol oxidizing activity. In contrast, all human ADH isoenzymes identified previously are cathodic and react equally well with either substrate. These new ADH forms have been separated from the other known ones by DEAE-cellulose chromatography and are then purified on Agarose-hexane-AMP. Although the physical characteristics of the new anodic ADH forms are similar to those of the known human ADH isoenzymes, the former are not inhibited by 12 mM 4-methyl pyrazole, oxidize ethanol very poorly and appear to prefer longer chain alcohols as substrates.     

χ alcohol dehydrogenase isozymes of horse liver

Horse liver contains previously unrecognized isozymes of alcohol dehydrogenase. In contrast to the molecular forms identified up to now, under the conditions employed these variants migrate toward the anode on starch gel electrophoresis and were separated from the cathodic isozymes by DEAE-cellulose chromatography. They were then purified on agarose-hexane-AMP. Their physicochemical and compositional characteristics are similar to those x alcohol dehydrogenases from human liver. Like these and similar ones from simian liver, they retain most of their activity in the presence of10 mm 4-methylpyrazole, oxidize short-chain primary alcohols very poorly, and appear to prefer longer chain primary alcohols and ω-hydroxy fatty acids as substrates.     

Organ-specific human alcohol dehydrogenase: isolation and characterization of isozymes from testis

Class III alcohol dehydrogenase (ADH) predominates in human testis. The two isozymes of this class were isolated jointly by affinity and conventional ion exchange chromatography. They display anodic electrophoretic mobility at pH 8.2, are completely insensitive to 4-methylpyrazole inhibition and oxidize ethanol and other short-chain primary alcohols very poorly. Thus, their kinetic and inhibition characteristics are identical to human liver class III ADH. In contrast, class I ADH is a barely detectable component of testicular alcohol dehydrogenase. The physicochemical characteristics of class III ADH are virtually identical to those of alcohol dehydrogenases found in other organs.     

Redox potential dependence of photophosphorylation and electron transfer in continuous illumination of Rhodopseudomonas sphaeroides chromatophores

The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.     

Isolation and characterization of shrew (Suncus murinus) liver alcohol dehydrogenases

1. Starch gel electrophoresis of adult shrew (Suncus murinus) liver extracts revealed five forms of alcohol dehydrogenase (ADH 1-5) and four of them were purified. 2. ADH-4 and ADH-5 resemble human class I ADH in terms of electrophoretic mobility, substrate specificity and sensitivity to pyrazole inhibition. 3. ADH-2 does not belong to any of the three classes of human ADHs but rather with catalytic properties similar to those of the class B ADH found in guinea pig liver. 4. ADH-1 prefers secondary alcohol over primary alcohol substrates and between the enantiomers tested, the enzyme favors the S isomers.     

Characterization and kinetics of native and chemically acitvated human liver alcohol dehydrogenases.

Acetimidylation of the amino groups of alcohol dehydrogenase from human and horse liver yields several modified enzyme forms, which differ in electrophoretic mobility and can be separated by ion exchange chromatography, but which are similar in kinetic characteristics. The acetimidylated, as well as the methylated, enzymes from human livers of the normal phenotype have increased activity and larger Michaelis and inhibition constants. These results suggest that the human enzyme has amino groups at the active sites, as was shown previously for the horse enzyme. The variant subunit occuring in the enzyme isolated from atypical human livers does not seem to be activated by acetimidylation, which may indicate that substitution of proline for Ala-230 or modifiction of Lys-228 is sufficient to fully activate the enzyme. Results of product inhibition studies of native and modified human enzymes are consistent with an Ordered Bi Bi mechanism. However, the major isoenzyme of native human liver alcohol, dehydrogenase exhibits nonlinear kinetics over a wide range of ethanol concentrations. This result may indicate that subunits with different kinetic characteristics are present or that there is negative cooperativity between subunits. After chemical modification, the kinetic patterns become linear, suggesting that the mechanism is altered.     

Purification and characterization of a new alcohol dehydrogenase from human stomach

Starch gel electrophoresis of homogenates from human stomach mucosa resolves three alcohol dehydrogenase (ADH) forms: the anodic chi-ADH (class III), the cathodic gamma-ADH (class I), and a new form of slow cathodic mobility that has not been previously characterized. In this work, we describe the purification in three chromatographic steps and the physical and kinetic characterization of this new human alcohol dehydrogenase, which we have named sigma-ADH. The enzyme exhibits the general physicochemical features (Mr, zinc content, subunit Mr, cofactor preference) of all mammalian alcohol dehydrogenases. The kinetic studies show a high Km value (41 mM) and a high kcat value (280 min-1) for ethanol at pH 7.5. The Km decreases as the alcohol increases its chain length. The aldehydes are better substrates than the corresponding alcohols, with m-nitrobenzaldehyde being the best substrate examined. sigma-ADH is strongly inhibited by 4-methylpyrazole, but with a Ki (10 microM) still higher than that for a class I isoenzyme. These properties suggest that sigma-ADH is a class II isoenzyme, different from pi-ADH and similar to that previously described by us in rat stomach. At the high ethanol concentrations in stomach after drinking, sigma-ADH is probably the ADH form with the largest contribution to human gastric ethanol metabolism.     

Isolation and characterization of an anodic form of human liver alcohol dehydrogenase

A form of human liver alcohol dehydrogenase previously identified on starch gel electrophoresis as the anodic band (Li, T.-K. and Magnes, L.J. Biochem. Biophys. Res. Commun. 63, 202, 1975) has now been separated from the other molecular forms of the enzyme by affinity chromatography on 4-[3-(N-6-aminocaproyl)-aminopropyl]-pyrazole-Sepharose and purified to homogeneity on Agarose-hexane-AMP. Its physical properties are similar to those of other molecular forms already known, suggesting that they may be related. In contrast to other forms, the anodic species is inactive towards methanol, and its K_M for ethanol is as much as 100 times that of the other forms. This anodic form of alcohol dehydrogenase may contribute significantly to alcohol elimination in man, particularly at high alcohol concentrations when the other enzyme species are saturated.     

Kinetic and mechanistic studies of methylated liver alcohol dehydrogenase.

Reductive methylation of lysine residues activates liver alcohol dehydrogenase in the oxidation of primary alcohols, but decreases the activity of the enzyme towards secondary alcohols. The modification also desensitizes the dehydrogenase to substrate inhibition at high alcohol concentrations. Steady-state kinetic studies of methylated liver alcohol dehydrogenase over a wide range of alcohol concentrations suggest that alcohol oxidation proceeds via a random addition of coenzyme and substrate with a pathway for the formation of the productive enzyme-NADH-alcohol complex. To facilitate the analyses of the effects of methylation on liver alcohol dehydrogenase and factors affecting them, new operational kinetic parameters to describe the results at high substrate concentration were introduced. The changes in the dehydrogenase activity on alkylation were found to be associated with changes in the maximum velocities that are affected by the hydrophobicity of alkyl groups introduced at lysine residues. The desensitization of alkylated liver alcohol dehydrogenase to substrate inhibition is identified with a decrease in inhibitory Michaelis constants for alcohols and this is favoured by the steric effects of substituents at the lysine residues.     

Amphibian alcohol dehydrogenase, the major frog liver enzyme. Relationships to other forms and assessment of an early gene duplication separating vertebrate class I and class III alcohol dehydrogenases

Submammalian alcohol dehydrogenase structures can be used to evaluate the origins and functions of the different types of the mammalian enzyme. Two avian forms were recently reported, and we now define the major amphibian alcohol dehydrogenase. The enzyme from the liver of the Green frog Rana perezi was purified, carboxymethylated, and submitted to amino acid sequence determination by peptide analysis of six different digests. The protein has a 375-residue subunit and is a class I alcohol dehydrogenase, bridging the gap toward the original separation of the classes that are observable in the human alcohol dehydrogenase system. In relation to the human class I enzyme, the amphibian protein has residue identities exactly halfway (68%) between those for the corresponding avian enzyme (74%) and the human class III enzyme (62%), suggesting an origin of the alcohol dehydrogenase classes very early in or close to the evolution of the vertebrate line. This conclusion suggests that these enzyme classes are more universal among animals than previously realized and constitutes the first real assessment of the origin of the duplications leading to the alcohol dehydrogenase classes. Functionally, the amphibian enzyme exhibits properties typical for class I but has an unusually low Km for ethanol (0.09 mM) and Ki for pyrazole (0.15 microM) at pH 10.0. This correlates with a strictly hydrophobic substrate pocket and one amino acid difference toward the human class I enzyme at the inner part of the pocket. Coenzyme binding is highly similar, while subunit-interacting residues, as in other alcohol dehydrogenases, exhibit several differences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of yeast alcohol dehydrogenase with protoberberine alkaloids

Oxidation of ethanol and reduction of aldehyde catalysed by yeast alcohol dehydrogenase is inhibited by several naturally occurring as well as semi-synthetic protoberberine alkaloids. The affinity of these compounds for the enzyme depends essentially on their hydrophobicity. Corysamine and coptisine are the most potent inhibitors among the natural alkaloids of this group. The kinetics of yeast alcohol dehydrogenase inhibition with coptisine were analysed and equilibrium measurements using optical methods were carried out. The results suggest that the binding site of the enzyme for protoberberines is not identical with those for coenzyme and substrate though it should be located near the nicotinamide ring of bound NAD. The binding of protoberberines seems to be limited to rather superficially located hydrophobic groups in the vicinity of the active site of the enzyme. The inability of these alkaloids to protrude deeply into the molecule of yeast alcohol dehydrogenase at the catalytically important region is the main difference in their behaviour towards alcohol dehydrogenases from yeast and horse liver.     

Simian liver alcohol dehydrogenase: isolation and characterization of isozymes from Macaca nemestrina

Three classes of hepatic alcohol dehydrogenase (ADH), analogous to those of human liver, are present in Macaca nemestrina. Their functional, compositional, and structural features have been established with isozymes purified to homogeneity by affinity and conventional ion-exchange chromatography. One unusual molecular form of M. nemestrina ADH is electrophoretically indistinguishable as it comigrates with one of the cathodic class I isozymes on starch gel electrophoresis. While its substrate and inhibitor specificity, a high Km value for ethanol (50 mM at pH 10), and lack of binding to the pyrazole affinity resin are consistent with the kinetics of class II ADH, the physiochemical and compositional properties are virtually identical with all other known mammalian alcohol dehydrogenases. The unexpected presence of this previously unknown ADH variant in livers of M. nemestrina demonstrates the need for prudence in assignment of ADH isozymes. Classification based solely on electrophoretic position in starch gels and enzymatic properties of human ADH but without isolation and characterization of individual isozymes may prove insufficient and inadequate. The genetic or phenotypic nature of this isozyme remains to be demonstrated.     

Active-site-specific zinc-depleted and reconstituted cobalt(II) human-liver alcohol dehydrogenase. Preparation, characterization and complexation with NADH and trans-4-(N,N-dimethylamino)-cinnamaldehyde

The active-site zinc atom of the beta 1 beta 1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From beta 1 gamma 1 and gamma 1 gamma 1 isozyme the active-site zinc is extracted much more slowly than from beta 1 beta 1 isozyme. Only partially active-site metal-depleted enzyme species were obtained from these isozymes. The active-site-specific reconstituted cobalt(II) derivative of the beta 1 beta 1 isozyme shows spectroscopic properties comparable to those of the active-site-specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme-induced conformational change of the protein leads to a red shift of the d-d band from 648 nm to 673 nm. The chromophoric substrate trans-4-(N,N-dimethylamino)-cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between beta 1 and gamma 1 subunits (threonine-48 instead of serine-48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal-bound chromophoric substrate.     

Inhibition by ethanol, acetaldehyde and trifluoroethanol of reactions catalysed by yeast and horse liver alcohol dehydrogenases.

1. Produced inhibition by ethanol of the acetaldehyde-NADH reaction, catalysed by the alcohol dehydrogenases from yeast and horse liver, was studied at 25 degrees C and pH 6-9. 2. The results with yeast alcohol dehydrogenase are generally consistent with the preferred-pathway mechanism proposed previously [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. The observed hyperbolic inhibition by ethanol of the maximum rate of acetaldehyde reduction confirms the existence of the alternative pathway involving an enzyme-ethanol complex. 3. The maximum rate of acetaldehyde reduction with horse liver alcohol dehydrogenase is also subject to hyperbolic inhibition by ethanol. 4. The measured inhibition constants for ethanol provide some of the information required in the determination of the dissociation constant for ethanol from the active ternary complex. 5. Product inhibition by acetaldehyde of the ethanol-NAD+ reaction with yeast alcohol dehydrogenase was examined briefly. The results are consistent with the proposed mechanism. However, the nature of the inhibition of the maximum rate cannot be determined within the accessible range of experimental conditions. 6. Inhibition of yeast alcohol dehydrogenase by trifluoroethanol was studied at 25 degrees C and pH 6-10. The inhibition was competitive with respect to ethanol in the ethanol-NAD+ reaction. Estimates were made of the dissociation constant for trifluoroethanol from the enzyme-NAD+-trifluoroethanol complex in the range pH6-10.     

Inactivation of alcohol and retinol dehydrogenases by acetaldehyde and formaldehyde.

The rapid and progressive inactivation of alcohol dehydrogenase from horse liver, rat liver and from human retina and of retinol dehydrogenase of rat liver by low concentrations of acetaldehyde or formaldehyde is illustrated. The inactivation of alcohol dehydrogenase can be largely prevented and partially reversed with glutathione. These findings are discussed as a model for better understanding of toxic effects of alcohol and in the context of the importance of protein turnover measurements for clarification of untoward alcohol effects.     

Properties of 3-aldoxime pyridine adenine dinucleotide, an NAD analogue

The NAD⁺ analogue, 3-aldoxime pyridine adenine dinucleotide, is prepared by transglycosidation. Contrary to the published data, this analogue shows no activity as coenzyme with alcohol dehydrogenase from horse liver or from yeast. This is demonstrated by three methods: no increase of absorption at 331 nm by the enzymic oxidation of ethanol; no increase at 290 nm with cinnamic alcohol; and no exchange reaction. The inhibition by this analogue of the oxidation of ethanol by NAD⁺ is competitive at pH 7.6 and 9.5 with yeast alcohol dehydrogenase; with liver alcohol dehydrogenase, it is of the mixed type at pH 7.6 and non-competitive at pH 9.5. The lack of activity of the analogue and inhibition of the competitive or mixed type may be explained by the fact that the binary complex does not bind the substrate or that in the ternary complex the hydride shift does not occur. The non-competitive inhibition at pH 9.5 with the horse liver alcohol dehydrogenase may be explained by the existence of binding sites specific for this analogue.     

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.     

A chemical and enzymological account of the multiple forms of human liver aldehyde dehydrogenase. Implications for ethnic differences in alcohol metabolism

Three major low-pI zones of aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) may be visualized with specific histochemical staining after starch gel electrophoresis at pH 7.4 of Caucasian human liver extracts, whereas about 50% of Chinese human liver extracts show only two such zones. The three zones of activity were purified to apparent homogeneity from Caucasian liver. The substrate specificity of each form was investigated by double reciprocal plots using 13 aldehydes of various chemistries. The acetaldehyde-preferring isozyme I lacking in 50% of Chinese livers had a slightly lower native and subunit molecular weight than the "universal' isozymes IIa and IIb. All forms were highly sensitive to disulfiram inhibition. This inhibition could be protected against, or reversed, by dithiothreitol. 2,2'-Dithiodipyridine was a slower inhibitor of isoenzyme I. All three purified forms of the enzyme, as well as crude extracts of normal and isozyme I-deficient Chinese livers, showed positive immunoreactivity to antibodies prepared in rabbits against type I enzyme. Tryptic peptide maps of forms IIa and IIb were almost identical, whereas that of form I, although showing some similarities, was clearly different. These results provide a consistent explanation for the acetaldehyde-mediated extreme sensitivity to moderate alcohol ingestion shown normally by about 50% of oriental subjects and during disulfiram (Antabuse) therapy by all subjects.     

Intrahepatic distribution of human glutathione-dependent formaldehyde dehydrogenase

By means of a microelectrophoretic separation technique the different forms of alcohol dehydrogenase can be detected in microdissected liver tissue samples of the nanogram range. Alcohol dehydrogenase 3 (the glutathione-dependent formaldehyde dehydrogenase) was demonstrated to be zonally distributed in the human liver parenchyma. A periportal/perivenous gradient is evident in both sexes, however, the periportal/perivenous ratio is higher in males. The biological functions of this enzymatic form and the possible role of the periportal maximum are discussed.