Human Liver Alcohol Dehydrogenase Research Articles

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.

A search for the Indianapolis-variant of human alcohol dehydrogenase in liver autposy samples from North Germany and Japan.

Human liver alcohol dehydrogenase (ADH) variants were screened in random autopsy specimens from 53 North German and 34 Japanese individuals. Based on pH-activity profile and electrophoretic pattern, only ADH2 and ADH3 variants were detected. In relatively fresh specimens, an "anodic band" or "pi-ADH" band was also observed. The recently reported new molecular forms collectively called "ADH Indianapolis" (Bosron et al. 1980) could not be demonstrated and therefore may be confined hitherto only to the American black population.

New molecular forms of human liver alcohol dehydrogenase: isolation and characterization of ADHIndianapolis.

The biochemical determinants of alcoholism and genetic correlates for the variability in man's response to alcohol have remained obscure until recently. The identification of genetically determined isoenzymes of alcohol dehydrogenase with different catalytic properties may bear importantly upon this problem. New molecular forms of human liver alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) have recently been identified in 16% of the liver specimens from an urban population from Indianapolis, Indiana [Bosron, W. F., Li, T.-K. & Vallee, B. L. (1979) Biochem. Biophys. Res. Commun. 91, 1549-1555]. The distinguishing features of these specimens were (i) they showed activity optima for ethanol oxidation at both pH 7.0 and 10.0 and (ii) they formed electrophoretic bands cathodic to the beta beta isoenzyme. From such livers, three new ADH forms have now been isolated, one of which has a single pH optimum at 7.0 and two of which have dual optima at pH 7.0 and 10.0. These new forms were designated ADHIndianapolis forms 1,2, and 3, respectively. They can be differentiated from previously described ADH isoenzymes, including the so-called "atypical" isoenzyme, by their electrophoretic mobility, pH optima, and Km for ethanol (approximately 60 mM at pH 7.5). Based upon the electrophoretic pattern of livers containing ADHIndianapolis and the mobility of the three isolated molecular forms, ADHIndianapolis may be the result of polymorphism at the ADH2 gene locus, which codes for the beta subunit.

Digitalis metabolism and human liver alcohol dehydrogenase.

Human liver alcohol dehydrogenase (alcohol: NAD" oxidoreductase, EC 1.1.1.1) catalyzes the oxidation of the 3 beta-OH group of digitoxigenin, digoxigenin, and gitoxigenin to their 3-keto derivatives, which have been characterized by high performance liquid chromatography and mass spectrometry. These studies have identified human liver alcohol dehydrogenase as the unknown NAD(H)-dependent liver enzyme specific for the free hydroxyl group at C3 of the cardiac genins; this hydroxyl is the critical site of the genins' enzymatic oxidation and concomitant pharmacological inactivation in humans. Several kinetic approaches have demonstrated that ethanol and the pharmacologically active components of the digitalis glycosides are oxidized with closely similar kcat/Km values at the same site on human liver alcohol dehydrogenase, for which they compete. Human liver alcohol dehydrogenase thereby becomes an important biochemical link in the metabolism, pharmacology, and toxicology of ethanol and these glycosides, structurally unrelated agents that are both used widely. Both the competition of ethanol with these cardiac sterols and the narrow margin of safety in the therapeutic use of digitalis derivatives would seem to place at increased risk those individuals who receive digitalis and simultaneously consume large amounts of ethanol or whose alcohol dehydrogenase function is impaired.

Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence.

We used phase fluorometry to investigate the wavelength dependence of the fluorescence lifetimes of N-acetyl-L-tryptophanamide (NATA) in solvents of varying viscosity and the lifetimes of tryptophan in human serum albumin, melittin, and liver alcohol dehydrogenase. In highly fluid solvents, and in completely vitrified solvents, the lifetime of NATA was constant across its emission spectrum. In viscous solvents, such as propylene glycol at -9 degrees C, the lifetimes of NATA increased across its emission spectrum, with the values being 3.3, 5.5, and 8.1 ns at 317, 344, and 400 nm, respectively. These wavelength-dependent lifetimes appear to be a result of reorientations of solvent dipoles around the excited state dipole moment of the indole moiety. For the three proteins investigated, the fluorescence lifetimes of tryptophan increased with increasing wavelength in a manner comparable to that observed for NATA in propylene glycol. These observations indicate that these protein matrices can reorientation around their tryptophan residues on the nanosecond timescale, and illustrate the potential of phase fluorometry for quantifying the details of these dipolar relaxation processes.

In vivo evaluation of ambivalent active-site-directed inactivators of liver alcohol dehydrogenase.

In order to decrease the rate of ethanol metabolism for the treatment of acute and chronic alcoholism it would be useful to inhibit liver alcohol dehydrogenase in vivo. Based on a knowledge of the three-dimensional structure of the horse enzyme, we designed active-site-directed inactivators [p-(XCH2CONH)C6H4(CH2)3COHN2] which bind to the enzyme-NAD or enzyme-NADH complex and alkylate methionine residue 306. In vitro, these reagents inactivated mouse, rat, horse and human liver alcohol dehydrogenases faster in the presence than in the absence of NAD or NADH, but with slightly different specificity. Mice and rats pretreated with the reagents eliminated ethanol in blood more slowly than those not treated, and the specific activity of alcohol dehydrogenase in liver homogenates of treated animals was decreased. It appears that the design of active-site-directed reagents is feasible, but these reagents must be improved so that they are more efficacious in vivo.

Human liver alcohol dehydrogenase an enzyme essential to the metabolism of digitalis

Human liver alcohol dehydrogenase oxidizes the 3β OH group of digitoxigenin, digoxigenin and gitoxigenin, the pharmacologically active principles of the corresponding cardiac glycosides. The oxidation products were identified by high performance liquid chromatography analysis. Ethanol and digitoxigenin are competitive, and the efficiency of their oxidation is virtually the same. Thus, liver alcohol dehydrogenase is a hitherto unknown NAD(H) dependent enzyme that performs the first and major step in the inactivation of these genins of the cardiac glycosides in the human. This could bear importantly both on the pharmacology and toxicology of digitalis therapy.

Heterogeneity and new molecular forms of human liver alcohol dehydrogenase

The distribution of the multiple molecular forms of human liver alcohol dehydrogenase and the specific enzymatic activity of homogenate supernatants were examined in 100 liver specimens from Indianapolis, IN. The specific enzymatic activities of livers with the ADH 3 2 phenotype were significantly higher than those with the ADH 3 1 or ADH 3 2−1 phenotype. By comparison of the electrophoretic pattern and the pH-activity profiles of the homogenate supernatants, sixteen percent of the specimens contained hitherto unknown enzyme forms exhibiting unusual electrophoretic mobility and a pH-optimum of 7.0 for ethanol oxidation. The data confirm that the molecular and catalytic properties of this enzyme are even more diverse than has been known.

Human liver alcohol dehydrogenase

Human liver alcohol dehydrogenase

Human liver alcohol dehydrogenase isoenzyme variations. Improved separation methods using prolonged high voltage starch-gel electrophoresis and isoelectric focusing.

Human liver alcohol dehydrogenase isozyme patterns were studied using prolonged high voltage starch-gel electrophoresis and gel-slab isoelectric focusing. Homo- and heterodimers of ADH2 locus were easily distinguished from each other. The gene frequencies of ADH2 2 and ADH2 3 in 46 random liver samples from Germany were found to be 0.044 and 0.424 respectively.

Isolation of II-alcohol dehydrogenase of human liver: Is it a determinant of alcoholism?

HUMAN LIVER ALCOHOL DEHYDROGENASE (ALCOHOL: NAD(+) oxidoreductase, EC 1.1.1.1), homogeneous by physicochemical criteria, has been available in quantity only recently [Lange, L. G. & Vallee, B. L. (1976) Biochemistry 15, 4681-4686]. Until now, the biochemical basis of human alcohol metabolism had to be extrapolated from the properties and behavior of enzymes from other species, primarily horses and yeast. The biological determinants of human alcoholism have remained obscure, although recent evidence indicates a genetic predisposition, requiring delineation. A functionally distinct form of human liver alcohol dehydrogenase (ADH), which we have designated II-ADH, is provocative since, thus far, it seems to be unique to human beings. It has a high K(m) for ethanol and is remarkably insensitive (apparent K(I), 500 muM) to pyrazole and its derivatives, which are usually potent ADH inhibitors (K(I), 1 muM), a property that is the basis for the isolation of II-ADH. The affinity resin 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole-Sepharose binds all other known forms of ADH but not II-ADH, thereby separating it selectively by affinity chromatography. In turn, this has led to the establishment of its identity with that enzyme form which was previously known as the anodic band and characterized by a high K(m) for ethanol (20 mM at pH 7.5). The remarkable insensitivity of II-ADH to pyrazole inhibition has also permitted quantitation of its role in hepatic ethanol oxidation. At 5 mM ethanol, a saturating concentration for virtually all other forms of ADH, II-ADH contributes less than 15% to total ethanol oxidation. However, at intoxicating concentrations, e.g., 60 mM, it can account for as much as 40% of the total ethanol oxidation rate of liver, indicating a seemingly unique role for this enzyme form in ethanol elimination. Thus far, we have found the amount of II-ADH varies from liver to liver of individuals and is considerably more labile than the other molecular forms, phenomena whose inter- or independence requires further study. The isolation of human II-ADH advances efforts to recognize and understand biochemical mechanisms that may be biological determinants of alcoholism and alcohol-related disease states, now generally approached and managed largely as psychosocial disorders.

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.

Metabolic consequences of alcohol consumption.

The metabolic effects of alcohol are closely related to organic as well as societal aspects of alcoholism. Individual differences in the biologic sensitivity to ethanol are thought to represent important features underlying these afflictions. The results of recent biomedical research provide evidence for a biochemical individuality with respect to the enzymes involved in alcohol metabolism. Human liver alcohol dehydrogenase, the principle ethanol oxidizing enzyme in man, exists in multiple molecular forms (isoenzymes). In addition, a genetic polymorphism ('atypical' variant) is observed. Large racial differences are found for the prevalence of the variant enzyme, which is characterized by a high specific activity. Possible relationships of the individual isoenzyme pattern of alcohol dehydrogenase and of other alcohol metabolizing enzymes to the nature and extent of metabolic effects of ethanol are discussed.

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.

Human liver alcohol dehydrogenase: purification, composition, and catalytic features.

Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.

Dissociation and Rate Constants of Some Human Liver Alcohol Dehydrogenase Isoenzymes.

ADH from human liver forms binary complexes with NADH, associated with a blue shift of the peak of the fluorescence emission of NADH. The wavelength shift is the same for all isoenzymes but the accompanying intensification of the fluorescence is different. The fluorescence is further increased by the formation of the very tight ternary enzyme-NADH-isobutyramide complexes. These properties are similar to those for the horse liver ADH, as well as the molecular weight of E=40 000 per active site of the dimer molecule (EE). "Stopped-flow" determined velocity constants (ER in equilibrium E+R) were found to be in good agreement with ethanol activity constants previously determined by activity measurement, confirming the validity of the ordered ternary complex mechanism also for the human ADH. No single isoenzyme activity as high as that reported by Mourad and Woronick or Drum has been found.

Human liver alcohol dehydrogenase—inhibition of methanol activity by pyrazole, 4-methylpyrazole, 4-hydroxymethylpyrazole and 4-carboxypyrazole

With human liver alcohol dehydrogenase of high purity at pH 7.0 and 500 μM NAD the K m for methanol is 7.0 mM (ten times greater than the K m for ethanol) and the turnover number 1.4/active site/min (about one-tenth of the turnover with ethanol in the same conditions). From secondary kinetic plots it can be calculated that at saturating concentrations of both substrates, namely methanol and NAD, these constants do not change appreciably: the K m for methanol is somewhat lower (5.2 mM) and the turnover number slightly higher (1.7/active site/min). The difference in turnover numbers with methanol and ethanol as substrates suggests that the kinetic mechanism for methanol is different from that for ethanol dehydrogenation. The dissociation constant between human alcohol dehydrogenase and NAD, determined kinetically with methanol as substrate, is 127 μM. The K i values for pyrazole, 4-methylpyrazole and 4-hydroxymethylpyrazole are 0.54, 0.09 and 6.6 μM respectively; 4-carboxypyrazole (100 μM) at 3mM methanol does not inhibit human ADH. The inhibitory effect of 4-methylpyrazole is therefore not likely to be enhanced by a possible metabolic conversion to 4-hydroxymethylpyrazole and 4-carboxypyrazole.

Identification of a distinctive molecular form of alcohol dehydrogenase in human livers with high activity

Summary Human liver alcohol dehydrogenase activity and isoenzyme pattern have been compared in specimens from different sources. Specific enzymatic activity was significantly higher in surgical biopsy samples than in autopsy specimens. Furthermore, tissues from individuals who died suddenly from trauma exhibited significantly higher activities than those obtained following disease-related deaths. All livers with high specific activity revealed on starch gel electrophoresis, in addition to the usual isoenzymes, a prominent, previously unrecognized, activity band. This molecular form, more anodic than the others at pH 8.6 and 7.7, was separated by isoelectric focusing. It exhibited kinetic properties characteristic of the alcohol dehydrogenase isoenzymes.

Steroid Oxidoreductase Activity of Alcohol Dehydrogenases from Horse, Rat, and Human Liver.

Alcohol dehydrogenase from horse (isoenzyme SS and ES, but not EE), rat and human liver were found to catalyze the NAD-dependent oxidation of 3beta-hydroxy groups in 5alpha- and 5beta-steroids of the C19, C21, and C24 series. The enzymes from horse and rat liver were more active on 5beta-than on 5alpha-steroids. This difference was most marked with the enzyme from rat liver, especially with 3beta-hydroxyandrostan-17-ones and 3beta-hydroxypregnan-20-ones as substrates. The Km of isoenzyme ES from horse liver was lower for 3beta-hydroxy-5alpha-cholanoic acid (0.4 muM) than for 3beta-hydroxy-5beta-cholanoic acid (0.9 muM). 3alpha-Hydroxysteroids were not substrates for the enzymes from horse and rat liver. Human liver alcohol dehydrogenase had low affinity for 3beta-hydroxy-5alpha (and 5beta)-cholanoic acids, but oxidation could be clearly demonstrated by gas chromatographic analysis of the products.

Structural studies of human-liver alcohol-dehydrogenase isoenzymes.

Alcohol dehydrogenase isoenzymes BE were isolated from human livers of both “normal” and “atypical” phenotypes [1]. Their pH‐rate profiles, their behaviour on CM‐cellulose and the number of hybrids formed with horse subunit A show that the “atypical” isoenzymes contain subunits B1 and B2 the “normal” one only subunit B1. These results indicate that most “atypical” individuals genetically represent heterozygotes. A high degree of homology between horse subunit A and human subunits B is found. However, the human isoenzymes have a molecular weight of 87000 and contain about 410 amino acid residues, 6 tyrosines and 3 tryptophans per subunit. The sequence of one tryptic peptide from subunit B1 is Phe‐Ala‐Lys and is altered to Phe‐Pro‐Lys in the subunit B2. This substituted residue is located in a region which corresponds to the coenzyme‐binding site of the horse enzyme.