Simian liver alcohol dehydrogenase: isolation and characterization of isoenzymes from Macaca mulatta

Like human liver alcohol dehydrogenase, that of Macaca mulatta can be purified and separated into anodic and cathodic pyrazole-insensitive and cathodic pyrazole-sensitive enzyme forms. Their inhibition by 4-methylpyrazole and their substrate specificities are analogous to those observed for the corresponding isoenzymes of human liver. However, on the basis of data available so far, the physiochemical and compositional characteristics, i.e., molecular weight, zinc content, and dimeric structure, of all simian alcohol dehydrogenase forms are virtually identical with those of other mammalian alcohol dehydrogenases studied up to now. Zinc is essential for their enzymatic function, as demonstrated by inhibition with chelating agents.     

Human class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase

The class III human liver alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, separates electrophoretically into a major anodic form (₁) of known structure, and at least one minor, also anodic but a slightly faster migrating form (₂). The primary structure of the minor form isolated by ion-exchange chromatography has now been determined. Results reveal an amino acid sequence identical to that of the major form, suggesting that the two derive from the same translation product, with the minor form modified chemically in a manner not detectable by sequence analysis. This pattern resembles that for the classical alcohol dehydrogenase (class I). Hence, the ₁/₂ multiplicity does not add further primary forms to the complex alcohol dehydrogenase system but shows the presence of modified forms also in class III.     

Identification of a human stomach alcohol dehydrogenase with distinctive kinetic properties

A new form of alcohol dehydrogenase, designated mu-alcohol dehydrogenase, was identified in surgical human stomach mucosa by isoelectric focusing and kinetic determinations. This enzyme was anodic to class I (alpha, beta, gamma) and class II (pi) alcohol dehydrogenases on agarose isoelectric focusing gels. The partially purified mu-alcohol dehydrogenase, specifically using NAD+ as cofactor, catalyzed the oxidation of aliphatic and aromatic alcohols with long chain alcohols being better substrates, indicating a barrel-shape hydrophobic binding pocket for substrate. mu-Alcohol dehydrogenase stood out in high Km values for both ethanol (18 mM) and NAD+ (340 microM) as well as in high Ki value (320 microM) for 4-methylpyrazole, a competitive inhibitor for ethanol. mu-Alcohol dehydrogenase may account for up to 50% of total stomach alcohol dehydrogenase activity and appeared to play a significant role in first-pass metabolism of ethanol in human.     

Horse liver alcohol dehydrogenase derivatives containing nickel(II) and cobalt(lI) in the noncatalytic metal binding site

The noncatalytic zinc in horse liver alcohol dehydrogenase was selectively replaced by nickel(II). This novel species, Zn(c)₂Ni(n)₂⁴ horse liver alcohol dehydrogenase (where c denotes the catalytic and n denotes the noncatalytic site) was compared to Zn(c)₂Co(n)₂ horse liver alcohol dehydrogenase with respect to its absorption, circular dichroism and magnetic circular dichroism spectra, as well as its magnetic moment. For Zn(c)₂Co(n)₂ horse liver alcohol dehydrogenase (prepared according to refs. 1 and 2) the extinction coefficients were redetermined in the UV, visible and near-infrared region and the molar ellipticities in the range 300-800 nm. The average magnetic moment was determined by the NMR method as 4.5-5.0 B.M. The results confirm a tetrahedral structure in the zinc-cobalt enzyme. In contrast, the spectroscopic data and the zero magnetic moment support a planar geometry for the nickel(Il) bound in the noncatalytic site. Zn(c)₂Ni(n)₂ horse liver alcohol dehydrogenase is very temperature-sensitive and precipitates after short exposure to room temperature. Stored in the cold it has the same activity as the native enzyme. The results indicate that the protein is flexible in the loop region binding the noncatalytic metal ion and that it may retain catalytic activity even in a partially distorted conformation.     

Identification of two dihydrodiol dehydrogenases associated with 3(17)alpha-hydroxysteroid dehydrogenase activity in mouse kidney

Dihydrodiol dehydrogenase activity was detected in the cytosol of various mouse tissues, among which kidney exhibited high specific activity comparable to the value for liver. The enzyme activity in the kidney cytosol was resolved into one major and three minor peaks by Q-Sepharose chromatography: one minor form cross-reacted immunologically with hepatic 3 alpha-hydroxysteroid dehydrogenase and another with aldehyde reductase. The other minor form was partially purified and the major form was purified to homogeneity. These two forms, although different in their charges, were monomeric proteins with the same molecular weight of 39,000 and had similar catalytic properties. They oxidized cis-benzene dihydrodiol and alicyclic alcohols as well as trans-dihydrodiols of benzene and naphthalene in the presence of NADP+ or NAD+, and reduced several xenobiotic aldehydes and ketones with NAD(P)H as a cofactor. The enzymes also catalyzed the oxidation of 3 alpha-hydroxysteroids and epitestosterone, and the reduction of 3- and 17-ketosteroids, showing much lower Km values (10(-7)-10(-6) M) for the steroids than for the xenobiotic alcohols. The results of mixed substrate experiments, heat stability, and activity staining on polyacrylamide gel electrophoresis suggested that, in the two enzymes, both dihydrodiol dehydrogenase and 3(17)alpha-hydroxysteroid dehydrogenase activities reside on a single enzyme protein. Thus, dihydrodiol dehydrogenase existed in four forms in mouse kidney cytosol, and the two forms distinct from the hepatic enzymes may be identical to 3(17)alpha-hydroxysteroid dehydrogenases.     

Occurrence of multiple forms of alcohol dehydrogenase in Penicillium supplemented with 2,3-butanediol

The NAD-dependent oxidation of ethanol, 2,3-butanediol, and other primary and secondary alcohols, catalyzed by alcohol dehydrogenases derived from Penicillium charlesii, was investigated. Alcohol dehydrogenase, ADH-I, was purified to homogeneity in a yield of 54%. The enzyme utilizes several primary alcohols as substrates, with K_m values of the order of 10^?4m. A K_m value of 60 mm was obtained for R,R,-2,3-butanediol. The stereospecificity of the oxidation of 2-butanol was investigated, and S-(+)-2-butanol was found to be oxidized 2.4 times faster than was R-(?)-2-butanol. The reduction of 2-butanone was shown to produce S-(+)-2-butanol and R-(?)-butanol in a ratio of 7:3. ADH-I is the primary isozyme of alcohol dehydrogenase present in cultures utilizing glucose as the sole carbon source. The level of alcohol dehydrogenase activity increased 7.6-fold in mycelia from cultures grown with glucose and 2,3-butanediol (0.5%) as carbon sources compared with the activity in cultures grown on only glucose. Two additional forms of alcohol dehydrogenase, ADH-II and ADH-III, were present in the cultures supplemented with 2,3-butanediol. These forms of alcohol dehydrogenase catalyze the oxidation of ethanol and 2,3-butanediol. These data suggest that P. charlesii carries out an oxidation of 2,3-butanediol which may constitute the first reaction in the degradation of 2,3-butanediol as well as the last reaction in the mixed-acid fermentation. Alcohol dehydrogenase activities in P. charlesii may be encoded by multiple genes, one which is expressed constitutively and others whose expression is inducible by 2,3-butanediol.     

Cytosolic alcohol dehydrogenases from yeast Torulopsis candida]

A comparative study of cell cytosol alcohol dehydrogenase (ADH) from yeast Torulopsis candida IBFM-Y-127 grown on glucose and hexadecane which were the only source of carbon, was made. In both cases ADH had a pH optimum within the range of 7.0--10.0, when various normal primary alcohols (C2--C16) were used. The enzyme was active only in the presence of NAD, which cannot be substituted by NADP. The total activity of ADH decreased approximately 8-fold when the length of hydrocarbon radicals was changed from C2 up to C16. When the cells were grown on hexadecane, only ethyl, n-buthyl, n-amyl and n-hexyl alcohols were active as substrates. The dehydration rate of each alcohol was far lower than that for the cytosol of glucose-grown cells. In the latter case the enzyme activity also decreased with an increase in the alcohol radical from C2 to C6. In all cases studied methyl alcohol and cyclic (cinnamyl alcohol--C8) alcohol were not dehydrated at all. Disc-electrophoresis in polyacrylamide gel, involving gel colouration for the assay of enzyme activity showed that glucose--grown cell cytosol contained three forms of ADH. One of those forms was highly active when short--chain normal primary alcohols were used; this form may be probably regarded as "classical" ADH (EC 1.1.1.1). The two other forms caused intensive dehydration of long-chain alcohols (the best substrates were C7--C10 alcohols for one form and C10--C14 for the others). The two forms of ADH are probably isoenzymes of octanol dehydrogenase (EC 1.1.1.73). Cytosol of cells grown on n-alcane, had a reduced number of ADH forms. The data obtained are discussed in terms of the regulatory role of carbon and energy source (glucose or hexadecane) in the redistribution of alcohol dehydrogenases between structural components of cells (mitochondria) and cytosol.     

Drosophila alcohol dehydrogenase activity in vitro and in vivo: Effects of acetone feeding

When adult Drosophila are placed on medium containing 0.5% acetone, their level of alcohol dehydrogenase activity drops rapidly. At the same time, the proportion of activity in the various electrophoretic forms of the enzyme shifts; most of the activity becomes localized in what is ordinarily a minor form of the enzyme. Moreover, the loss of enzyme activity occurs in vivo as well, as shown by sensitivity to ethanol poisoning, insensitivity to pentenol treatment, and inability to utilize ethanol as an energy source. These observations are discussed in light of a model advanced for the origin of the multiple forms of alcohol dehydrogenase in Drosophila.This research was supported by NIH Grants GM-18254 and ES-1527 and by DOE Contract No. EY-76-5-02-2965. Publication No. 1016 from the Department of Biology, Johns Hopkins University.     

Expressivity of the gene-enzymatic system and viability indicators in the ontogenesis of Drosophila inbred lines and hybrids

The activity, physico-chemical properties and multiple molecular forms of enzymes (alcohol dehydrogenase, superoxide dismutase, nonspecific alpha- and beta-esterases, hydroxide peptidohydrolase) were studied in ontogenesis of Drosophila inbred lines and their hybrids under conditions of high temperature (37-41 degrees C) and the presence in food of 10% ethanol. It was established that resistance of individuals to the effect of high temperature and alcohol, including manifestation of adaptive heterosis in hybrids not always depends on the level of the activity of enzymes analysed and is rather determined by allelic state of the appropriate structural genes. So, in conditions of the alcohol stress the individuals containing highly active F form of alcohol dehydrogenase have selective advantage and flie with hybrid F/S enzyme of higher activity and heat stability are more stable to the effect of high temperature. It is supposed that the complexes of adaptation genes (CGA) are formed in individuals of populations in response to the regular action of unfavourable environmental factors. These complexes condition optimal allelic control and most efficient regulation of enzyme activity in environment. Genotypic adaptation of individuals as well as occurrence of adaptive heterosis in hybrids seem likely to be connected with formation of CGA.     

Glucose-6-phosphate dehydrogenase of Anabaena sp.

The kinetic and molecular properties of cyanobacterial glucose-6-phosphate dehydrogenase, partly purified from Anabaena sp. ATCC 27893, show that it undergoes relatively slow, reversible transitions between different aggregation states which differ in catalytic activity. Sucrose gradient centrifugation and polyacrylamide gel electrophoresis reveal three principal forms, with approximate molecular weights of 120 000 (M ₁), 240 000 (M ₂) and 345 000 (M ₃). The relative catalytic activities are: M ₁M ₂<M ₃. In concentrated solutions of the enzyme, the equilibrium favors the more active, oligomeric forms. Dilution in the absence of effectors shifts the equilibrium in favor of the M ₁ form, with a marked diminution of catalytic activity. This transition is prevented by a substrate, glucose-6-phosphate, and also by glutamine. The other substrate, nicotinamide adenine dinucleotide phosphate (NADP⁺), and (in crude cell-free extracts) ribulose-1,5-diphosphate are negative effectors, which tend to maintain the enzyme in the M ₁ form. The equilibrium state between different forms of the enzyme is also strongly dependent on hydrogen ion concentration. Although the optimal pH for catalytic activity is 7.4, dissociation to the hypoactive M ₁ form is favored at pH values above 7; a pH of 6.5 is optimal for maintenace of the enzyme in the active state. Reduced nicotamide adenine dinucleotide phosphate (NADPH) and adenosine 5-triphosphate (ATP), inhibit catalytic activity, but do not significantly affect the equilibrium state. The relevance of these findings to the regulation of enzyme activity in vivo is discussed.Abbreviations G6PD glucose-6-phosphate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - RUDP ribulose-1,5-diphosphate - G6P glucose-6-phosphate - 6PG 6-phosphogluconate     

Alcohol dehydrogenase from Methylobacterium organophilum.

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 x 10(-5) M for methanol and 8.2 x 10(-5) M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.     

Amino Acid Sequence and Molecular Weight of Native NADP Malate Dehydrogenase from the C(4) Plant Zea mays

N-terminus amino acid analysis of purified corn (Zea mays) NADP malate dehydrogenase showed that the mature protein begins at serine-41 of the preprotein sequence and not threonine-58 as previously concluded; therefore, the transit peptide consists of 40 amino acids. The theoretical molecular weight of the mature subunit protein (392 amino acids) is 42,564, agreeing with an experimental value of about 43,000. The molecular weight of the native unactivated (dark form) and activated (light form) of NADP malate dehydrogenase, determined by analytical ultracentrifugation analysis, was about 84,000, indicating that both forms are dimers. However, conventional and high performance liquid chromatography gel filtration procedures indicated apparent molecular weights of about 110,000 to 120,000 for the unactivated native enzyme and about 143,000 to 150,000 for the active enzyme; in these cases, the molecular weight may be overestimated due to the effect of an unusual molecular conformation on the mobility of the enzyme.     

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.     

Alcohol dehydrogenase of Biomphalaria glabrata (Mollusca: Pulmonata): Polymorphism,genetic analysis,and interspecific variations

Alcohol dehydrogenase of Biomphalaria glabrata has been characterized by electrophoresis, substrate specificities, and other physicochemical means. It exists as a multiple molecular form possessing a minimum number of three bands in ovotestis, five in digestive gland, and six in albumen gland. Each organ shows characteristic electrophoretic forms which differ in substrate specificities and the response to the organomercurial inhibitor p-hydroxymercuribenzoate. Mercaptoethanol treatment has no effect on any electrophoretic form. Genetic analyses of the electrophoretic variants show that three different loci are responsible for the synthesis of the various electrophoretic forms observed in this species. Different species vary in their electrophoretic patterns. A possible role of alcohol dehydrogenase isozymes in the phylogenetic relationship among three species, B. glabrata, B. tenagophila, and B. straminea, has been discussed.This work was supported by a grant from the Conselho Nacional de Pesquisas, Brazil.     

Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens)

A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.     

Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides

Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase–gum Arabic complex formed under optimized conditions showed 93 % retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase–gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase–gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K _m and V _max) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.     

Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling

Abstract Dye-linked alcohol dehydrogenase from Rhodopseudomonas acidophila strain M402, able to oxidize polyethylene glycols, was purified to homogeneity. The monomeric enzyme, having a molecular mass of 72 kDa, contains one PQQ and one haem c per enzyme molecule. In other respects also, the enzyme is very similar to the type I quinohaemoprotein alcohol dehydrogenases known to occur in Comamonas testosteroni, Comamonas acidovorans , and Pseudomonas putida species. However, dissimilarities exist with respect to the isoelectric points and the substrate specificities. On reinvestigating the substrate specificity of the C. testosteroni enzyme, it also appeared to exhibit good activity towards polyethylene glycols. Based on what has been reported for the polyethylene glycol-oxidizing alcohol dehydrogenase of Sphingomonas macrogoltabidus , this enzyme is quite different from that of R. acidophila . Keywords: Polyethylene glycol dehydrogenase activity; Alcohol dehydrogenase; PQQ; Haem c ; Rhodopseudomonas acidophila     

The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc

Insect alcohol dehydrogenase is highly different from the well-known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonensis has now been characterized by protein analysis and was found to have a 254-residue protein chain with an acetyl-blocked N-terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinc-containing alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.     

Molecular forms of guanine aminohydrolase (author's transl)

Guanine aminohydrolase (E.C. 3.5.4.3) has been purified 11-fold from the supernatant fraction of guinea-pig liver homogenates in 0.25 M sucrose (centrifuged at 50,000 X g) through thermic denaturation at 60 degrees C and ammonium sulphate fractionation (30--60% saturation). The enzyme in the homogenates and purified preparations exhibited two Km values. In both preparations four enzymatic electrophoretic bands have been detected. Purified guanine aminohydrolase is chromatographically resolved on DEAE-sephadex in three components whose active forms appeared separately on their pherograms. The enzymatic form eluted at lower ionic strength has the least anodic mobility, is inhibited by guanine (4 X 10(-5) M) and presents only one Km value (1.5 X 10(-5) M). The enzymatic form eluted at greater ionic strength exhibits the highest anodic mobility, is also inhibited by guanine (7 X 10(-5) M) and its Km value seems to be 6.3 X 10(-6) M. Molecular weight of enzymatics forms determined by Sephadex G-200 chromatography, is 120,000 +/- 5,000. The preceding results, correlated with the chromatographic homogeneity of guanine aminohydrolase, purified in Sephadex G-100, suggests that the four molecular forms of the native enzyme may be considered as isozymes.     

Steady-state kinetics of horse-liver alcohol dehydrogenase with a covalently bound coenzyme analogue

The steady-state kinetics of the enzyme modified by affinity labelling with NAD analogue, nicotinamide-N6-[N-(6-aminohexyl)carbamoylmethyl]-adenine dinucleotide, has been investigated using a recycling reaction with p-nitrosodimethylaniline and n-butanol as substrates and compared to the kinetics of native alcohol dehydrogenase. The modified enzyme obeys a ping-pong mechanism involving two inactive enzyme forms (enzyme-NAD and enzyme-NADH complexes in the 'open' conformations, the nicotinamide moieties of the coenzymes being out of the active center). The rate of p-nitrosodimethylaniline reduction in the reaction catalyzed by the modified enzyme is comparable to that observed in the presence of the native enzyme. On the other hand, the oxidation of butanol by the modified enzyme is essentially slower under our experimental conditions (pH 8.5). The measurements in the presence of specific alcohol dehydrogenase inhibitors competing with substrates and coenzymes (isobutyramide, pyrazole and AMP) revealed that the relative portion of the inactive 'open' form of the enzyme-NADH complex is negligible, whereas the 'open' form of the enzyme-NAD complex seems to represent a more significant portion (about 30%) under the conditions used.