Assay of enzymatic activity by electrochemical detection of reduced coenzyme.

A highly sensitive electrochemical assay of the enzymatic activities of aqueous samples of lactate dehydrogenase, alcohol dehydrogenase, and malate dehydrogenase has been developed using an improved amperometric determination of NADH concentration in the test solution. An anode current sensitivity of 750 μA/mmol of NADH was obtained with a platinum-mesh electrode in an H cell modified to permit vigorous stirring of the anolyte. Fouling of the platinum anode was significantly decreased by working at a pH ≥ 8.1. The rate of increase in net anode current in substrate solutions containing as little as 2 × 10⁻³ unit of enzyme/ml correlated well with the rate of change in absorbance at 340 nm for each sample. The reproducibility of the assay of enzyme activity was about ± 10%.     

Effect of carbon source and pH of the growth medium on spore germination inPhycomyces blakesleeanus

Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD⁺-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.     

A high pH discontinuous buffer system for resolution of isozymes in starch gel electrophoresis

A Tris-citrate pH 9.5 gel/borate pH 8.2 electrode discontinuous buffer system for starch gel electrophoresis of proteins was developed to resolve iso- and allozymes of aspartate aminotransferase in frogs (Hyla crucifer). This buffer system also enhanced resolution of NADP-dependent malate dehydrogenase and the L-lactate dehydrogenase-A locus in this species. It provided good resolution of NAD-dependent malate dehydrogenase in esocid fishes, and esterases, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, and S-aconitate hydratase in ambystomatid salamanders. Variation suppressed by other buffers was revealed by this buffer for some enzyme encoding loci, while at other loci, this buffer suppressed electromorph variability. The concentration of tris(hydroxymethyl)aminomethane in gels made with this buffer was much higher than in pH 8.7 "Poulik" gels, but running characteristics of the two gel types were similar. Gels made with this new buffer were less prone to splitting and "warping" than Poulik gels, and were easier to handle. When screening a given taxon for enzyme variability, tests using multiple buffers are essential to maximize the amount of electrophoretically detectable variation.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Construction and characterization of a multi-layer enzyme electrode: Covalent binding of quinoprotein glucose dehydrogenase onto gold electrodes

Multilayers of quinoprotein glucose dehydrogenase were assembled onto modified gold electrodes. As a primary modifier the bifunctional 3,3′-dithiodipropionic acid bis(N-hydroxysuccinimide ester) was chemisorbed. Glucose dehydrogenase was covalently bound to this activated electrode in a stepwise procedure. In the presence of glucose, the electrode functions as a sensor for electron acceptors. Catalytic current, as observed for p-aminophenol, was used to characterize electrode performance. The dependence of the electrode response on the number of enzyme layers showed that the transition from a kinetic to a diffusion-limited sensor is reached at 6–7 enzyme layers. The response of multilayer electrode is stable over a broad range of pH and ionic strength of the bulk solution. It also shows good stability: after 2 months, 75% of its original activity remained.     

Kinetic effect of some aliphatic amines on yeast alcohol dehydrogenase.

Initial rate studies of ethanol oxidation catalyzed by yeast alcohol dehydrogenase (EC 1.1.1.1) were carried out in the presence of varying concentrations of aliphatic amines over the pH range from 8.0 to 10.5. Aliphatic amines either activate or inhibit the enzyme depending on whether the pH is greater or less than 9.5 suggesting that the protonated amines activate and the nonprotonated amines inhibit the enzyme. Aliphatic amines activate yeast alcohol dehydrogenase by decreasing Kb while they inhibit the enzyme by increasing both Ka and Kia. When both protonated and nonprotonated amines are present in solution, either overall activation or inhibition will be observed depending on the relative concentration of the two amine species.     

Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.     

A High Ph Discontinuous Buffer System for Resolution of Isozymes in Starch Gel Electrophoresis

A Tris-citrate pH 9.5 gel/borate pH 8.2 electrode discontinuous buffer system for starch gel electrophoresis of proteins was developed to resolve iso- and allozymes of aspartate aminotransferase in frogs (Hyla crucifer).- This buffer system also enhanced resolution of NADP-dependent malate dehydrogenase and the L-lactate dehydrogenase-A locus in this species. It provided good resolution of NAD-dependent malate dehydrogenase in esocid fishes, and esterases, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phospbate dehydrogenase, alcohol dehydrogenase, and S-aconitate hydratase in ambystomatid salamanders. Variation suppressed by other buffers was revealed by this buffer for some enzyme encoding loci, while at other loci, this buffer suppressed electromorph variability. The concentration of tris(hydroxymethyl)aminomethane in gels made with this buffer was much higher than in pH 8.7 “Poulik” gels, but running characteristics of the two gel types were similar. Gels made with this new buffer were less prone to splitting and “warping” than Poulik gels, and were easier to handle. When screening a given taxon for enzyme variability, tests using multiple buffers are essential to maximize the amount of electrophoretically detectable variation.     

Mechanism of action of Drosophila melanogaster alcohol dehydrogenase.

Reported kinetic pH dependence data for alcohol dehydrogenase from Drosophila melanogaster are analyzed with regard to differences in rate behaviour between this non-metallo enzyme and the zinc-containing liver alcohol dehydrogenase present in vertebrates. For the Drosophila enzyme a mechanism of action is proposed according to which catalytic proton release to solution during alcohol oxidation occurs at the binary-complex level as an obligatory step preceding substrate binding. Such proton release involves an ionizing group with a pKa of about 7.6 in the enzyme.NAD+ complex, tentatively identified as a tyrosyl residue. The ionized form of this group is proposed to participate in the binding of alcohol substrates and to act as a nucleophilic catalyst of the subsequent step of hydride ion transfer from the bound alcohol to NAD+. Herein lie fundamental mechanistic differences between the metallo and non-metallo short chain alcohol dehydrogenases.     

Potential applications of an alcohol-aldehyde/ketone oxidoreductase from thermophilic bacteria

Practical uses of a novel alcohol dehydrogenase from Thermoanaerobium brockii have been examined in crude and purified form. Stoichiometric reduction of NADP (50 mg) was demonstrated with agarose-immobilized enzyme and 0.3 (v/v) 2-propanol solution as reductant. A coenzyme recycle number of 20000 was achieved in enzymatic reactions that employed the alcohol dehydrogenase for NADPH/NADP regeneration. Gram-scale synthesis of chiral R(+) 2-pentanol was shown in a system composed of enzyme, 2-pentanone and 2-propanol as reductant. The effect of temperature, reaction time and substrate concentration on alcohol optical purity was examined. An optical purity of 80% was achieved in the enzymatic synthesis of R(+) 2-pentanol. The enzyme was easily immobilized and stable on an enzyme electrode for analytical detection of alcohols and carbonyls. T. brockii enzyme has potential applications as a commercial alcohol dehydrogenase because of broad substrate specificity and activity at high temperature or high solvent concentration, rare carbonyl si-face stereo-specificity in hydrogen transfer, and high stability and activation of immobilized enzyme.     

Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator 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.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization.

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.     

Mediatorless voltammetric oxidation of NADH and sensing of ethanol

A simple, selective and sensitive method for the detection of NADH and ethanol is presented. Self-assembled monolayers (SAMs) of mercaptopyrimidine (MPM) and their derivatives, thiocytosine (TC) and 4,6-diamino-2-mercaptopyrimidine (DMP) on gold (Au) electrode are used for the voltammetric detection of NADH and ethanol in neutral aqueous solution. A decrease of 200-300 mV in the overpotential associated with an observable increase in the peak current was obtained for the oxidation of NADH on MPM and TC monolayer-modified electrodes without any redox mediator. The facilitated electron transfer for the oxidation of NADH at the TC monolayer is ascribed to the existence of stable cationic p-quinonoid form of TC. The electrode modified with DMP monolayer could not exhibit stable response for NADH owing to the fouling of electrode surface. The MPM and TC monolayer-modified electrodes show high selectivity and excellent sensitivity (MPM: 0.633+/-0.005 microA cm(-2) microM(-1); TC: 0.658+/-0.008 microA cm(-2) microM(-1)) towards NADH with detection limit (3sigma) of 2.5 and 0.5 microM, respectively. Presence of large excess of ascorbate (AA) does not interfere the detection of NADH and the monolayer-modified electrode shows individual voltammetric peaks for AA and NADH. Voltammetric sensing of ethanol using alcohol dehydrogenase on MPM and TC monolayer-modified electrode is successfully demonstrated and these electrode can detect as low as 0.5 mM ethanol in neutral pH. The sensitivity of the MPM and TC monolayer-modified electrodes toward ethanol was found to be 3.24+/-0.03 and 3.435+/-0.04 microA cm(-2) mM(-1), respectively.     

Immobilization of horse liver alcohol dehydrogenase on copolymers of glycidyl methacrylate and ethylene dimethacrylate

Alcohol dehydrogenase has been immobilized to the basic copolymer and its several derivatives using various techniques. Enzyme coupling to the supports with amino groups by means of glutaraldehyde was found the most suitable. Activity of alcohol dehydrogenase coupled to these amino supports was comparable to that of the enzyme bound to Sepharose. Thermal and pH stability of alcohol dehydrogenase increased essentially upon immobilization. Kinetic properties of the immobilized enzyme differed from those of free alcohol dehydrogenase, pH optimum shifted to alkaline range, and apparent Michaelis constants for substrates and coenzymes increased. Curvatures observed in Lineweaver-Burk plots for coenzymes suggest an involvement of diffusion effects in the reaction catalyzed by alcohol dehydrogenase linked to these polymers.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Overexpression, purification and properties of alcohol dehydrogenase IV from Saccharomyces cerevisiae

We have purified ADHIV, a novel alcohol dehydrogenase (ADH) isozyme in the yeast Saccharomyces cerevisiae, after increasing the normally low amount of ADHIV protein in laboratory strains. This was done by overexpression of the structural gene (ADH4) on a 2micro-based multicopy vector. Characterization of the purified enzyme revealed a dimeric structure as well as a different substrate specificity and pH profile as compared to other alcohol dehydrogenase isozymes. On the other hand, we could demonstrate that ADHIV is activated by zinc ions, like the other yeast alcohol dehydrogenase isozymes, and not by ferrous ions, like a structurally similar alcohol dehydrogenase from the bacterium Zymomonas mobilis.     

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.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.