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.     

The half-of-the-sites reactivity of horse liver alcohol dehydrogenase in the presence of alcohol substrates

We investigated by stopped-flow techniques the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase varying the concentration of the reagents, pH and temperature. The course of the reaction under enzymelimiting conditions is biphasic and the measured amplitude of the initial step corresponds under saturation conditions to half of the total enzyme concentration (half-burst). The fast initial step (with a maximum rate of 20 s^?1 at pH 7.0) shows an isotope effect of approximately 2, which indicates that this rate contains a contribution from a hydrogen transfer. It is also shown that this rate differs by at least one order of magnitude with respect to that of the hydrogen transfer during benzaldehyde reduction. The half-of-the-sites reactivity of alcohol dehydrogenase in the initial transient process is obtained independent of reagent concentration, pH and/or temperature. It is obtained also when coenzyme analogues are substituted for NAD, and when different alcohols are substituted for benzyl alcohol. These data are taken to demonstrate unequivocally that the half-of-the-sites reactivity of alcohol dehydrogenase cannot be due to an interplay of rate constants (as proposed by various authors) and must rather be ascribed to a kinetic non-equivalence of the two subunits when active ternary complexes are being formed. When oxidation of benzyl alcohol is carried out in the presence of 0.1 m-isobutyramide (which makes a very tight complex with NADH at the enzyme active site), reaction stops after formation of an amount of NADH product that is equivalent to one half of the enzyme active site concentration.This is considered in the light of the pyrazole experiment designed by McFarland &; Bernhard (1972), in which reduction of benzaldehyde is carried out in the presence of pyrazole (which forms a very tight ternary complex with NAD at the enzyme active site). In this case, reaction stops after formation of an amount of NAD-product which is equivalent to the total enzyme active site concentration. It is shown that accommodation of these two seemingly contradictory sets of data poses severe restrictions on the alcohol dehydrogenase mechanism. In particular, it is shown that the only mechanism that adheres to such requirements is one in which the two subunits have distinct and alternating functions in each enzyme cycle. These two functions are the triggering of the chemical transformation and the chemical transformation itself. It is also shown that binding of NAD-substrate to one subunit triggers chemical reactivity in the other NAD-alcohol-containing subunit, whereas on aldehyde reduction, the triggering event is desorption of alcohol product from the first reacted subunit.     

Limiting rates of ligand association to alcohol dehydrogenase.

Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s^?1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD⁺ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.     

Inhibitory monoclonal antibodies against rat liver alcohol dehydrogenase

Eleven hybridoma clones which secrete monoclonal antibodies against purified rat liver alcohol dehydrogenase (EC 1.1.1.1) were isolated. Antibodies (R-1-R-11) were identified by their ability to bind to immobilized pure alcohol dehydrogenase in an enzyme-linked immunoadsorbent assay, in which antibody R-9 showed the highest binding capacity. Except for R-1 and R-7, all antibodies inhibited catalytic activity of the enzyme isolated from inbred (Fischer-344) or outbred (Sprague-Dawley) strains (R-11 greater than R-9 greater than R-4 greater than R-6 greater than R-10 greater than R-8 greater than R-2 = R-3 = R-5). The inhibition of enzyme activity by antibodies was noncompetitive for ethanol and NAD+, and was dependent on antibody concentration and incubation time. Antibodies R-4, R-9, and R-11 were most effective when enzyme activity was assayed below pH 7.7-7.8, a condition thought to protonate the enzyme's active center. These three antibodies did not inhibit horse liver alcohol dehydrogenase activity, indicating their species specificity. Such antibodies will be useful to delineate structural and functional roles of rat liver alcohol dehydrogenase.     

Inactivation of alcohol dehydrogenase by 3-butyn-1-ol

Horse liver and yeast alcohol dehydrogenases are rapidly inactivated during their catalysis of the oxidation of 3-butyn-1-ol. In the case of the horse liver enzyme, the inactivation is secondary to covalent modification of the apoenzyme by an electrophilic product that accumulates in the reaction solution and that can also react with water, glutathione, and other enzymes. The modified protein exhibits enhanced ultraviolet absorbance, which is not bleached upon dialysis of the denatured enzyme at pH 7.4 for 24 h. The inactivation by 3-butyn-1-ol is more rapid than that which is afforded by the related alcohols 2-propyn-1-ol and 2-propen-1-ol under identical conditions and no inactivation is seen upon incubation with 3-hydroxypropanoic nitrile plus nicotinamide-adenine dinucleotide.     

Binding of ligands to the catalytic zinc ion in horse liver alcohol dehydrogenase

The affinity of nitrogen and sulfur ligands for the catalytic zinc ion in horse liver alcohol dehydrogenase has been investigated by their influence on the affinity labeling reaction with iodoacetate. All the nitrogen compounds including ammonia, a primary and a secondary amine, and heterocycles containing a pyridine-type nitrogen with the exception of 2,2-dipyridyl were found to activate the affinity labeling reaction. Activation results from inner-sphere ligand coordination to the catalytic zinc ion. Closely related pyridine compounds gave a regular increase in affinity for the enzyme with increasing basicity, as expected for coordination to a metal ion. The sulfur compounds penicillamine and mercaptoethanol also activated the affinity labeling reaction, but dimercaptopropanol bound very tightly as a bidentate inhibited the reaction. The anions hydrosulfide, diethyldithiocarbamate, and cyanide coordinated to the catalytic zinc ion, whereas azide, thiocyanate, tetrazole, and iodide complexed the anion-binding site. The anionic metal ligands increased the rate of inactivation of the enzyme with iodoacetamide by binding to the catalytic zinc ion, while the binding of iodoacetate to the anion-binding site was prevented.     

The inhibition of alcohol dehydrogenase in vitro and in isolated hepatocytes by 4-substituted pyrazoles

As a means of comparing the functional properties of an enzyme in dilute solution in vitro with those for the same enzyme acting in its normal cellular environment, a study was conducted with 4-substituted pyrazoles as inhibitors of rat liver alcohol dehydrogenase in vitro and ethanol oxidation in isolated rat hepatocytes. Inhibitor constants (Ki's) for the same set of pyrazole derivatives were also determined for human liver alcohol dehydrogenase. The best-fitting equations were derived to relate the Ki's to the chemical nature of substituents. These quantitative structure-activity relationships show that pyrazoles with stronger electron-withdrawing substituents are weaker inhibitors both for the enzyme in vitro and, to an equal extent, for ethanol oxidation by intact cells. Inhibitor effectiveness is also dependent on substituent hydrophobicity, but, while increasing hydrophobicity makes stronger inhibitors of the enzyme in vitro, it can diminish the effectiveness in vivo by decreasing permeability through the cell membrane. A structure-activity analysis of published Ki's for pyrazoles acting against human pi-ADH indicates that its active site differs from those in other alcohol dehydrogenases.     

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.     

Stability of immobilized alcohol dehydrogenase prepared using the thermostable enzyme from yeast mitochondria

Alcohol dehydrogenase from yeast was partially purified by heat treatment (70°C, 30 min) and immobilized on porous glass, Enzacryl-TI0 and hornblende. The stabilities of these preparations were studied at 30°C and in the case of Enzacryl-TI0 and hornblende at 50°C also. These stabilities were compared with those of immobilized alcohol dehydrogenase from yeast cytosol. In all cases the mitochondrial enzyme provided the more stable bound enzyme conjugates. However, at 50°C the soluble mitochondrial enzyme was more stable than any of the immobilized derivatives: half-life values were 40, 14 and 8 h for the soluble, Enzacryl-TI0 and hornblende samples, respectively.     

2-Mercaptoethanol as a substrate for liver alcohol dehydrogenase.

An activity was identified in a phosphate buffer extract of calf liver acetone powder which utilized 2-mercaptoethanol and NAD⁺ as substrates and formed NADH as one product. The activity responsible for catalyzing this reaction is associated with calf liver alcohol dehydrogenase based on copurification, similarity in pH optima, and similarity in response to chelating agents and other inactivating agents. Crystalline horse liver alcohol dehydrogenase also catalyzes the formation of NADH from NAD⁺ using 2-mercaptoethanol as the substrate. Although the K_m for mercaptoethanol is much lower than that for ethanol, 30 μm as compared to 0.625 mm, the maximum velocity with mercaptoethanol as the substrate is only 7% of that when ethanol is the substrate. Because of this difference in maximum velocity, 2-mercaptoethanol is an apparent competitive inhibitor with respect to ethanol with crystalline horse liver alcohol dehydrogenase, consistent with ethanol and 2-mercaptoethanol binding at the same site. The apparent K_i for 2-mercaptoethanol is 14 μm. 2-Butanethiol is a competitive inhibitor with respect to both 2-mercaptoethanol and ethanol with horse and beef liver alcohol dehydrogenases.     

Preparations of holodehydrogenases by covalent fixation of NAD+-analogs to alcohol and lactate dehydrogenase

Diazoniumaryl residues which are connected to the adenosine part of the coenzyme NAD⁺ react with amino acid residues of dehydrogenases. These coenzyme analogs bind to the active site of the enzymes. The binary complexes are stabilized by the formation of ternary dead-end complexes with pseudosubstrates. After removal of these pseudosubstrates, the coenzyme analogs remain attached in the vicinity of the active sites by azo bridges. Addition of enzyme substrates to the synthesized holoenzymes causes an immediate reduction of the covalent-bound analogs. Reoxidation can be achieved by pH changes or by addition of adequate substrates. This modification does not cause a strong loss of enzymatic activity of the enzymes. The optical properties of the holoenzymes are in accordance with that of binary NAD⁺ (NADH)-enzyme complexes.     

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.     

Separation and partial characterization of multiple forms of rat liver alcohol dehydrogenase

Rat liver alcohol dehydrogenase was purified and four isoenzyme forms, demonstrated by starch gel electrophoresis, were separated by O-(carboxymethyl)-cellulose chromatography. Each of the isoenzymes had a distinct isoelectric point. All isoenzymes were active with both ethanol (or acetaldehyde) and steroid substrates, and had similar Michaelis-Menten constants for each of the substrates and coenzymes studied. The three isoenzymes with the lowest migration toward the cathode exhibited the same pH optimum of 10.7 for ethanol oxidation, a greater activity with 5 beta-androstan-3 beta-ol-17-one than with ethanol as a substrate, and an unchanged electrophoretic mobility following storage in the presence of 100 microM dithiothreitol. By contrast the isoenzyme with the highest mobility toward the cathode exhibited a pH optimum of 9.5 for ethanol oxidation, a low steroid/ethanol ratio of activity, and converted to the migrating pattern of the two isoenzymes with intermediate mobility when stored. The similarities between the isoenzymes of rat liver alcohol dehydrogenase differ considerably from differences in substrate specificity exhibited by isoenzymes of horse liver alcohol dehydrogenase.     

Preparation of 2,3-seco-5 alpha-cholestane-2,3-diol and 4 alpha-methyl-2,3-seco-5 alpha-cholestane-2,3-diol and its reactions with o-nitrophenyl selenocyanate

The reaction of 2,3-seco-5 alpha-cholestane-2,3-diol and 4 alpha-methyl-2,3-seco-5 alpha-cholestane-2,3-diol with o-nitrophenyl selenocyanate was studied. The diols were synthesized from cholesterol.     

The manipulation of fatty acid composition in L-M cell monolayers supplemented with cyclopentenyl fatty acids

Mouse L-M fibroblasts, grown in a serum-free medium, were supplemented with fatty acids of 16 and 18 carbon chain lengths that contain a cyclopentene ring in the ω position. These fatty acids, unnatural to mammalian systems, were incorporated into the major lipid classes of L-M fibroblasts. Supplementation with the cyclopentenyl fatty acids caused an accumulation of neutral glycerolipids and marked inhibition of cell growth. Following the addition of supplement, the cells became more rounded. Of particular interest was the fact that the phospholipid fraction isolated from treated cells contained cyclic fatty acids that accounted for as much as 24% of the total phospholipid acyl groups. Unlike the pattern of distribution displayed by endogenous natural monoenes, the majority of the cyclic acid present was esterified in the sn-1 position of both phosphatidylcholine and phosphatidylethanolamine. The 18-carbon cyclic fatty acid [chaulmoogric acid, 13-(2-cyclopenten-1-yl)tridecanoic acid] was incorporated at the expense of the endogenous C-16:0, C-18:0, and C-18:1 fatty acids of the glycerophospholipids. The esterification altered the ratio of saturated to unsaturated acyl groups in the cellular phospholipids. No biochemical modification of chaulmoogric acid was detected.Our results imply that incorporation of unnatural fatty acid analogs, such as chaulmoogric acid, into cellular membranes would alter the functional properties of biological membranes that are dependent on membrane fluidity and structural organization.     

Inhibition of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase by glyoxylate

The overall reaction catalyzed by the pyruvate dehydrogenase complex from rat epididymal fat tissue is inhibited by glyoxylate at concentrations greater than 10 μm. The inhibition is competitive with respect to pyruvate; K_i was found to be 80 μm. Qualitatively similar results were observed using pyruvate dehydrogenase from rat liver, kidney, and heart. Glyoxylate also inhibits the pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat, with the inhibition being readily detectable using 50 μm glyoxylate. These effects of glyoxylate are largely reversed by millimolar concentrations of thiols (especially cysteine) because such compounds form relatively stable adducts with glyoxylate. Presumably these inhibitions by low levels of glyoxylate had not been previously observed, because others have used high concentrations of thiols in pyruvate dehydrogenase assays. Since the inhibitory effects are seen with suspected physiological concentrations, it seems likely that glyoxylate partially controls the activity of pyruvate dehydrogenase in vivo.     

Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3.

Kidney homogenates from chicks fed a vitamin D-deficient diet for 10 days and supplemented with 6.5 nmol of vitamin D₃ 48 hr prior to sacrifice metabolized $\text{in}\text{vitro}\text{[}{}^3\text{H}\text{]}$-25-hydroxyvitamin D₃ (25-OH-D₃) to 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified [³H]-24,25-(OH)₂-D₃, 3 similar metabolites (peaks A′, C′ and E′) were produced. On high pressure liquid chromatography, peaks A, C and E migrated to exactly the same respective positions as peaks A′, C′ and E′. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [³H]-25-OH-D₃ or [³H]-24,25-(OH)₂-D₃. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)₂-D₃ in the kidney of chicks supplemented with vitamin D₃.     

Differences in the polymorphic forms of metallothionein

Metallothionein induced in rat liver by metal ions can be resolved into two forms, referred to as isoforms I and II. These polymorphic forms differ in several properties. Sequence analysis of the CNBr-digested thionein isoforms revealed seven differences in the first 25 amino acids between isoforms I and II of Cd-induced thionein. The sequence of the first 25 amino acids in isoform II of Cu-induced thionein was identical to that of isoform II of Cd-induced thionein, suggesting that the same polymorphic forms are induced with different metal ions. Isoform II of Cd,Zn-thionein exhibited a reproducibly greater Stokes radius (17 Å) by gel filtration compared to isoform I (16.2 Å), whereas isoform II of Cu-thionein eluted with a Stokes radius of 16.2 Å. The isoforms also differ in Zn-binding affinity. Isoform I of several preparations of Cd,Zn-thionein and Zn-thionein invariably reconstituted greater esterase activity in apo-carbonic anhydrase than did isoform II. In Cd,Zn-thionein samples, only the Zn²⁺ ions were transferred to apo-carbonic anhydrase. Similarity, the Zn²⁺ ions in isoform I were more reactive with EDTA compared to isoform II. The greater reactivity of isoform I with EDTA was evident kinetically from spectrophotometric assays as well as thermodynamically from equilibrium dialysis experiments. The significance of these differences is unclear, but it is conceivable that the dissimilarities in the conformational and Zn-binding affinity of rat liver metallothionein may correlate with a functional distinction.