Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

A Metaphosphate-dependent Nicotinamide Adenine Dinucleotide Kinase from Brevibacterium ammoniagenes

The enzyme utilizing metaphosphate for nicotinamide adenine dinucleotide phosphorylation was purified 500-fold from B. ammoniagenes and its properties were studied. The isolated enzyme appeared homogeneous on disc gel electrophoresis; its molecular weight was determined to be 9.0 × 10⁴ by gel filtration. This enzyme specifically phosphorylated nicotinamide adenine dinucleotide at the optimum pH at 6.0. Of phosphoryl donors tested, metaphosphate was most effective for the reaction, and adenosine-5′-triphosphate was less effective. The activity was inhibited by adenosine-5′-monophosphate, adenosine-5′-diphosphate or reduced pyridine nucleotides. The enzyme did not exhibit catalytic activity in the absence of a divalent cation. We concluded that the enzyme phosphorylating nicotinamide adenine dinucleotide in the presence of metaphosphate is distinct from adenosine-5′-triphosphate-dependent nicotinamide adenine dinucleotide kinase, and tentatively designated it metaphosphate-dependent nicotinamide adenine dinucleotide kinase.     

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate-Dependent Enzyme

Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5′-phosphate-containing enzyme, but the requirement of pyridoxal-5′-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5′-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5′-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5′-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5′-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5′-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5′ phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5′-phosphate that is present in the mammalian and bacterial enzymes.     

Energy-linked Functions of Submitochondrial Particles Prepared from Mung Bean Mitochondria

Submitochondrial particles from mung bean mitochondria (Phaseolus aureus) are able to catalyze an energy-linked reduced nicotinamide adenine dinucleotide-nicotinamide adenine dinucleotide phosphate transhydrogenase reaction supported by ATP or by aerobically generated high energy intermediates. The energy transfer pathway appears to differ from that utilized for oxidative phosphorylation.     

Nuclear magnetic resonance studies on pyridine dinucleotides. The pH dependence of the carbon-13 nuclear magnetic resonance of NAD+ analogs.

The pH dependence of the ¹³C chemical shifts for nicotinamide adenine dinucleotide (NAD⁺), thionicotinamide adenine dinucleotide (TNAD⁺), pyridine adenine dinucleotide (PyrAD⁺), N-methyl-nicotinamide adenine dinucleotide (N-Me-NAD⁺), acetylpyridine adenine dinucleotide (AcPyAD⁺), nicotinamide hypoxanthine dinucleotide (NHD⁺), and nicotinamide adenine dinucleotide phosphate (NADP⁺) are reported. In these analogs the ¹³C chemical shifts of the pyridinium moiety reflect the pK_a of the opposing purine base, while the ¹³C chemical shift dependence on pD for the pyridinium carbons of nicotinamide mononucleotide (NMN⁺) and adenosine monophosphate (AMP), 1,4-dihydronicotinamide adenine dinucleotide (NADH), 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH), and nicotinic acid adenine dinucleotide (N(a)AD⁺) are not influenced by the adenine ring in the pD range tested. Through the use of ¹³C-labeled NAD⁺, the source of the pH dependence of the ¹³C chemical shifts was shown to be intramolecular in origin. However, serious doubt is cast on the utility of employing the pD dependence of chemical shift data to determine the nature of solution conformers or their relative populations.     

Effects induced by rotenone during aerobic growth ofParacoccus denitrificans in continuous culture

Paracoccus denitrificans was grown aerobically in chemostat culture in the presence of rotenone. After 6 to 10 generation times, cells showed an oxygen uptake which was completely rotenone-insensitive after removal of rotenone by washing with bovine serum albumin containing medium.The H⁺/O ratio of these cells for endogenous substrates decreased from about 7.50 to 3.95. The latter ratio was similar to the value obtained for starved cells oxidizing exogenous succinate, indicating that site I phosphorylation was absent in these rotenone-insensitive cells.Membrane particles prepared from these cells showed an 80% decrease in activity of reduced nicotinamide adenine dinucleotide oxidase and reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase, while also the kinetic behaviour of the reduced nicotinamide adenine dinucleotide dehydrogenase in the reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase assay was changed. Moreover the reduced nicotinamide adenine dinucleotide oxidase activity was practically rotenone-insensitive.Electron paramagnetic resonance spectroscopy on membrane particles from rotenone-insensitive cells at 15 K revealed that the resonance lines atg _z 2.05 andg _y g _x 1.92 arising from iron-sulfur center 2 were undetectable. The intensities of the other electron paramagnetic resonance signals originating from reduced nicotinamide adenine dinucleotide dehydrogenase linked iron-sulfur centers were only slightly diminished.These observations confirm our previous suggestion that site I phosphorylation, rotenone sensitivity and the presence of iron-sulfur center 2 are correlated.Abbreviations EPR electron paramagnetic resonance - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - ATP adenosine triphosphate     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Effects of 2-Butylmalonate, 2-Phenylsuccinate, Benzylmalonate, and p-Iodobenzylmalonate on the Oxidation of Substrates by Mung Bean Mitochondria

The effect of inhibitors of carboxylic acid anion transport on the oxidation of substrates by mung bean (Phaseolus aureus) mitochondria was investigated. The oxidation of malate in the presence of either glutamate or cysteine sulfinate was inhibited by 2-butylmalonate, 2-phenylsuccinate, benzylmalonate, and p-iodobenzylmalonate in both intact and broken mitochondria. The oxidation of succinate, on the other hand, was inhibited in intact but not in broken mitochondria. The oxidation of reduced nicotinamide adenine dinucleotide was inhibited only by p-iodobenzylmalonate. This inhibition occurred only in coupled mitochondria and could be reversed by the addition of adenosine diphosphate.     

The primary structure of sheep liver cytosolic serine hydroxymethyltransferase and an analysis of the evolutionary relationships among serine hydroxymethyltransferases

The complete amino-acid sequence of sheep liver cytosolic serine hydroxymethyltransferase was determined from an analysis of tryptic, chymotryptic, CNBr and hydroxylamine peptides. Each subunit of sheep liver serine hydroxymethyltransferase consisted of 483 amin-acid residues. A comparison of this sequence with 8 other serine hydroxymethyltransferases revealed that a possible gene duplication event could have occurred after the divergence of animals and fungi. This analysis also showed independent duplication of SHMT genes in Neurospora crassa. At the secondary structural level, all the serine hydroxymethyltransferases belong to the α/β category of proteins. The predicted secondary structure of sheep liver serine hydroxymethyltransferase was similar to that of the observed structure of tryptophan synthase, another pyridoxal 5′-phosphate containing enzyme, suggesting that sheep liver serine hydroxymethyltransferase might have a similar pyridoxal 5′-phosphate binding domain. In addition, a conserved glycinerich region, G L Q G G P, was identified in all the serine hydroxymethyltransferases and could be important in pyridoxal 5′-phosphate binding. A comparison of the cytosolic serine hydroxymethyltransferases from rabbit and sheep liver with other proteins sequenced from both these sources showed that serine hydroxymethyltransferase was a highly conserved protein. In was slightly less conserved than cytochrome c but better conserved than myoglobin, both of which are well known evolutionary markers. C67 and C203 were specifically protected by pyridoxal 5′-phosphate against modification with [¹⁴C]iodoacetic acid, while C247 and C261 were buried in the native serine hydroxymethyltransferase. However, the cysteines are not conserved among the various serine hydroxymethyltransferases. The exact role of the cysteines in the reaction catalyzed by serine hydroxymethyltransferase remains to be elucidated.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration–activity relationship is the hyperbolic type and the apparent K_m and K_cat for the tested substrates were calculated.     

A new spin-labelled analogue of nicotinamide adenine dinucleotide

The preparation of a spin-labelled analogue of nicotinamide adenine dinucleotide, 3-(4′,4′,5′,5′-tetramethyl-3′-oxide-1′-oxyl-2′-imidazolinyl) pyridine adenine dinucleotide, is descrebed. This compound was obtained by treatment of 3-carboxaldehyde pyridine adenine dinucleotide with 2,3-dimethyl-2,3-dihydroxylaminobutane followed by oxidation with lead dioxide. The interpretation of the particular electron spin resonance spectra of this nitronylnitroxide (five lines) in terms of the rotational correlation time of the radical is shown to be possible. The high stability of this compound makes its use in NAD⁺-dependent biological systems feasible.     

Evidence for a physiologically active nicotinamide phosphoribosyltransferase in cultured human fibroblasts

Nicotinamide phosphoribosyltransferase, (EC 2.4.2.12) was examined in extracts of diploid human fibroblasts grown in culture. The enzyme was found to have an apparent Km for nicotinamide of 1.6 × 10^?6M, to be specific for nicotinamide, stimulated by adenosine triphosphate (ATP) and inhibited by nicotinamide adenine dinucleotide (NAD). In these respects it is very similar to rat liver nicotinamide phosphoribosyltransferase but not like the enzyme previously observed in human tissue extracts which had a Km for nicotinamide of approximately 0.1 M and was insensitive to ATP. Discovery of this enzyme activity supports previous studies using radiolabeled nicotinamide which show that human fibroblasts can incorporate nicotinamide into NAD directly through nicotinamide mononucleotide.     

Role of molybdenum in nitrate reduction by chlorella

Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The whole enzyme nicotinamide adenine dinucleotide nitrate reductase is formed by cells grown in the absence of added molybdate, but only its first activity (nicotinamide adenine dinucleotide diaphorase) is functional. The second activity of the complex, which subsequently participates also in the enzymatic transfer of electrons from nicotinamide adenine dinucleotide to nitrate (FNH₂-nitrate reductase), depends on the presence of molybdenum. Neither molybdate nor nitrate is required for nitrate reductase synthesis de novo, but ammonia acts as a nutritional repressor of the complete enzyme complex. Under conditions which exclude de novo synthesis of nitrate reductase, the addition of molybdate to molybdenum-deficient cells clearly increases the activity level of this enzyme, thus suggesting in vivo incorporation of the trace metal into the pre-existing inactive apoenzyme.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline dehydrogenases in hyperthermophilic archaea: distribution,function, structure,and application

Dye-linked l-proline dehydrogenase (ProDH) catalyzes the oxidation of l-proline to ∆¹-pyrroline-5-carboxylate (P5C) in the presence of artificial electron acceptors. The enzyme is known to be widely distributed in bacteria and eukarya, together with nicotinamide adenine dinucleotide (phosphate)-dependent P5C dehydrogenase, and to function in the metabolism of l-proline to l-glutamate. In addition, over the course of the last decade, three other types of ProDH with molecular compositions completely different from previously known ones have been identified in hyperthermophilic archaea. The first is a heterotetrameric αβγδ-type ProDH, which exhibits both ProDH and reduced nicotinamide adenine dinucleotide dehydrogenase activity and includes two electron transfer proteins. The second is a heterooctameric α₄β₄-type ProDH, which uses flavin adenine dinucleotide, flavin mononucleotide, adenosine triphosphate, and Fe as cofactors and creates a new electron transfer pathway. The third is a recently identified homodimeric ProDH, which exhibits the greatest thermostability among these archaeal ProDHs. This minireview focuses on the functional and structural properties of these three types of archaeal ProDH and their distribution in archaea. In addition, we will describe the specific application of hyperthermostable ProDH for use in a biosensor and for DNA sensing.     

Purification and some properties of carbon monoxide dehydrogenase from Pseudomonas carboxydohydrogena

A soluble yellow CO dehydrogenase from CO-autotrophically grown cells of Pseudomonas carboxydohydrogena was purified 35-fold in seven steps to better than 95% homogeneity with a yield of 30%. The final specific activity was 180 μmol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, nicotinamide adenine dinucleotide (phosphate), flavin mononucleotide, and flavin adenine dinucleotide were not reduced by the enzyme, but methylene blue, thionin, and toluylene blue were reduced. The molecular weight of native enzyme was determined to be 4 × 10⁵. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed at least three nonidentical subunits of molecular weights 14,000 (α), 28,000 (β), and 85,000 (γ). The ratio of densities of each subunit after electrophoresis was about 1:2:6 (α/β/γ), suggesting an α₃β₃γ₃ structure for the enzyme. The purified enzyme was free of formate dehydrogenase and nicotinamide adenine dinucleotide-specific hydrogenase activities, but contained particulate hydrogenase-like activity with thionin as electron acceptor. Known metalchelating agents tested had no effect on CO dehydrogenase activity. No divalent cations tested stimulated enzyme activity. The native enzyme does not contain Ni since cells assimilated little ⁶³Ni during growth, and the specific ⁶³Ni content of the enzyme declined during purification. The isoelectric point of the native enzyme was found to be 4.5 to 4.7. The K_m for CO was found to be 63 μM. The spectrum of the enzyme and its protein-free extract revealed that it contains bound flavin. The cofactor was flavin adenine dinucleotide based on enzyme digestion and thin-layer chromatography. One mole of native enzyme contains at least 3 mol of noncovalently bound flavin adenine dinucleotide.     

Catabolism of Phloroglucinol by the Rumen Anaerobe Coprococcus

A rumen isolate, Coprococcus, sp. Pe₁5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K_m values of 3.0 × 10⁻⁵ M and 29.0 × 10⁻⁵ M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.     

Purification and Some Properties of Adenosine (Phosphate) Deaminase from the Liver of the Squid Todarodes pacificus

An enzyme that catalyzed the deamination of adenosine 3′-phenylphosphonate was purified from squid liver to homogeneity as judged by SDS-PAGE. The molecular weight of the enzyme was estimated to be 60,000 by SDS-PAGE and 140,000 by Sephadex G-150 gel filtration. The enzyme deaminated adenosine, 2′-deoxyadenosine, 3′-AMP, and 2′,3′-cyclic AMP, but not adenine, 5′-AMP, 3′,5′-cyclic AMP, ADP, or ATP. The apparent K_m and V_max at pH 4.0 for these substrates were comparable (0.11-0.34mM and 179-295 μmol min^?1 mg^?1, respectively). The enzyme had maximum activity at pH 3.5-4.0 for adenosine 3′-phenylphosphonate, at pH 5.5 for adenosine and 2′-deoxyadenosine, and at pH 4.0 for 2′,3′-cyclic AMP and 3′-AMP when the compounds were at concentration of 0.1 mM. The K_m at 4.0 and 5.5 for each substrate varied, but the Vmax were invariant. These results indicated that the squid enzyme was a novel adenosine (phosphate) deaminase with a unique substrate specificity.     

Purification and regulatory properties of mung bean (vigna radiata L.) serine hydroxymethyltransferase

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C₁ fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto.     

A new ternary complex between horse liver alcohol dehydrogenase, NAD+, and acetone

Acetone was found to form a dead-end ternary complex with horse liver alcohol dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD⁺) when the reactants were incubated for a long time at relatively high concentrations. The complex formation was demonstrated by measuring the increase in absorbance at 320 nm, the quenching of protein fluorescence, and the loss of enzyme activity. Since acetone is a substrate of liver alcohol dehydrogenase, and the presence of acetaldehyde or pyrazole prevents acetone from forming the dead-end complex with liver alcohol dehydrogenase and NAD⁺, the acetone molecule in the complex may be bound to the substrate binding site of liver alcohol dehydrogenase. The dissociation of the complex was demonstrated by prolonged dialysis or by addition of reduced nicotinamide adenine dinucleotide (NADH) and iso-butyramide. A modified nicotinamide adenine dinucleotide was obtained as a main product from the dead-end complex after dissociation of the complex or denaturation of the apoenzyme. The modified nicotinamide adenine dinucleotide was found to exhibit an absorption spectrum similar to that of NADH; however, it was not oxidizable by liver alcohol dehydrogenase in the presence of acetaldehyde and exhibited no fluorescence.