The enthalpy change for the reduction of nicotinamide–adenine dinucleotide

The heat of the reaction NAD(+)+propan-2-ol=NADH+acetone+H(+) was determined to be 42.5+/-0.6kJ/mol (10.17+/-0.15kcal/mol) from equilibrium measurements at 9-42 degrees C catalysed by yeast alcohol dehydrogenase. With the aid of thermochemical data for acetone and propan-2-ol the values of DeltaH=-29.2kJ/mol (-6.99kcal/mol) and DeltaG(0)=22.1kJ/mol (5.28kcal/mol) are derived for the reduction of NAD (NAD(+)+H(2)=NADH+H(+)). These values are consistent with analogous but less accurate data for the ethanol-acetaldehyde reaction. Thermodynamic data for the reduction of NAD and NADP are summarized.     

Relation between the oxidation state of nicotinamide–adenine dinucleotide and the metabolism of spermatozoa

1. The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD(+) and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP(+) and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100x[NAD(+)/(NAD(+)+NADH)], without altering the total content of NAD(+)+NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25 degrees C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25 degrees C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD(+)+NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ;Cold shock', resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14 degrees C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater.     

Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD(+) through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD(+) (probably through the 8 position of the adenine residue) to a number of different spacer-arm-agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD(+) derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD(+). Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD(+)-binding site of this enzyme. Problems associated with the use of immobilized derivatives for enzyme binding and mechanistic studies are briefly discussed.     

Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm

1. A study has been made of the ability of rat liver in vivo to maintain equilibrium in the combined glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactate dehydrogenase reactions, i.e. in the system: [Formula: see text] Attempts were made to upset equilibrium. The [lactate]/[pyruvate] ratio was rapidly changed by injection of ethanol or crotyl alcohol, and the value of [ATP]/[ADP][HPO(4) (2-)] was rapidly changed by injection of ethionine or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. 2. The concentrations of the metabolites occurring in the above equation were measured in freeze-clamped liver. 3. Although the injected agents caused large changes in the concentrations of the individual components, near-equilibrium in the system was maintained, as indicated by the fact that the value of [ATP]/[ADP][HPO(4) (2-)], referred to as the phosphorylation state of the adenine nucleotides, measured directly agreed with the value calculated for equilibrium conditions from the above equation. 4. The results are discussed and taken to confirm that the order of magnitude of the value of the redox state of the cytoplasmic NAD couple in rat liver is controlled by the phosphorylation state of the adenine nucleotide system.     

The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

1. Superovulated rat ovary was found to contain high activities of NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD-malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP-citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective K(m) values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP-malate dehydrogenase or NADP-isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed.     

Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide–adenine dinucleotide in barley

1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

The redox state of free nicotinamide–adenine dinucleotide phosphate in the cytoplasm of rat liver

1. The concentrations of the oxidized and reduced substrates of the ;malic' enzyme (EC 1.1.1.40) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in freeze-clamped rat livers. By assuming that the reactants of these dehydrogenase systems are at equilibrium in the cytoplasm the [free NADP(+)]/[free NADPH] ratio was calculated. The justification of the assumption is discussed. 2. The values of this ratio obtained under different nutritional conditions (well-fed, 48hr.-starved, fed with a low-carbohydrate diet, fed with a high-sucrose diet) were all of the same order of magnitude although characteristic changes occurred on varying the diet. The value of the ratio fell on starvation and on feeding with the low-carbohydrate diet and rose slightly on feeding with the high-sucrose diet. 3. The mean values of the ratio were calculated to be between 0.001 and 0.015, which is about 100000 times lower than the values of the cytoplasmic [free NAD(+)]/[free NADH] ratio. 4. The differences in the redox state of the two nicotinamide-adenine dinucleotide couples can be explained on a simple physicochemical basis. The differences are the result of equilibria that are determined by the equilibrium constants of a number of highly active readily reversible dehydrogenases and transaminases and the concentrations of the substrates and products of these enzymes. 5. The decisive feature is the fact that the NAD and NADP couples share substrates. This sharing provides a link between the redox states of the two couples. 6. The application of the method of calculation to data published by Kraupp, Adler-Kastner, Niessner & Plank (1967), Goldberg, Passonneau & Lowry (1966) and Kauffman, Brown, Passonneau & Lowry (1968) shows that the redox states of the NAD and NADP couples in cardiac-muscle cytoplasm and in mouse-brain cytoplasm are of the same order as those in rat liver. 7. The determination of the equilibrium constant at 38 degrees , pH7.0 and I 0.25 (required for the calculation of the [free NADP(+)]/[free NADPH] ratio), gave a value of 3.44x10(-2)m for the ;malic' enzyme (with CO(2) rather than HCO(3) (-) as the reactant) and a value of 1.98x10(-2)m(-1) for glutathione reductase.     

Reduced nicotinamide–adenine dinucleotide–nitrite reductase from Azotobacter chroococcum

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.     

The purification and properties of the respiratory-chain reduced nicotinamide–adenine dinucleotide dehydrogenase of Torulopsis utilis

1. An NADH-ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH-ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH-ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH-ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes.     

Transport of reduced nicotinamide–adenine dinucleotide into mitochondria of rat white adipose tissue

Mitochondria from rat white adipose tissue were prepared, exhibiting good respiratory control and P/O ratios. They would not oxidize NADH unless NNN'N'-tetramethyl-p-phenylenediamine was added as a carrier of reducing equivalents. These mitochondria were found to oxidize neither l-glycerol 3-phosphate nor l-glutamate plus l-malate at significant rates. The activity of aspartate aminotransferase in these mitochondria was found to be low compared with that found in rat liver mitochondria. As a consequence of this, the adipose-tissue mitochondria exhibited very low rates of cytoplasmic NADH oxidation in a reconstituted Borst (1962) cycle compared with liver mitochondria.     

Reactions of d-glyceraldehyde 3-phosphate dehydrogenase facilitated by oxidized nicotinamide–adenine dinucleotide

Transient kinetic methods have been used to study the influence of NAD(+) on the rate of elementary processes of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase. In the pH range 5-8 NAD(+) is bound to the enzyme during the following elementary processes of the mechanism: phosphorolysis of the acyl-enzyme, its formation from 1,3-diphosphoglycerate and the enzyme and the formation and breakdown of the glyceraldehyde 3-phosphate-enzyme complex. The rates of these four elementary processes only equal or exceed the turnover rate of the enzyme when NAD(+) is bound and are as much as 10(4) times the rates in the absence of NAD(+). Autocatalysis of the reductive dephosphorylation of 1,3-diphosphoglycerate occurs when glyceraldehyde 3-phosphate release is rate determining because NAD(+) is a reaction product. An important feature of the enzyme mechanism is that the negative-free-energy change of a chemical reaction, acyl-enzyme formation, is linked in a simple way to the positive-free-energy change of a dissociation reaction, NAD(+) release.     

The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH₂ of high purity, the maximal specific rates were similar to those obtained with NAD and NADH₂, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH₂, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH₂ do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH₂. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH₂ than NAD and NADH₂. 5. Phosphate behaves as a competitive inhibitor towards NADP.     

The activities of nicotinamide mononucleotied adenylyltransferase and of nicotinamide–adenine dinucleotide kinase in the livers of rats subjected to different hormonal treatments

1. The activities of NMN adenylyltransferase and of NAD(+) kinase have been measured in the livers of adrenalectomized or alloxan-diabetic rats and in the livers of rats treated with glucagon, pituitary growth hormone or thyroxine. 2. The activities of these enzymes have been compared with the effects of the same treatments on the nicotinamide nucleotide concentrations in the liver. 3. Alloxandiabetes (+37%) and thyroxine (+27%) both increased the activity of NMN adenylyltransferase. The other treatments were without effect on this enzyme. 4. Only thyroxine increased the activity of NAD(+) kinase significantly (+26%) although both adrenalectomy and glucagon tended to increase its activity. 5. The activity of NAD(+) glycohydrolase was measured in the livers of diabetic rats, and in the livers of rats treated with either growth hormone or thyroxine. Of these treatments, only growth hormone altered the enzyme activity (+26%, calculated on a total hepatic activity basis). 6. Female rats had a greater hepatic NAD(+)-kinase activity than males but there was no sex difference with respect to NMN adenylyltransferase. 7. The lack of correlation between the maximum potential activity of these three enzymes and the known changes of the nicotinamide nucleotides in each of the hormone conditions is discussed.     

The isocitrate dehydrogenases of Acinetobacter lwoffi. Studies on the regulation of nicotinamide–adenine dinucleotide phosphate-linked isoenzyme

Of the two NADP-linked isocitrate dehydrogenases in Acinetobacter lwoffi the higher-molecular-weight form, isoenzyme-II, is reversibly stimulated sixfold by low concentrations of glyoxylate or pyruvate. Kinetic results indicate that this stimulation of activity involves both an increase in V(max.) and a decrease in the apparent K(m) values for substrates, most markedly that for NADP(+). Other changes brought about by glyoxylate or pyruvate include a shift in the pH optimum for activity and an increased stability to inactivation by heat or urea. Mixtures of glyoxylate plus oxaloacetate, known to inhibit isocitrate dehydrogenases from other organisms, produce inhibition of both A. lowffi isoenzymes, and do not reflect the stimulatory specificity of glyoxylate for isoenzyme-II. Isoenzyme-II is also stimulated by AMP and ADP, but the activation by glyoxylate or pyruvate is shown to be quite independent of the adenylate activation. Differential desensitization of the enzyme by urea to the two types of activator further supports the view that the enzyme possesses two distinct allosteric regulatory sites. The metabolic significance of the activations is discussed.     

Oxidized and reduced nicotinamide–adenine dinucleotide phosphate in tissue suspensions of rat liver

1. The concentrations of NADP and NADPH(2) in homogenates of rat liver (expressed as mug./g. wet wt. of tissue homogenized) were compared with values obtained from intact samples of liver taken from the same female rat. With 0.25m-sucrose alone as the suspending medium, or in combination with tris buffer or 0.01-0.1m-nicotinamide, considerable decreases in the sum of the NADP+NADPH(2) concentrations were occasionally observed during 30min. storage of homogenates at 0 degrees . However, addition of 0.5m-nicotinamide+5mm-tris buffer to 0.25m-sucrose for use as a suspending medium maintained the sum of the NADP+NADPH(2) concentrations in homogenates at the level found in intact tissue for at least 30min. at 0 degrees . 2. The effects of freezing intact tissue and homogenates in liquid nitrogen before the extraction of NADP and NADPH(2) were studied. Freezing alone appears to convert a significant amount (approx. 30%) of liver NADPH(2) into an equivalent amount of NADP in intact tissue. This is discussed in terms of the ;bound NADP' reported by Burch, Lowry & Von Dippe (1963). 3. The intracellular distributions of NADP and NADPH(2) in intracellular fractions of rat liver were studied by using a modified centrifuging scheme that allows extraction of the isolated fractions to be performed within 45min. of killing the animal. Approx. 50% of the total NADP+NADPH(2) was found in the large-particle fractions and the remaining 50% was mostly in the soluble fraction of the cell. 4. Further investigations are reported on the nature of ;bound NADP' in rat liver. Most of this material appears associated with the ;nuclear' (containing nuclei, debris, erythrocytes etc.) or large-mitochondrial fractions, or both, obtained by low-speed centrifuging of rat-liver homogenates. 5. Although in some experiments the variations produced in the concentration of NADPH(2) present in large-particle fractions were followed by similar changes in that of ;bound NADP', in other cases no such direct relationship was obtained. Addition of phenazine methosulphate, for example, consistently lowered the concentration of NADPH(2) yet raised the concentration of ;bound NADP' in rat-liver mitochondrial fractions.     

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate-NAD(+) compound was followed by the change in E(325); thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6.0 at different temperatures and ionic strengths as functions of pyruvate, NAD(+) and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD(+). It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme-NAD(+) compound involved in the normal catalytic process.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).     

Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron−sulfur clusters of human mitoNEET

The outer mitochondrial membrane protein mitoNEET is a cellular target of the antidiabetic drug pioglitazone. Binding of pioglitazone stabilizes the protein against [2Fe-2S] cluster release. Here, we report that reduced nicotinamide adenine dinucleotide phosphate (NADPH) can bind to homodimeric mitoNEET, influencing the stability of the [2Fe-2S] cluster that is bound within a loop region (Y71?H87) in each subunit. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry experiments demonstrated that NADPH binds weakly to mitoNEET(44?108), a soluble domain of mitoNEET containing residues 44?108. Visible?UV absorption measurements revealed the destabilizing effect of NADP binding on the [2Fe-2S] clusters. Disruption of the three-dimensional structure of mitoNEET(44?108) as a result of decomposition of the iron?sulfur clusters was observed by NMR and circular dichroism experiments. Binding of NADPH facilitated release of the iron?sulfur clusters from the protein at pH≤7.0. Residues K55 and H58 of each subunit of mitoNEET were shown to be involved in NADPH binding. NADPH binding may perturb the interactions of K55 and H58 from one subunit with H87′ and R73′, respectively, from the other subunit, thereby interfering with [2Fe-2S] cluster binding. This may account for the destabilization effect of NADPH binding on the [2Fe-2S] clusters.     

The characterization of two reduced nicotinamide–adenine dinucleotide phosphate-linked aldehyde reductases from pig brain

1. NADPH-linked aldehyde reductase from pig, ox and rat brain exhibits non-linear reciprocal plots when partially purified enzyme preparations are studied. 2. In pig brain this non-linearity is due to the presence of two distinct aldehyde reductases, which can be separated by DEAE-cellulose chromatography. 3. These two enzymes can be distinguished by several criteria, including pH optima, Michaelis constants for substrates and their inhibitor sensitivity. 4. The probable role of these enzymes in the metabolism of the aldehydes derived from the biogenic amines is discussed.