Degradation of nicotinamide adenine dinucleotide in cell suspension cultures

Metabolites of [carbonyl-¹⁴C]-NAD in cell suspension cultures of mung bean, soybean and garbanzo bean are trigonelline and compounds of the pyridine nucleotide cycle. Degradation of nicotinate does not occur. In parsley cell cultures nicotinate degradation and formation of nicotinic acid N-α-l-arabinoside were observed. These conjugates are alternative reservoir forms of nicotinic acid. The adenine moiety of NAD is degraded in cell cultures via hypoxanthine-xanthine-allantoin-allantoic acid, with accumulation of the latter two compounds.     

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities.

Extracts of Salmonella typhimurium were chromatographed by using Sephadex G-150 to separate the various enzymes involved with pyridine nucleotide cycle metabolism. This procedure revealed a previously unsuspected nicotinamide adenine dinucleotide (NAD) glycohydrolase (EC 3.2.2.5) activity, which was not observed in crude extracts. In contrast to NAd glycohydrolase, NAD pyrophosphatase (EC 3.6.1.22) was readily measured in crude extracts. This enzyme possessed a native molecular weight of 120,000. Other enzymes examined included nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.00), molecular weight of 43,000; NMN glycohydrolase (EC 3.2.2.14), molecular weight of 67,000; nicotinic acid phosphoribosyl transferase (EC 2.4.2.11), molecular weight of 47,000; and nicotinamide deamidase (EC 3.5.1.19), molecular weight of 35,000. NMN deamidase and NMN glycohydrolase activities were both examined for end product repression by measuring their activities in crude extracts prepared from cells grown with and without 10(-5) M nicotinic acid. No repression was observed with either activity. Both activities were also examined for feedback inhibition by NAD, reduced NAD, and NADP. NMN deamidase was unaffected by any of the compounds tested. NMN glycohydrolase was greatly inhibited by NAD and reduced NAD, whereas NADP was much less effective. Inhibition of NMN glycohydrolase was found to level off at an NAD concentration of ca. 1 mN, the approximate intracellular concentration of NAD.     

Light-induced conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide phosphate in higher plant leaves

Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.     

Electrochemical regeneration of nicotinamide adenine dinucleotide.

Reduced nicotinamide adenine dinucleotide (NADH) has been characterized electrochemically by solid electrode voltammetry and controlled potential electrolysis. Photometric and enzymatic assay showed that enzymatically active nicotinamide adenine dinucleotide (NAD-+) could be regenerated electrolytically from its reduced form without the use of so-called electron mediators. Complete regeneration of enzymatically active NAD can be expected in pyrophosphate buffers and phosphate buffers during the electrolysis. Advantages of electrochemical regeneration of coenzymes are discussed, especially with regard to immobilization of enzymes.     

Effect of nicotinamide adenine dinucleotide on the membrane-associated reduced nicotinamide adenine dinucleotide oxidase of Mycobacterium phlei.

NAD+ had a biphasic effect on the NADH oxidase activity in electron transport particles from Mycobacterium phlei. The oxidase was inhibited competitively by NAD+ at concentrations above 0.05 mM. NAD+ in concentrations from 0.02 to 0.05 mM resulted in maximum stimulation of both NADH oxidation and oxygen uptake with concentrations of substrate both above and below the apparent K-M. Oxygen uptake and cyanide sensitivity indicated that the NAD+ stimulatory effect was linked to the terminal respiratory chain. The stimulatory effect was specific for NAD+. NAD+ was also specific in protecting the oxidase during heating at 50 degrees and against inactivation during storage at 0 degrees. NAD+ glycohydrolase did not affect stimulation nor heat protection of the NADH oxidase activity if the particles were previously preincubated with NAD+. Binding studies revealed that the particles bound approximately 3.6 pmol of [14C1NAD+ per mg of electron transport particle protein. Although bound NAD+ represented only a small fraction of the total added NAD+ necessary for maximal stimulation, removal of the apparently unbound NAD+ by Sephadex chromatography revealed that particles retained the stimulated state for at least 48 hours. Further addition of NAD+ to stimulated washed particles resulted in competitive inhibition of oxidase activity. Desensitization of the oxidase to the stimulatory effect of NAD+ was achieved by heating the particles at 50 degrees for 2 min without appreciable loss of enzymatic activity. Kinetic studies indicated that addition of NADH to electron transport particles prior to preincubation with NAD+ inhibited stimulation. In addition, NADH inhibited binding of [14C]NAD+. The utilization of artificial electron acceptors, which act as a shunt of the respiratory chain at or near the flavoprotein component, indicated that NAD+ acts as at the level of the NADH dehydrogenase at a site other than the catalytic one resulting in a conformational change which causes restoration as well as protection of oxidase activity.     

Bull semen nicotinamide adenine dinucleotide nucleosidase. VI. Fluorescence studies of the enzyme with reduced nicotinamide adenine dinucleotide

The binding of NADH to bull semen NAD nucleosidase was observed to be accompanied by a considerable enhancement of the fluorescence of NADH. The fluorescence enhancement observed in the binding of NADH to the enzyme was utilized to study the stoichiometry of binding of this compound to the enzyme. Results obtained from the fluorescence titration of the enzyme with NADH indicated the binding of one mole of NADH per mole of enzyme (36,000 g). The dissociation constant for the enzyme-NADH complex was determined to be 2.52 × 10^?6m. NADH was also found to be a very effective competitive inhibitor of the NADase-catalyzed hydrolysis of NAD, and the inhibitor dissociation constant (K_I) for the enzyme-NADH complex was determined to be 2.99 × 10^?6m which was in good agreement with the value obtained from the fluorescence titration experiments.     

The proton-translocating nicotinamide adenine dinucleotide transhydrogenase

H⁺-transhydrogenase couples the reversible transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. There are separate sites on the enzyme for the binding of NAD(H) and of NADP(H). There are some indications of the position of the binding sites in the primary sequence of the enzymes from mitochondria andEscherichia coli. Transfer of hydride ion equivalents only proceeds when a reduced and an oxidized nucleotide are simultaneously bound to the enzyme. When p=0 the rate of interconversion of the ternary complexes of enzyme and nucleotide substrates is probably limiting. An increase in p accelerates the rate of interconversion in the direction of NADH NADP⁺ until another kinetic component, possibly product release, becomes limiting. The available data are consistent with either direct or indirect mechanisms of energy coupling.Abbreviations DCCD N N¹-dicyclohexylcarbodiimide - FSBA 5¹-[p-(fluorosulfonyl)benzoyl] adenosine - FCCP carbonylcyanide-p-fluoromethoxyphenylhydrazone - H⁺-Thase H⁺-transhydrogenase - thio-NADP⁺ thionicotinamide adenine dinucleotide phosphate - AcPdAd⁺ 3-acetylpyridine adenine dinucleotide - p proton electrochemical gradient - membane potential - pH pH difference across the membrane     

Electrochemical regeneration of nicotinamide adenine dinucleotide

Reduced nicotinamide adenine dinucleotide (NADH) has been characterized electrochemically by solid electrode voltammetry and controlled potential electrolysis. Photometric and enzymatic assay showed that enzymatically active nicotinamide adenine dinucleotide (NAD⁺) could be regenerated electrolytically from its reduced form without the use of so-called electron mediators. Complete regeneration of enzymatically active NAD can be expected in pyrophosphate buffers and phosphate buffers during the electrolysis. Advantages of electrochemical regeneration of coenzymes are discussed, especially with regard to immobilization of enzymes.     

Protein binding of nicotinamide adenine dinucleotide and regulation of nicotinamide adenine dinucleotide glycohydrolase activity in homogenates of rabbit skeletal muscle

Gel-permeation chromatography and ultrafiltration have been used to study the free and bound forms of NAD in crude extracts prepared from rabbit muscle. Both techniques indicate that over 80% of the endogenous NAD is free.Nicotinamide inhibits the destruction of NAD in muscle homogenates (50% inhibition at 1.6 mm nicotinamide). In the absence of nicotinamide, there is a rapid destruction of free NAD, but a more gradual destruction of bound NAD. The latter result confirms earlier findings that bound NAD is protected from the hydrolytic action of NADase. However, this protection is unlikely to constitute an important mechanism for controlling NADase activity in muscle homogenates because such a small proportion of the endogenous NAD is bound.In the absence of nicotinamide, NAD also disappears rapidly from minced muscle. Interestingly, the NAD/NADH ratio remains constant (NAD/NADH = 18.1–18.5) during the disappearance of NAD in minced muscle. Upon homogenization of the mince, the NAD/NADH ratio abruptly decreases, then slowly increases during subsequent incubation. The latter rise in NAD/NADH ratio appears to be independent of absolute changes in NAD concentration brought about by the action of NADase or the addition of exogenous NAD.     

Isolation and characterization of yeast nicotinamide adenine dinucleotide kinase.

NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23) from yeast has been purified utilizing ion-exchange and NAD+-agarose affinity chromatography to give a 2100-fold purification. The apparent homogeneity of the enzyme preparation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a subunit molecular weight of 31,000, and a native molecular weight of 124,000, and is, thus, probably a tetramer. The single form of the enzyme has an apparent isoelectric point of 5.85. Initial velocity studies in the forward direction with both substrates gave intersecting Lineweaver-Burk plots, and this suggests a sequential mechanism in which both substrates are bound before products are released. Replots of these data were linear and gave Km values for NAD+ and ATP of 0.68 mM and 2.3 mM, respectively.     

Formation of reduced nicotinamide adenine dinucleotide peroxide

Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.     

Cytochrome c-induced cytosolic nicotinamide adenine dinucleotide oxidation,mitochondrial permeability transition,and apoptosis

A catalytic amount of cytochrome c (cyto-c) added to the incubation medium of isolated mitochondria promotes the transfer of reducing equivalents from extramitochondrial nicotinamide adenine dinucleotide in its reduced state (NADH) to molecular oxygen inside the mitochondria, a process coupled to the generation of a membrane potential. This mimics in many aspects the early stages of those apoptotic pathways characterized by the persistence of mitochondrial membrane potential but with cyto-c already exported into the cytosol. In cyclosporin-sensitive and calcium-induced mitochondrial permeability transition (MPT) a release of cyto-c can also be observed. However, in MPT uncoupled respiration associated with mitochondrial swelling and preceded by the complete dissipation of the membrane potential which cannot be restored with ATP addition or any other source of energy is immediately activated. The results obtained and discussed with regard to intactness of mitochondrial preparations indicate that MPT could be an apoptotic event downstream but not upstream of cyto-c release linked to the energy-requiring processes. In the early stages of apoptosis cytosolic cyto-c participates in the activation of caspases and at the same time can promote the oxidation of cytosolic NADH, making more energy available for the correct execution of the cell death program. This hypothesis is not in contrast with available data in the literature showing that cyto-c is present in the cytosol of both control and apoptosis-induced cultured cell lines.     

Spectral characterization of a fluorescent nicotinamide adenine dinucleotide analog: 3-Aminopyridine adenine dinucleotide

Both absorption and fluorescence properties of 3-amino pyridine adenine dinucleotide (AAD) were examined. The shape of the AAD fluorescence emission spectrum, maximal at 425 nm, remains unchanged over the pH range 2 to 11, indicating that there is only one detectable emitting species. AAD fluorescence increases as the pH decreases, with an apparent pK_a of about 3.5. The absorption-pH profile indicates a pK_a of about 3.3 for the ground state of AAD. Effects of organic solvents on AAD fluorescence are somewhat diverse. The low fluorescence quantum yield of 0.022 corresponds well with the short lifetime of 1.15 ns at 23 °C in neutral aqueous solution. The steady-state polarization of AAD in water and that at infinite viscosity were determined at 23 °C to be 0.037 and 0.083, respectively. Since a smaller value of polarization for either donor or acceptor leads to a better estimate of the orientation factor for dipole-dipole interaction, AAD appears to be particularly suitable for energy transfer studies. Similar to NADH, AAD also assumes a folded conformation in aqueous solution. This is evident from effects of temperature and hydrolysis by phosphodiesterase on absorption and/or fluorescence properties of AAD. Energy transfer from the adenine group to the 3-aminopyridine ring has been detected to occur in aqueous solution at 23 °C with an efficiency of about 0.12, corresponding to a distance of 7.5 Å between these two ring moieties.     

Pyridine nucleotide cycle of Salmonella typhimurium: in vivo recycling of nicotinamide adenine dinucleotide

The relative contribution of the two known pyridine nucleotide cycles of Salmonella typhimurium towards the intracellular recycling of nicotinamide adenine dinucleotide was determined. The results indicate that intracellular nicotinamide adenine dinucleotide is recycled by both the four-membered pyridine nucleotide cycle (PNC IV) and the six-membered pyridine nucleotide cycle (PNC VI) with a relative contribution of 60 to 69% and 31 to 40%, respectively. These studies also revealed a nicotinic acid mononucleotide-degradative activity which converts nicotinic acid mononucleotide to nicotinic acid. This represents the first demonstration of a functional PNC IV pathway in S. typhimurium.     

Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogues.

The rate of the primary acid modification reaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analogues has been studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent deuterium isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a proton transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary first step in the case of the primary acid modification reaction of beta-NADH, in which beta to alpha anomerization takes place.     

Turnover at nicotinamide adenine dinucleotide in cultures of human cells.

The rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C-adenine. In one set of experiments, cells were labeled by growth in medium containing 14C-adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C-adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C-adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C-adenine and the rate of entry of 14C-adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C-adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse-chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half-life of an intact NAD molecule is 60 +/- 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.