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Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time.

In addition to its role as a redox coenzyme, NAD is a substrate of various enzymes that split the molecule to either catalyze covalent modifications of target proteins or convert NAD into biologically active metabolites. The coenzyme bioavailability may be significantly affected by these reactions, with ensuing major impact on energy metabolism, cell survival, and aging. Moreover, through the activity of the NAD-dependent deacetylating sirtuins, NAD behaves as a beacon molecule that reports the cell metabolic state, and accordingly modulates transcriptional responses and metabolic adaptations. In this view, NAD biosynthesis emerges as a highly regulated process: it enables cells to preserve NAD homeostasis in response to significant NAD-consuming events and it can be modulated by various stimuli to induce, via NAD level changes, suitable NAD-mediated metabolic responses. Here we review the current knowledge on the regulation of mammalian NAD biosynthesis, with focus on the relevant rate-limiting enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.

Nicotinate phosphoribosyltransferase (NaPRT, EC 2.4.2.11) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, the first reaction of the Preiss-Handler pathway for the biosynthesis of NAD+. Even though NaPRT activity has been described to be responsible for the ability of Na to increase NAD+ levels in human cells more effectively than nicotinamide (Nam), so far a limited number of studies on the human NaPRT have appeared. Here, extensive characterization of a recombinant human NaPRT is reported. We determined its major kinetic parameters and assayed the influence of different compounds on its enzymatic activity. In particular, ATP showed an apparent dual stimulation/inhibition effect at low/high substrates saturation, respectively, consistent with a negative cooperativity model, whereas inorganic phosphate was found to act as an activator. Among other metabolites assayed, including nucleotides, nucleosides, and intermediates of carbohydrates metabolism, some showed inhibitory properties, i.e. CoA, several acyl-CoAs, glyceraldehyde 3-phosphate, phosphoenolpyruvate, and fructose 1,6-bisphosphate, whereas dihydroxyacetone phosphate and pyruvate exerted a stimulatory effect. Furthermore, in light of the absence of crystallographic data, we performed homology modeling to predict the protein three-dimensional structure, and molecular docking simulations to identify residues involved in the recognition and stabilization of several ligands. Most of these residues resulted universally conserved among NaPRTs, and, in this study, their importance for enzyme activity was validated through site-directed mutagenesis.

NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed.

Several oat brans (crunchy oat bran, oat bran alone, and oat breakfast cereal) and wheat brans (wheat bran alone, wheat bran powder, wheat bran with malt flavor, bran breakfast cereal, tablet of bran, and tablet of bran with cellulose) used as dietary fiber supplements by consumers were evaluated as alternative antioxidant sources (i) in the normal human consumer, preventing disease and promoting health, and (ii) in food processing, preserving oxidative alterations. Products containing wheat bran exhibited higher peroxyl radical scavenging effectiveness than those with oat bran. Wheat bran powder was the best hydroxyl radical (OH*) scavenger. In terms of hydrogen peroxide (H2O2) scavenging, wheat bran alone was the most effective, while crunchy oat bran, oat bran alone, and oat breakfast cereal did not scavenge H2O2. The shelf life of fats (obtained by the Rancimat method for butter) increased most in the presence of crunchy oat bran. When the antioxidant activity during 28 days of storage was measured by the linoleic acid assay, all of the oat and wheat bran samples analyzed showed very good antioxidant activities. The Trolox equivalent antioxidant capacity (TEAC) assay was used to provide a ranking order of antioxidant activity. The wheat bran results for TEAC (6 min), in decreasing order, were wheat bran powderwheat bran with malt flavoror = wheat bran aloneor = bran breakfast cerealtablet of brantablet of bran with cellulose. The products made with oat bran showed lower TEAC values. In general, avenanthramide showed a higher antioxidant level than each of the following typical cereal components: ferulic acid, gentisic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid, vanillic acid, vanillin, and phytic acid.

Summary Adenosine 5'-tetraphosphate (Ap4) is a ubiquitous metabolite involved in cell signaling in mammals. Its full physiological significance remains unknown. Here we show that two enzymes committed to NAD biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPT), can both catalyze the synthesis and degradation of Ap4 through their facultative ATPase activity. We propose a mechanism for this unforeseen additional reaction, and demonstrate its evolutionary conservation in bacterial orthologs of mammalian NAMPT and NAPT. Furthermore, evolutionary distant forms of NAMPT were inhibited in vitro by the FK866 drug but, remarkably, it does not block synthesis of Ap4. In fact, FK866-treated murine cells showed decreased NAD but increased Ap4 levels. Finally, murine cells and plasma with engineered or naturally fluctuating NAMPT levels showed matching Ap4 fluctuations. These results suggest a role of Ap4 in the actions of NAMPT, and prompt to evaluate the role of Ap4 production in the actions of NAMPT inhibitors.

In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in various stress conditions and the consequent NAD decline is generally accompanied by the activation of NAD biosynthetic pathways to guarantee NAD homeostasis. In the bacterial world a similar scenario has only recently begun to surface. Here we review the current knowledge on the involvement of NAD homeostasis in bacterial stress response mechanisms. In particular, we focus on the participation of both NAD-consuming enzymes (DNA ligase, mono(ADP-ribosyl) transferase, sirtuins, and RNA 2'-phosphotransferase) and NAD biosynthetic enzymes (both de novo, and recycling enzymes) in the response to DNA/RNA damage. As further supporting evidence for such a link, a genomic context analysis is presented showing several conserved associations between NAD homeostasis and stress responsive genes.

NMN deamidase (PncC) is a bacterial enzyme involved in NAD biosynthesis. We have previously demonstrated that PncC is structurally distinct from other known amidohydrolases. Here, we extended PncC characterization by mutating all potential catalytic residues and assessing their individual roles in catalysis through kinetic analyses. Inspection of these residues’ spatial arrangement in the active site, allowed us to conclude that PncC is a serine-amidohydrolase, employing a Ser/Lys dyad for catalysis. Analysis of the PncC structure in complex with a modeled NMN substrate supported our conclusion, and enabled us to propose the catalytic mechanism.

The enzyme NMN adenylyltransferase, leading to NAD synthesis, has been observed for the first time in soluble extracts from the extreme acidothermophilic archaeon Sulfolobus solfataricus. Comparison of its molecular and kinetic properties with those of the enzyme isolated from prokaryotes and eukaryotes revealed significant differences, knowledge of which may contribute to the understanding of metabolic evolutionary mechanisms. The thermophilic enzyme shows a molecular mass of about 66,000 and an isoelectric point of 5.4. The Km values for ATP, NMN and nicotinic acid mononucleotide are 0.08 microM, 1.4 microM and 17 microM, respectively. The enzyme shows a remarkable degree of thermophilicity, with an activation energy of 95 kJ/mol.

A general procedure is described for developing HPLC-based assays for nucleotidases by using a 20-mm C-18 reversed-phase column. The method is applied on the determination of pyrimidine 5′-nucleotidase activity in human erythrocytes lysate and plasma. The activity is detected using isocratic conditions which separate the product (nucleoside) from the substrate (nucleotide) in less than 1 min with 100 mM potassium phosphate, pH 6.0, at a 2 ml/min flow rate. Nucleoside products of the enzymatic reactions are directly quantitated from their ultraviolet absorbances. The activity value closely agreed with that determined by measuring the production of inorganic phosphate. The method does not require prior dialysis of the sample and is 50-fold more sensitive than that based on colorimetric measurements of inorganic phosphate. Furthermore, avoiding the use of radioactive materials offers significant advantages with respect to commonly adopted assays and can be conveniently used for the determination of nucleotidase activity from different sources.

We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.

Beyond its role as an essential coenzyme in numerous oxidoreductase reactions as well as respiration, there is growing recognition that NAD+ fulfills many other vital regulatory functions both as a substrate and as an allosteric effector. This review describes the enzymes involved in pyridine nucleotide metabolism, starting with a detailed consideration of the anaerobic and aerobic pathways leading to quinolinate, a key precursor of NAD+. Conversion of quinolinate and 5'-phosphoribosyl-1'-pyrophosphate to NAD+ and diphosphate by phosphoribosyltransferase is then explored before proceeding to a discussion the molecular and kinetic properties of NMN adenylytransferase. The salient features of NAD+ synthetase as well as NAD+ kinase are likewise presented. The remainder of the review encompasses the metabolic steps devoted to (a) the salvaging of various niacin derivatives, including the roles played by NAD+ and NADH pyrophosphatases, nicotinamide deamidase, and NMN deamidase, and (b) utilization of niacins by nicotinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase.

This review describes the enzymes involved in human pyridine nucleotide metabolism starting with a detailed consideration of their major kinetic, molecular and structural properties. The presentation encompasses all the reactions starting from the de novo pyridine ring formation and leading to nicotinamide adenine dinucleotide (NAD(+)) synthesis and utilization. The regulation of NAD(+) homeostasis with respect to the physiological role played by the enzymes both utilizing NAD(+) through the nonredox NAD(+)-dependent reactions and catalyzing the recycling of the common product, nicotinamide, is discussed. The salient features of other enzymes such as NAD(+) pyrophosphatase, nicotinamide mononucleotide 5'-nucleotidase, nicotinamide riboside kinase and nicotinamide riboside phosphorylase, described under 'miscellaneous', are likewise presented.

This chapter illustrates the way in which kinetic analysis provides convincing evidence for a covalent enzyme-substrate intermediate and also shows that the pyrimidine nucleotidases-I (PN-I) phosphotransferase activity displays higher affinity for oxynucleosides with respect to deoxynucleosides, whereas the contrary seems to be true for pyrimidine nucleotidases-II (PN-II). The essential feature of each intermediate species in the above reactions is that the intermediate must be of sufficient reactivity to be returned to a respective monoester species. Despite the fact that the putative phosphoryl-enzyme intermediate remains to be chemically characterized, the chapter presents evidence that human red cell pyrimidine nucleotidases operate by so-called “branched mechanisms,” with water and pyrimidine nucleosides competing as phosphoryl acceptors. Beyond the interesting catalytic features of PN-I and PN-II, the spectrum of substrates with which phosphotransfer activity can be observed may also prove to be invaluable in comprehending the action of antineoplastic agents and other unphosphorylated prodrugs. Although most cells presumably possess the ability to prevent the formation of undesirable metabolic by-products, the transfer reactions described in the chapter may represent opportunities for therapeutic intervention.

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

In this study, the antioxidant properties of Mediterranean food spices (annatto, cumin, oregano, sweet and hot paprika, rosemary, and saffron) at 5% concentration and of common food additives (butylated hydroxyanisole [BHA], butylated hydroxytoluene [BHT], and propyl gallate) at 100 microg/g are compared. The ability of these compounds to inhibit lipid peroxidation was, in decreasing order, rosemary > oregano > propyl gallate > annatto > BHA > sweet paprika > cumin > hot paprika > saffron > BHT. Deoxyribose damage is partially inhibited in the presence of cumin extract that exhibits the strongest protective action. The rest of the spices also protect deoxyribose better than the BHA and BHT used in the assay. Finally, the results obtained in the assay point to the prooxidant effect of propyl gallate. Hydrogen peroxide scavenging activity is measured by using peroxidase-based assay systems. In aqueous medium, the spice extracts show lower antioxidant activity than propyl gallate, the decreasing order being cumin > oregano > annatto > rosemary > hot paprika > sweet paprika. BHA and BHT did not scavenge H2O2 Spices are able to scavenge HOCl and protect alpha1-antiproteinase. The results indicate that rosemary and oregano are more effective HOCl scavengers than the other substances analyzed, which, in decreasing order, were propyl gallate, annatto, sweet and hot paprika, saffron, and cumin. The effect of Mediterranean food spices on the oxidative stability of refined olive oil tested by the Rancimat method was compared with common food additives during storage (72 h, 2, 4, and 6 months) at room temperature. The results showed that the spice extracts analyzed have significant stabilizing effects (P < 0.05).

The first identification and characterization of a catalytic activity associated with NadR protein is reported. A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases. The Escherichia coli nadR gene was cloned into a T7-based vector and overexpressed. In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.

Two cytoplasmic pyrimidine 5′-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5′-monophosphates and 3′-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5′-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.

Synechocystis sp. slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa. Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii. The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP. The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the `Nudix' hydrolase family. The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells. The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities. Both activities have been characterized and compared to their archaeal counterparts.

Publisher Summary Nicotinamide-mononucleotide adenylyltransferase is a widely distributed enzyme, which reversibly catalyzes the formation of nicotinamide adenine dinucleotide (NAD). The enzyme has been purified to homogeneity from several sources, including yeast, bovine testis, human placenta, and archaebacterial cells. This chapter describes the purification procedure and the major molecular and enzymatic properties of the enzyme from human placenta. Three different nicotinamide–mononucleotide adenylyltransferase (NMNAT) assays are also described in the chapter. Two of them are spectrophotometric and the third is a high-performance liquid chromatography (HPLC)-based assay. The enzyme preparation corresponding to the last step of the purification procedure is quite stable if stored at 4°, in the presence of 1 M KCl. However, it is rapidly inactivated on freezing and thawing. A detailed product inhibition study, performed on both reaction directions, indicates an ordered Bi–Bi mechanism for the reaction. Among a large variety of compounds tested as possible effectors of the enzyme activity, including pyrimidine and purine bases, oxy- and deoxynucleosides, oxy- and deoxynucleotides, cyclic AMP, several halogenated and methylated nucleobases, oxy- and deoxyribose, oxy- and deoxyribose 5-phosphate, glucose, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-diphosphate, fructose, and fructose 1,6-diphosphate, only reduced β -NMN ( β -NMNH) and ADP-ribose effectively depress the enzyme activity.

Publisher Summary The enzyme nicotinamide–mononucleotide adenylyltransferase plays a key role in pyridine nucleotide turnover, being involved in both de novo and salvage synthesis of nicotinamide adenine dinucleotide (NAD). Sustained degradation and resynthesis of NAD is a well-recognized phenomenon both in eukaryotic and in prokaryotic microorganisms. Sulfolobus solfataricus is a thermophilic archaeon living under extreme conditions of temperature and pH. Comparison of the molecular and catalytic properties of nicotinamide–mononucleotide adenylyltransferase from mesophilic and thermophilic microorganisms can provide significant information for the elucidation of the significance of NAD turnover in an evolutionary perspective. In addition, the identification of the molecular determinants of the enzyme thermostability and thermophilicity appears to have possible biotechnological interest. Owing to the high reaction temperature, a continuous spectrophotometric assay cannot be used. Therefore, the activity of the enzyme from Sulfolobus solfataricus is measured with two different discontinuous assays. The effect of temperature on the enzyme activity shows Arrhenius activation energy of 95 kJ/mol, indicating a remarkable thermophilicity, even when compared with that of other enzymes from the same microorganism.

A pyrimidine nucleotidase with unique specificity has been obtained for the first time as an homogeneous protein from the cytosolic fraction of human erythrocytes. Both conventional chromatography and f.p.l.c. techniques have been used in the purification procedure. The final enzyme preparation gave a single protein band of M(r) = 23,500 on SDS/PAGE under both reducing and non-reducing conditions. The native enzyme was eluted at M(r) = 45,000 in gel filtration chromatography on Superose 12, suggesting a dimeric structure. Amino acid analysis was consistent with an acidic isoelectric point and revealed the presence of six half-cystine and two methionine residues per subunit. The enzyme was active on a variety of pyrimidine nucleoside monophosphates, being most active on the 3′-monophosphates. Km values for 3′dUMP, 3′UMP, 5′dUMP, 5′UMP, 5-fluoro-2′dUMP, ranged from 192 microM to 1.15 mM. The enzyme activity was inhibited by both reaction products, orthophosphate, and the nucleoside formed. Product inhibition studies suggested an Ordered Uni-Bi mechanism for the reaction. The enzyme required Mg2+ for its activity, while heavy metals cations were strongly inhibitory. The enzyme activity was inhibited by metal chelating agents and it was sensitive to thioreactive reagents. The isoelectric point was 5.4 and the optimum activity pH was 6.5.

The nuclear location of NMN adenylyltransferase (NMNAT), the last enzyme in the main biosynthetic pathway of NAD+, prompted us to investigate about a possible involvement of this enzyme in the regulation of the cellular activity by hypothesizing its eventual relationship with poly(ADP-ribose) polymerase (ADPRP), another chromatin bound enzyme. This hypothesis was based on the discovery of an inhibitory effect exerted by NMNAT on ADPRP in vitro in reconstituted systems, composed by enzymes purified both from different sources (heterologous system) (1) and from identical sources (homologous system) (2). In order to verify such a phenomenon in man and to better elucidate its functional significance we carried out the purification of NMNAT from human placenta, where the system involved in NAD+ comsumption has been fully characterized (3).

This paper presents data about the presence of the NMN adenylyltransferase at the nuclear matrix level of human placenta nuclei. It was found that 40-45% of the activity (depending on the extraction procedure) referred to the total nuclear NMN adenylyltransferase was tightly associated with this subnuclear compartment. The matrices purified by two different procedures exhibited DNA, RNA and protein contents comparable with those described in literature. Extensive digestion of human placenta nuclei with DNase I was not able to solubilize the NMN adenylyltransferase activity. Therefore, the data we present are consistent with the conclusion that a part of the total nuclear NMN adenylyltransferase is associated with the nuclear matrix.

Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting.

NAD pyrophosphorylase (ATP:NMN adenylyltransferase) activity has been measured in the skeletal muscle of dystrophic mice. The amount of this enzyme in the dystrophic mice, as determined by three different methods, was about one half of that in the controls. In addition, the concentration of ATP was too low to be detected in crude extracts of dystrophic mouse skeletal muscle, which were prepared using Tris buffer alone or Tris buffer containing either 3 M KCl, or 1 mM PMSF.

Two cytoplasmic forms of pyrimidine nucleotidase (PN-I and PN-II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5′-monophosphates and 3′-monophosphates respectively. PN-I and PN-II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3′-azido-3′-deoxy-thymidine (AZT), cytosine-β-d-arabinofuranoside (AraC) and 5-fluoro-2′-deoxy-uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN-I and PN-II. PN-I phosphotransferase activity revealed higher affinity for oxy-nucleosides with respect to deoxy-nucleosides, whereas the contrary seems to be true for PN-II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine-specific phosphotransferase activity.

Since the discovery that nicotinamide mononucleotide adenylyltransferase (NMN:ATP adenylyltransferase; EC 2.7.7.1) is exclusively located in the cell nucleus (1), various authors have suggested that NAD fulfills a role in addition to that of respiratory coenzyme in eukaryotic cells. Morton (2) hypothesized that NAD might play a key role in the regulation of cell proliferation in that the nucleus may sense changes in intracellular NAD concentrations. Thereafter the discovery that another nuclear enzyme, poly(ADP)ribose synthetase, utilizes NAD as a substrate for the modification of nuclear proteins (3) suggested that such an enzyme may provide the mechanism by which NAD concentrations are sensed by the nucleus. The involvement of this modification in DNA metabolism in eukaryotes in fact is now generally accepted (4). In prokaryotes NAD is known to be a substrate in the DNA ligase-catalyzed joining of DNA fragments (5). Both ADP ribosylation and DNA ligase reactions result in the breakdown of the NAD molecule, yielding free nicotinamide and nicotinamide mononucleotide, respectively, which are then recycled back to dinucleotide through appropriate salvage pathways known as “pyridine nucleotide cycles” (6, 7). Figure 43.1 represents the overall pathway of NAD metabolism taking place in eukaryotic cells.

Summary A new spectrophotometric assay for human erythrocyte pyrimidine 5′-nucleotidase using a continuous spectrophotometric method is described. The activity is detected by measuring uracil, which is produced by a coupled assay including cytidine deaminase and uridine nucleosidase. The results obtained with the continuous spectrophoto-metric method have been confirmed by high pressure liquid chromatography (HPLC) analysis. This procedure, not based upon inorganic phosphate formation and avoiding the use of radioactive material, offers significant advantages with respect to commonly adopted assays.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4.

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is subjected in yeast to inactivation. An inactivating fraction has been isolated and purified to homogeneity with a procedure which includes gel filtration, adsorption chromatography, and electrofocusing techniques. The molecular weight of the enzyme, estimated either by sodium dodecyl sulfate disc gel electrophoresis or by gel filtration is approximately 44,000. No quaternary structure was evidenced. The inactivating activity possesses proteolytic activity against casein and hemoglobin with pH optima of 2.5 and 3.2, respectively. The optimal pH for uridine nucleosidase inactivation is around 4.7. The inactivating activity as well as the proteolytic activity of the preparation can be inhibited by IA but not by IB2 and IC, yeast macromolecular inhibitors for proteinase A (EC 3.4.23.8), B (EC 3.4.22.9), and C (EC 3.4.12.8), respectively. The apparent isoelectric point is pH 4.03. The carbohydrate content is 8.5%. A comparison of the properties of the inactivating protein with those of known yeast proteinases leads to the conclusion that it is identical with the enzyme previously designated as proteinase A, which for the first time has been obtained homogeneous and characterized. It has been shown that proteinase A could play a physiological role in the uridine nucleosidase inactivation process when it is associated, as a complex, with proteinase B.

The enzymatic properties of homogeneous bakers' yeast uridine nucleosidase, prepared as previously described (G. Magni et al., J. Biol. Chem. 1975 250 , 9–13) have been further investigated, and in addition to glucose-6-phosphate and ribose the enzyme activity was inhibited by ribose-5-phosphate and ribulose-5-phosphate. The curves describing this inhibitions were sigmoidal and when the data were plotted according to Hill, n′ values different from 1 were observed suggesting the existence of interactions among the inhibitory molecules binding sites. Furthermore the percentage of inhibition exerted by glucose-6-phosphate, ribose and ribose-5-phosphate on the enzyme activity varied at different pH values. The addition of various chelating agents to the activity assay mixture caused a strong inhibition of the enzyme activity and metal analysis by atomic absorption spectrophotometry, colorimetric methods and electronic paramagnetic resonance, indicated the presence of 1 copper atom per enzyme molecule. Finally the inhibition exerted by metal ions on the enzyme activity was described.

Uridine 5′-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.

Mounting evidence attests to the paramount importance of the non-redox NAD functions. Indeed, NAD homeostasis is related to the free radicals-mediated production of reactive oxygen species responsible for irreversible cellular damage in infectious disease, diabetes, inflammatory syndromes, neurodegeneration and cancer. Because the cellular redox status depends on both the absolute concentration of pyridine dinucleotides and their respective ratios of oxidized and reduced forms (i.e., NAD/NADH and NADP/NADPH), it is conceivable that an altered regulation of the synthesis and degradation of NAD impairs the cell redox state and likely contributes to the mechanisms underlying the pathogenesis of the above mentioned diseases. Taking into account the recent appearance in the literature of comprehensive reviews covering different aspects of the significance of NAD metabolism, with particular attention to the enzymes involved in NAD cleavage, this monograph includes the most recent results on NAD biosynthesis in mammals and humans. Due to recent findings on nicotinamide riboside as a nutrient, its inclusion under "niacins" is proposed. Here, the enzymes involved in the de novo and reutilization pathways are overviewed.