Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C. The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate. S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.     

Purification and characterization of a pyridine nucleotide glycohydrolase from rabbit spleen

A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.     

Mode of action of melinacidin, an inhibitor of nicotinic acid biosynthesis

Melinacidin, a new antibacterial agent, blocked the synthesis of nicotinic acid and its amide in Bacillus subtilis cells. The inhibitory activity of the agent was reversed by nicotinic acid, its amide, or nicotinamide adenine dinucleotides, but not by l-kynurenine, l-3-hydroxykynurenine, l-hydroxyanthranilic acid, or quinolinic acid. These properties indicated that the antibiotic interferes with the conversion of quinolinic acid to nicotinate ribonucleotide by the enzyme quinolinate phosphoribosyl-transferase. However, the activity of a purified preparation of this enzyme derived from a Pseudomonas strain was not impaired by the antibiotic. This suggested that, in B. subtilis, melinacidin interferes with a reaction which occurs before the formation of quinolinic acid in the biosynthetic pathway leading to nicotinic acid. Failure of quinolinic acid to reverse melinacidin inhibition in B. subtilis cultures might be due to insufficient penetration of the cell membranes by quinolinate.     

Regulation of Tryptophan Pyrrolase Activity in Xanthomonas pruni

Tryptophan pyrrolase was studied in partially purified extracts of Xanthomonas pruni. The dialyzed enzyme required both heme and ascorbate for maximal activity. Other reducing agents were able to substitute for ascorbate. Protoporphyrin competed with heme for the enzyme, suggesting that the native enzyme is a hemoprotein. The enzyme exhibited sigmoid saturation kinetics. Reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), nicotinic acid mononucleotide, and anthranilic acid enhanced the sigmoid kinetics and presumably bound to allosteric sites on the enzyme. The sigmoid kinetics were diminished in the presence of alpha-methyltryptophan. NAD, NADP, nicotinic acid, nicotinamide, nicotinamide mononucleotide, and several other related compounds were without effect on the activity of the enzyme. These data indicate that the activity of the enzyme is under feedback regulation by the ultimate end products of the pathway leading to NAD biosynthesis, as well as by certain intermediates of this pathway.     

Inhibition of Hog Liver Crystalline Quinolinate Phosphoribosyltransferase by Nucleotides,Quinolinate Analogues and Sulfhydryl Reagents

Effects of the precursors and intermediates of the NAD biosynthetic pathway, and of quinolinate analogues etc. on hog liver crystalline quinolinate phosphoribosyltransferase (an intermediary enzyme in the de novo NAD biosynthetic pathway) activity were investigated. The enzyme activity was inhibited by many kinds of nucleotides, phthalic acid and SH reagents. But amino acids, nicotinic acid and nicotinamide had practically no effect. The apparent inhibition by ATP removed by raising Mg²⁺ concentration. Phthalic acid was proved to be a competitive inhibitor to quinolinic acid. The Ki value for phthalic acid was calculated at 1.7 × 10^?4 m by a Dixon plot.     

Kinetic analysis of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase

Properties of the transglycosidation reaction catalyzed by rabbit spleen pyridine nucleotide glycohydrolase were characterized using a modified cyanide addition method by which initial velocities of the transglycosidation (vT) and hydrolysis (vH) of pyridine nucleotides could be monitored simultaneously. (1) The vT was routinely determined with NMN and nicotinic acid used as substrates and was observed to be maximal at pH 6. Arrhenius plots of vT and vH indicated that the activation energies for transglycosidation and hydrolysis were 8.7 and 10.7 kcal/mol, respectively. (2) The enzyme showed a broad spectrum of substrate specificity with respect to both pyridine nucleotides and bases. Of the compounds tested, NMN and nicotinic acid were shown to be the best substrates when compared on the basis of Vmax/Km values. Kinetic constants for the enzyme-catalyzed transglycosidation reaction were as follows; Km(NMN) = 0.53 mM, Km(nicotinic acid), as acid form = 15 mM, apparent Vmax = 7.8 mumol/min/mg protein, in the presence of 0.2 M nicotinic acid. (3) The ratio of vT/vH was shown to be dependent on both pH and nicotinic acid concentration. However, transglycosidation versus hydrolysis partition at a fixed pH was constant regardless of the nicotinic acid concentration employed and approximated to be 1.2 x 10(4) at the maximal pH. (4) Nicotinamide, one of the most potent inhibitors for the enzyme-catalyzed hydrolysis, was shown to function as an antagonist for the transglycosidation reaction with NMN and nicotinic acid used as substrates. The inhibition mechanism with nicotinamide was purely noncompetitive with respect to nicotinic acid; on the other hand, the double reciprocal plot of the transglycosidation velocity against NMN concentration at a fixed concentration of nicotinamide was concave downwards. (5) The equilibrium constant of the reaction, NMN + 3-acetylpyridine----3-acetylpyridine mononucleotide + nicotinamide, was 0.61, whereas the conversion of NMN with nicotinic acid to nicotinic acid mononucleotide was essentially irreversible. These enzymatic properties of rabbit spleen pyridine nucleotide glycohydrolase suggested that the enzyme should not function as a glycohydrolase but as a transglycosidase and could serve in an important mechanism for an alternative biosynthetic pathway of nicotinic acid mononucleotide, one of the precursors for NAD synthesis, when nicotinic acid is supplied.     

Nitrile Hydratase-Catalyzed Production of Nicotinamide from 3-Cyanopyridine in Rhodococcus rhodochrous J1

Nitrile hydratase, which occurs abundantly in cells of Rhodococcus rhodochrous J1 isolated from soil samples, catalyzes the hydration of 3-cyanopyridine to nicotinamide. By using resting cells, the reaction conditions for nicotinamide production were optimized. Under the optimum conditions, 100% of the added 12 M 3-cyanopyridine was converted to nicotinamide without the formation of nicotinic acid, and the highest yield achieved was 1,465 g of nicotinamide per liter of reaction mixture containing resting cells (1.48 g as dry cell weight) in 9 h. The nicotinamide produced was crystallized and then identified physicochemically. The further conversion of the nicotinamide to nicotinic acid was due to the low activity of nicotinamide as a substrate for the amidase(s) present in this organism.     

Negative modulation of Escherichia coli NAD kinase by NADPH and NADH.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2+ of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyme was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme. These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2+ and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Hydration of cyanopyridine to nicotinamide by whole cell nitrile hydratase

Summary 3-cyanopyridine was hydrated to nicotinamide by whole cells ofBrevibacterium R-312 containing nitrile hydratase. Cells used for kinetic studies had an initial activity of 0.30 mg nicotinamide/mg cells(dry)-min and storage half-lives (pH 8) of approximately 100 days, 10 days, 5 days and less than 1 day at 4°C, 10°C, 25°C, and 30°C respectively. Temperature and pH maxima were 35°C and 8.0, respectively. Fermentations gave a maximum total hydratase activity of 1.25 mg nicotinamide/min, but at this maximum the amidase activity was unacceptably high (25% of the hydratase activity): nicotinamide was converted too rapidly to nicotinic acid. But systematic fermentation studies (7 1) showed that harvesting at mid-log phase (18–20 h) prior to the attainment of maximum total activity gave reasonably high levels of hydratase (0.3 mg nicotinamide/mg cells-min) and acceptable levels of amidase (0.03 mg nicotinic acid/mg cells-min).     

NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties

NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent K _m for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn²⁺ and Ca²⁺ could be substituted for Mg²⁺. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a K _i of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP.This paper is dedicated to Sydney C. Rittenberg on the occassion of his retirement, with respect and much affection, in appreciation for his friendship and years of distinguished service as a teacher and scientist     

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.     

Vitamin PP, nicotinamide nucleotides and neuro-muscular transmission in guinea pig cecum longitudinal muscle (taenia coli)

The membrane potential, inhibition postsynaptic potentials, resistance of the guinea pig taenia coli membrane were studied as affected by exogenic vitamin PP and its derivatives of nucleotide nature. It is shown that nicotinic acid, nicotinamide, NAD+, NADH, NADP+, NADPH evoke the membrane hyperpolarization and a decrease in the amplitude of the inhibition postsynaptic potentials. Nicotinamid dinucleotides cause a decrease in the membrane resistance, whereas nicotinic acid and nicotinamide do not affect this parameter. The character of the observed effects does not depend on the degree of nicotinamide dinucleotides oxidation.     

Bioprocess development for nicotinic acid hydroxamate synthesis by acyltransferase activity of Bacillus smithii strain IITR6b2

In this work, acyltransferase activity of a new bacterial isolate Bacillus smithii strain IITR6b2 was utilized for the synthesis of nicotinic acid hydroxamate (NAH), a heterocyclic class of hydroxamic acid. NAH is an important pyridine derivative and has found its role as bioligand, urease inhibitor, antityrosinase, antioxidant, antimetastatic, and vasodilating agents. Amidase having acyltransferase activity with nicotinamide is suitable for nicotinic acid hydroxamate production. However, amidase can also simultaneously hydrolyze nicotinamide and nicotinic acid hydroxamate to nicotinic acid. Nicotinic acid is an undesirable by-product and thus any biocatalytic process involving amidase for nicotinic acid hydroxamate production needs to have high ratios of acyltransferase to amide hydrolase and acyltransferase to nicotinic acid hydroxamate hydrolase activity. Isolate Bacillus smithii strain IITR6b2 was found to have 28- and 12.3-fold higher acyltransferase to amide and hydroxamic acid hydrolase activities, respectively. This higher ratio resulted in a limited undesirable by-product, nicotinic acid (NA) synthesis. The optimal substrate/co-substrate ratio, pH, temperature, incubation time, and resting cells concentration were 200/250 mM, 7, 30 °C, 40 min, and 0.7 mg_DCW ml^?1, respectively, and 94.5 % molar conversion of nicotinamide to nicotinic acid hydroxamate was achieved under these reaction conditions. To avoid substrate inhibition effect, a fed-batch process based on the optimized parameters with two feedings of substrates (200/200 mM) at 40-min intervals was developed and a molar conversion yield of 89.4 % with the productivity of 52.9 g h^?1 g _DCW ^?1 was achieved at laboratory scale. Finally, 6.4 g of powder containing 58.5 % (w/w) nicotinic acid hydroxamate was recovered after lyophilization and further purification resulted in 95 % pure product.     

Influence of dietary deficiency of nicotinamide on lead toxicity in young rats

The influence of dietary nicotinamide deficiency on lead intoxication in young developing rats was investigated. The Pb induced an increase in brain dopamine and noradrenaline, inhibition in blood δ-aminolevulinic acid dehydratase activity, an elevation in urinary excretion of δ-aminolevulinic acid and blood and tissue uptake of Pb were significantly more marked in animals maintained on a nicotinamide-deficient diet than those fed a nicotinamide-sufficient diet. The nicotinamide deficiency may enhance the susceptibility to Pb intoxication possible by enhancing the absorption of Pb and altering nicotinic acid metabolism.     

A nuclear magnetic resonance-based functional assay for nicotinamide adenine dinucleotide synthetase

Nicotinamide adenine dinucleotide synthetase (NadE) is an essential enzyme for bacterial pathogens and is thus a promising antibacterial target. It catalyzes the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. Changes in chemical shifts that occur in the nicotinic acid ring as it is converted to nicotinamide can be used for monitoring the reaction. A robust nuclear magnetic resonance-based activity assay was developed using robotically controlled reaction initiation and quenching. The single-enzyme assay has less potential for false positives compared to a coupled activity assay and is especially well suited to the high concentration of compounds in fragment screens. The assay has been used to screen fragment libraries for NadE inhibitors.     

Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production

NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.     

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.     

Control of the steady-state concentrations of the nicotinamide nucleotides in rat liver

1. The effects of injecting nicotinamide, 5-methylnicotinamide, ethionine, nicotinamide+5-methylnicotinamide and nicotinamide+ethionine on concentrations in rat liver of NAD, NADP and ATP were investigated up to 5hr. after injection. 2. Nicotinamide induced three- to four-fold increases in hepatic NAD concentration even in the presence of 5-methylnicotinamide or ethionine, whereas 5-methylnicotinamide or ethionine alone did not cause marked changes in hepatic NAD concentration. 3. Nicotinamide alone also induced a twofold increase in hepatic NADP concentration. However, in the presence of 5-methylnicotinamide+nicotinamide, the NADP concentration decreased by 25% after 5hr., and in the presence of nicotinamide+ethionine by 30% in the same time. In the presence of 5-methylnicotinamide or ethionine alone hepatic NADP concentrations fell by 50% after 5hr. 4. 5-Methylnicotinamide inhibited the microsomal NAD(+) glycohydrolase (EC 3.2.2.6) by 60% at a concentration of 1mm and the NADP(+) glycohydrolase by 40% at the same concentration. 5. The rat liver NAD(+) kinase (EC 2.7.1.23) was found to have V(max.) 4.83mumoles/g. wet wt./hr. and K(m) (NAD(+)) 5.8mm. This enzyme was also inhibited by 5-methylnicotinamide in a ;mixed' fashion. 6. The results are discussed with respect to the control of NAD synthesis. It is suggested that in vivo the NAD(P)(+) glycohydrolases are effectively inactive and that the increased NAD concentrations induced by nicotinamide are due to increased substrate concentration available to both the nicotinamide and nicotinic acid pathways of NAD formation.