Nicotinic Acid Mononucleotide Research Articles

Effect of NAD, nicotinamide and nicotinic acid on cyclic AMP accumulation by fat cells

The addition of exogenous nicotinamide adenine dinucleotide (NAD) to isolated rat fat cells at concentrations between 1 and 10 μmol markedly inhibited the rise in cyclic AMP accumulation due to norepinephrine. Catabolism of NAD to adenosine did not account for the inhibitory effect since NAD was effective in the presence of adenosine deaminase. The bovine fraction V albumin in which fat cells were incubated contained a substantial amount of glycohydrolase which cleaved NAD to nicotinamide. Incubation of fat cells in albumin-free medium did not abolish the inhibitory effect of NAD, and under these conditions there was little degradation of NAD to nicotinamide during the first 10 min of incubation. Fat cells cleave NAD, in the medium to nicotinamide mononucleotide which does not inhibit cyclic AMP accumulation. NADH, NADP and nicotinic acid mononucleotide were less effective as inhibitors of cyclic AMP accumulation than NAD or deamido NAD. Ineffective as inhibitors were NADPH, nicotinamide mononucleotide, and 1-methyl nicotinamide. N′-methyl nicotinamide was as potent as nicotinamide while nicotinic acid was 100- to 500fold more effective than either compound as an inhibitor of cyclic AMP accumulation. Incubation of fat cells with 100 μM but not 10 μM NAD for 5 min resulted in a decrease in the subsequent accumulation of cyclic AMP by rat fat cell ghosts.

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.

Niacin nucleotide synthesis and glycolytic activity in erythrocytes of patients suffering from pellagra

Erythrocytes of subjects with pellagra exhibit impaired synthesis of NAD from nicotinic acid in vitro. A detailed time course study of in vitro incorporation of 14C-nicotinic acid into NAD and its intermediates namely nicotinic acid mononucleotide (NaMN), nicotinic acid adenine dinucleotide (NaAD) was made in erythrocytes of patients with pellagra and normal subjects. The incorporation of 14C-nicotinic acid into the total nucleotides (NAD + NaAD + NaMN) was significantly reduced in the erythrocytes of pellagrins at 6 and 21 hours. Relatively more radioactivity was found in NaMN during these periods indicating thereby, that the conversion of NaMN to NaAd may be defective in pellagra. This was confirmed by label chasing experiment, in which NaMN of the intact cells was prelabeled with 14C-nicotinic acid, and its further conversion to NaAD was followed in vitro. The activity of nicotinate phosphoribosyl transferase, was normal in the erythrocytes of pellagrins. Concentration of ATP and glycolytic activity of the erythrocytes were investigated under conditions for NAD synthesis. A significant reduction in the basal levels of ATP was observed in pellagrins. As compared to normals, the rate of glycolytic activity, and the levels of ATP during NAD synthesis, was significantly lower in the erythrocytes of pellagrins. From the data obtained, it was concluded that the fall in ATP concentration found in the erythrocytes of pellagrins may not be the limiting factor for the optimal synthesis of NAD. Instead, the defect in lowered NAD synthesis, may be due to reduced activity of the enzyme NaMN adenyl transferase, which converts NaMN to NaAD.

Purification and properties of quinolinate phosphoribosyltransferase from the "shitake" mushroom (Lentinus edodes).

A considerably high activity of quinolinate phosphoribosyltransferase, an intermediary enzyme in the de novo NAD biosynthetic pathway, was found in a soluble fraction of the “Shiitake” mushroom extract. Highly purified enzyme (approximately 1, 000-fold, chromatography-cally pure) was extracted for the first time from mushrooms and its general properties were investigated. An apparent molecular weight of 1.6×105 was estimated by the gel filtration method. The optimum pH for the reaction was 6.5. The reaction required a divalent cation in addition to quinolinic acid and PRPP. Michaelis constants for quinolinic acid, PRPP and Mg++ were 1.1×10-5 M, 2.3×10-5M and 2.0×10-4M, respectively. The reaction product was identified as nicotinic acid mononucleotide by KCN addition reaction and by paper partition chromatography.

Metabolism of the pyridine nucleotides involved in nicotinamide adenine dinucleotide biosynthesis by Clostridium butylicum.

In order to elucidate the mechanism of the accumulation of considerable amounts of free nicotinic acid (NA) in the culture medium of Clostridium butylicum, this organism was investigated with regard to its ability to metabolize nicotinamide adenine dinucleotide (NAD) and its immediate biosynthetic precursors, nicotinic acid mononucleotide (NAMN) and nicotinic acid adenine dinucleotide (deamido-NAD). Cell-free extracts of C. butylicum were found to degrade NAMN and deamido-NAD to NA. NAMN, in the presence of adenosine triphosphate (ATP), was converted to deamido-NAD, but only at high concentrations of ATP (20 mM) was significant synthetic activity observed in competition with NAMN degradation. Degradation of both NAMN and deamido-NAD was activated by ATP at concentrations of 5 and 10 mm. Anaerobiosis markedly enhanced the degradation of the nucleotides. The data indicate that the synthesis of NAMN and deamido-NAD prevails over their degradation only in the presence of high concentrations of ATP. NAD was degraded to nicotinamide mononucleotide (NMN) by a pyrophosphatase. Phosphate markedly inhibited both the deamido-NAD and NAD pyrophosphatases. Under anaerobic conditions there was practically no further degradation of NMN to NA, whereas barely measurable amounts of NA were formed under aerobic conditions. All of these observations suggest that, under the given conditions of anaerobiosis and physiological phosphate concentrations, there is very little degradation of NAD to NMN and practically no degradation to NA by C. butylicum. Thus, NAD represents an insignificant source of the NA accumulated in the culture medium. The intermediates in the biosynthetic pathway (NAMN and deamido-NAD) have been shown to be the major source of the NA which is accumulated by C. butylicum.

Pyridine Nucleotide Metabolism in Escherichia coli: III. BIOSYNTHESIS FROM ALTERNATIVE PRECURSORS IN VIVO

Abstract In Escherichia coli, the nicotinamide moiety of pyridine nucleotides may be derived from de novo synthesis of the pyridine ring or through the uptake of either nicotinic acid or nicotinamide. De novo synthesis is the least preferred pathway; it is suppressed if an exogenous source is available. Nicotinamide is kinetically preferred over nicotinic acid; the data are consistent with the hypothesis that nicotinamide can pass through the bacterial membrane much faster than can the charged nicotinic acid, and once inside the cell, the amide is efficiently converted to the acid. Thus even at low concentrations nicotinamide is instantly taken up by cells, but at concentrations of less than 8 x 10-6 m nicotinic acid in the medium, the rate of entry of the nicotinic acid into the cell appears to be the rate-limiting step in the synthesis of pyridine nucleotide from exogenous niacin. Under conditions where sufficient exogenous niacin or nicotinamide is present so that entry into the cell is not limiting, the rate of pyridine nucleotide biosynthesis is apparently regulated by the rate of conversion of nicotinic acid → nicotinic acid mononucleotide, as suggested by Imsande and Pardee ((1962) J. Biol. Chem. 237, 1305). Since the rate of conversion of nicotinamide → nicotinic acid is not regulated, excess nicotinic acid that is formed from nicotinamide is continually excreted into the medium.

Pyridine Nucleotide Metabolism in Escherichia coli: II. NIACIN STARVATION

Abstract The effect of niacin starvation has been studied in a niacin-requiring auxotroph of Escherichia coli. If a culture is totally deprived of niacin, cells continue to divide until the total pyridine nucleotide content has fallen from 1.9 x 106 to 1.2 x 105 molecules per cell. During starvation, the relative proportion of the pyridine nucleotides changes greatly: the TPN: DPN ratio increases from 0.30 to over 2.0 and nicotinic acid mononucleotide accumulates until it is present at concentrations comparable to DPN. The changes in the distribution of the pyridine nucleotides suggest that DPN is turning over during niacin starvation and that the normally observed breakdown of TPN to DPN during exponential growth is inhibited. If cells are starved for niacin by balanced growth under limiting concentrations of niacin, less disproportion in the TPN: DPN ratio is observed, and growth occurs with a pyridine nucleotide content as low as 4 x 104 molecules per cell.

Metabolism of nicotinamide adenine dinucleotide in human and bovine strainsof Mycobacterium tuberculosis.

A marked difference was found to exist between the nicotinamide adenine dinucleotide (NAD) glycohydrolase activity of human strains of Mycobacterium tuberculosis as compared with bovine strains. Human strains had from 6- to 20-fold higher NAD glycohydrolase activity than bovine strains. This finding explains the accumulation of free nicotinic acid in the culture medium by human strains and not by bovine strains. The biosynthetic intermediates nicotinic acid mononucleotide and deamido-NAD were not degraded by either human or bovine strains of M. tuberculosis; hence these nucleotides do not represent a source of the nicotinic acid accumulated by the human strains.

Inhibition of Nicotinate Phosphoribosyl Transferase by Nonsteroidal Anti-Inflammatory Drugs: A Possible Mechanism of Action

When incubated with 14C-nicotinic acid and phosphorylribose-1-pyrophosphate, human platelet lysate incorporates radioactivity into nicotinic acid mononucleotide. Under these conditions, the apparent Km of nicotinic acid for nicotinate phosphoribosyl transferase is 2.4 × 10−5M. Along with 2-hydroxynicotinic acid (Ki = 2.3 × 10−4M), the following nonsteroidal anti-inflammatory compounds competed reversibly with nicotinic acid for the enzyme: flufenamic acid (Ki = 4.6 × 10−5M), mefenamic acid (Ki = 7.6 × 10−5M), salicylic acid (Ki = 1.6 × 10−4M), phenylbutazone (Ki = 1.6 × 10−4M), and indomethacin (Ki = 4.2 × 10−4M). Such inhibition may explain the decreases in nicotinamide adenine dinucleotide phosphate content of rat liver after administration of salicylate. Furthermore, suppression of nicotinamide adenine dinucleotide biosynthesis by anti-inflammatory drugs may restrain mucopolysaccharide biosynthesis and, thereby, reduce inflammatory responsiveness. Hence, this antagonism of niacin may be involved in the pharmacologic activity and, particularly, the toxicity of anti-inflammatory drugs.

Nicotinate Phosphoribosyltransferase of Yeast: PURIFICATION AND PROPERTIES

Abstract Nicotinate phosphoribosyltransferase from bakers' yeast has been purified about 1,000-fold; the purified material showed only one band on cellulose acetate electrophoresis (pH 5.0 and 8.6). In addition to 5-phosphoribosyl 1-pyrophosphate, adenosine triphosphate (ATP) or an analogous nucleotide is required for enzyme activity and the stoichiometric hydrolysis of ATP to adenosine diphosphate (ADP) and orthophosphate with formation of products, nicotinic acid mononucleotide and pyrophosphate, was verified with this purified preparation. The nucleotide specificity, however, is broader than that of the liver form of this enzyme. The molecular weight of the yeast enzyme was estimated from Sephadex chromatography to be 43,000 (about one-half that of the liver enzyme) whether in the presence or absence of ATP. Phosphate ions greatly stimulate the enzyme activity, giving optimum activity in the range of 10 to 100 mm phosphate in the presence of 5 mm Mg++. At 1 and 5 mm Mg++, optimum enzyme activities are observed with equimolar concentration of ATP. The enzyme catalyzes isotope exchange between ATP and ADP at rates approximately 8 times its molecular activity with all substrates, although no net ATP hydrolysis occurs in the absence of other substrates. ATP, guanosine triphosphate, and the substrates, nicotinic acid and 5-phosphoribosyl 1-pyrophosphate, protect the enzyme from heat inactivation.

Nicotinamide adenine dinucleotide metabolism in plants. I. Intermediates of biosynthesis in wheat leaves and the effect of benzimidazole

Leaves of wheat (Triticum aestivum L. var. Selkirk) were incubated with nicotinic acid-7-14C and nicotinamide-7-14C for varying time periods from 5 min to 12 h. Aliquots of alcoholic extracts of leaves were subjected to paper chromatography and radioautography to isolate the intermediates of the synthesis and breakdown of nicotinamide adenine dinucleotide. Nine compounds were isolated quantitatively and identified as intermediates in the pathway of NAD metabolism. All the intermediates were labeled rapidly and the rapidity of labeling became a problem in rigorously proving the sequential operation of the pathway. The results indicate that the Preiss-Handler pathway: nicotinic acid→nicotinic acid mononucleotide→nicotinic acid adenine dinucleotide→NAD operates in wheat leaves. The degradation of NAD proceeded from NAD→nicotinamide mononucleotide→nicotinamide riboside→nicotinamide. Deamidation of the nicotinamide to nicotinic acid initiated a fresh cycle of biosynthesis. The total radioactivity recovered in the intermediates indicates that no measurable amount was lost to other metabolic pathways. Nicotinamide is recovered without significant loss and recycled. The rapid appearance of labeled nicotinamide indicates a possible interconversion of nicotinic acid and nicotinamide. About 80% of the radioactivity accumulated was present in trigonelline which is considered, on the basis of other evidence, to be a non-toxic form of nicotinic acid. Benzimidazole treatment of the leaves increased the incorporation of 14C into NADP.

NAD biosynthesis in polymophonuclear leucocytes of guinea pigs comparative studies on different NAD precursors in intact cells

The following NAD precursors were studied: tryptophan, quinolinic acid, nicotinic acid, nicotinamide and nicotinic acid mononucleotide. Tryptophan and quinolinic acid seem not to be utilized in the NAD synthesis by polymorphonuclear leucocytes, a finding which is supported by the absence of quinolinate transphosphorribosylase in cell-free extracts of the cells. Nicotinamide is about 2.5 times less efficient than nicotinic acid as NAD precursor. The efficacy of nicotinic acid mononucleotide is close to that of nicotinamide. Intact polymorphonuclear leucocytes are able to degrade labeled nicotinic acid mononucleotide. Outside the cells, most of the label was identified as nicotinic acid. This degradation may be inhibited in cell-free extracts by the presence of NAD. Nicotinamide at high concentrations inhibits the incorporation of labeled nicotinic acid into NAD, and strikingly higher concentrations of NAD appear in the cells.

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.

Chromosomal location of the C gene involved in the biosynthesis of nicotinamide adenine dinucleotide in Escherichia coli K-12.

A gene involved in the synthesis of nicotinamide adenine dinucleotide has been found to be cotransducible with the genes involved in the utilization of arabinose (ara) and the biosynthesis of leucine (leu) and pantothenate (pan). Cotransduction frequency analysis places this nadC locus between leu and pan at approximately minute 1.5 on the genetic map of Escherichia coli. This gene codes for the enzyme, quinolate phosphoribosyl transferase, which catalyzes the conversion of quinolinic acid to nicotinic acid mononucleotide.

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.

Allosteric Properties of Bovine Liver Nicotinate Phosphoribosyltransferase

Abstract Nicotinate phosphoribosyltransferase has been partially purified from bovine liver and some of its kinetic properties studied. This enzyme is easily separable from niacin ribonucleotidase. ATP is not a required substrate for bovine liver nicotinate phosphoribosyltransferase, as has been reported, but activates the enzyme by lowering the apparent Michaelis constants for both nicotinate and ribosylpyrophosphate 5-phosphate by approximately 10-fold. The stimulation by ATP appears to be highly specific. The kinetics of ATP activation indicate that ATP is binding at only one site on the enzyme. When ATP is present in the reaction, it is cleaved to form ADP in an amount approximately stoichiometric with nicotinic acid mononucleotide formation. It is suggested that ATP cleavage may be required for the activation of the enzyme.

Uptake of Nicotinic Acid and Nicotinamide by Rat Erythrocytes

Abstract The uptake of nicotinic acid and nicotinamide by rat erythrocytes consists of two processes, diffusion and enzyme-catalyzed conversion to nucleotides which do not readily diffuse from the cell. These two processes provide for a very rapid removal of these compounds, especially nicotinic acid, from the external medium. The two processes have been investigated separately with the use of 14C-labeled substrates and fluoride to prevent the formation of nucleotides. Inorganic phosphate stimulates the formation of nucleotides 2- to 3-fold at an optimum concentration of 20 mm. Glycolytic inhibitors other than fluoride, including arsenate and iodoacetate, also arrest the formation of nucleotides. Replacement of glucose with 2-deoxyglucose likewise prevents nucleotide formation. The passage of nicotinic acid and its amide into and out of fluoride-inhibited erythrocytes differed greatly. The passage of the acid ceased when the temperature was lowered to 0° and exhibited a Q10 of about 2 for uptake between 10° and 30°, suggesting that it was transported by facilitated diffusion. On the other hand, free diffusion can account for the rapid uptake and removal of nicotinamide from the cells. Equilibration occurred within 1 min at temperatures between 0° and 37°. Quinolinic acid was taken up only very slightly, and there was no evidence of nicotinic acid mononucleotide formation.

Studies of Nicotinic Acid Metabolism in Astasia longa: II. PATHWAY AND REGULATION OF NICOTINAMIDE ADENINE DINUCLEOTIDE BIOSYNTHESIS IN CELL-FREE PREPARATIONS

Abstract Cell-free extracts of Astasia longa possess the following enzymes at the indicated relative activities: (a) quinolinic acid phosphoribosyl transferase, 0.5; (b) nicotinic acid mononucleotide pyrophosphorylase, 1; (c) nicotinic acid dinucleotide pyrophosphorylase, 1; (d) nicotinamide adenine dinucleotide synthetase, 0.003; (e) nicotinamide dinucleotide kinase, 1; (f) nicotinamide deamidase, 1; (g) nicotinic acid methyltransferase, 0.0001. The presence of Enzymes a, c, d, and e, in conjunction with data on the intracellular levels of deamido-NMN, deamido-NAD, NAD, and NADP, establishes this sequence of compounds as the pathway of biosynthesis of pyridine nucleotides in Astasia and indicates that the conversion of deamido-NAD to NAD is a rate-limiting step. Cell-free extracts also catalyzed the degradation and pyrophosphorolysis of deamido-NAD and of NAD, and catalyzed an exchange reaction between nicotinic acid and the nicotinamide moiety of NAD. These reactions, in conjunction with the presence of Enzymes b and f, and with data showing a high rate of turnover of NAD in exponentially growing cultures, indicate the operation of a nicotinic acid salvage pathway in Astasia. The finding that nicotinic acid is an inhibitor of Enzyme a and that quinolinic acid is an inhibitor of Enzyme b suggests a possible means of control of the relative rates of the biosynthetic and the salvage pathways. The finding that deamido-NAD, present in relatively high concentrations in Astasia, is an inhibitor of Enzyme b suggests another possible control mechanism. Growth in the presence of nicotinic acid or quinolinic acid did not lead to repression of either Enzyme a or b. Nicotinamide and high concentrations of nicotinic acid inhibited the growth of Astasia and nicotinamide repressed the level of Enzymes a and b.

Production of Nucleic Acid-Related Substances by Fermentative Processes

A new preparative method of NAD was provided by the finding that a large amount of NAD accumulated with AMP, ADP and ATP in the culture broth when Brevibacterium ammoniagenes ATCC 6872 was incubated in the medium containing adenine and nicotinic acid or nicotinamide. A large amount of nicotinic acid mononucleotide (NaMN) with a small amount of NAD accumulated when nicotinic acid or nicotinamide was singly added. NAD was isolated from the culture broth by ion exchange chromatography, and identified by paperchromatography, ultraviolet and infrared spectra, analyses of ribose and phosphate, and reduction by alcohol dehydrogenase from yeast. An example of the time course of the fermentation for the production of NAD was shown. The biosynthetic pathway of NAD from nicotinic acid or nicotinamide in the organism was discussed. The amount of NAD accumulated reached to several mg per ml.

A simple method for the synthesis of nicotinic acid mononucleotide

A rapid and efficient method for the preparation of nicotinic acid mononucleotide from nicotinamide mononucleotide is described. Deamidation is effected by the use of nitrosyl chloride at room temperature. The starting material is quantitatively converted to the corresponding acid. During the deamidation some hydrolysis of the ribosyl bond occurs. Yields of 75 to 85% are regularly obtained.