The hydrolysis of nicotinamide adenine nucleotide by brush border membranes of rat intestine.

The hydrolysis of NAD by rat intestine was studied to determine the subcellular site of this hydrolysis and to identify the niacin-containing products that are formed. Using [nicotinamide-14C]NAD as substrate, and high pressure liquid chromatography for identification and quantification of products, the present study demonstrates two independent reactions for the hydrolysis of NAD; one that forms nicotinamide through hydrolysis of the ribosyl-pyridinium bond and one that forms nicotinamide mononucleotide through the hydrolysis of the pyrophosphate bond. The nicotinamide mononucleotide is subsequently dephosphorylated to nicotinamide riboside. Enzymes which release nicotinamide mononucleotide and nicotinamide riboside are associated with the brush border membrane as determined by analysis of fractionated intestinal homogenates. The enzyme activity which releases nicotinamide from NAD is associated with the brush border membrane fraction and also with a second cellular particulate fraction. Between pH5 and pH6 NAD is hydrolysed principally to nicotinamide. At pH 7.0 rates of nicotinamide and nicotinamide mononucleotide formation are the same. Above pH 7.0 the formation of nicotinamide mononucleotide is preferred.     

Metabolism of NAD and N1-methylnicotinamide in growing and growth-arrested cells

Nicotinamide is metabolized primarily into NAD and N1-methylnicotinamide in cultured cells of normal rat kidney. The metabolic pathways for the nicotinamide metabolites are independently regulated and are influenced by the growth stage of the cells. N1-Methylnicotinamide levels are 1.5--2-fold elevated in cells growth-arrested by treatment with histidinol, thymidine, or picolinic acid, or by serum starvation. This increase is due to a more rapid rate of synthesis rather than decrease in excretion. The rates of both synthesis and degradation of NAD are increased in serum-starved cells so that the NAD concentration is the same as it is in growing cells. NAD and N1- methylnicotinamide levels are not significantly increased when the intracellular nicotinamide concentration is increased 20-fold by addition of excess nicotinamide to the culture medium, demonstrating that the size of the nicotinamide pool does not limit synthesis of these compounds. In medium containing normal amounts of nicotinamide, the apparent first-order rate constant for the decay of NAD, radioactively labeled in the nicotinamide moiety, is about 4 h-1. Labeled N1-methylnicotinamide is not metabolized, but rather is excreted into the medium with a first-order rate constant of 3.9 h-1. The rate of loss of label from NAD, but not from N1-methylnicotinamide, is increased about twofold by addition of excess nicotinamide to the culture medium. This could be explained by a dilution of a labeled nicotinamide pool which is formed during NAD degradation and which is recycled into NAD but not into N1-methylnicotinamide. The results demonstrate a rapid turnover of NAD at the bond joining nicotinamide and ADP-ribose, in agreement with previous studies. In addition, the results show that nicotinamide is metabolized into N1-methylnicotinamide with what appears to be a carefully regulated synthetic mechanism. The existence of significant amounts of N1-methylnicotinamide in cultured cells raises the question of the physiological importance of this compound.     

Pyridine nucleotide synthesis in 3T3 cells.

The biosynthesis of NAD has been examined in 3T3 cells. The net synthesis of pyridine nucleotides does not occur when cells are cultured in the absence of performed pyridine ring compounds; however, growth continues normally for up to four cell doublings resulting in cells with a total pyridine nucleotide content that is reduced by as much as 12-fold. The mechanism that adjust the relative amounts of NADP and NAD are also altered such that the amount of NADP relative to NAD increases 5-fold. Both nicotinate and nicotinamide can be used as a precursor for NAD biosynthesis, however nicotinate is utilized less efficiently than nicotinamide. The presence of functional pathways for the biosynthesis of NAD from nicotinate via nicotinate mononucleotide and nicotinate adenine dinucleotide and from nicotinamide via nicotinamide mononucleotide has been demonstrated by identification of biosynthetic intermediates following short term exposure of cells to radiolabelled precursors. When cells are grown in Dulbecco's modified Eagle's medium which contains 33 μM nicotinamide the biosynthesis of NAD proceeds by a single pathway with nicotinamide mononucleotide as the only intermediate. Nicotinamide ribonucleoside which previously has been postulated to be an intermediate in the conversion of nicotinamide to NAD is not an intermediate in NAD biosynthesis.     

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.     

Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1

NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.     

End-Product Regulation of the Tryptophan-Nicotinic Acid Pathway in Neurospora crassa

The regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa was examined with mutants (nic-2, nic-3) which require nicotinamide for growth. The accumulation of N-acetylkynurenin and 3-hydroxyanthranilic acid by these mutants served to estimate the level of function of the early reactions in the pathway. In still cultures, maximal accumulation occurred with media containing growth-limiting amounts of nicotinamide; the accumulation of intermediates was almost negligible with nicotinamide in excess. Only nicotinamide and closely related compounds which also supported the growth of these mutants inhibited the accumulation of intermediates. The site of inhibition was assessed to be between tryptophan and kynurenin (or N-acetylkynurenin). The synthesis of N-acetylkynurenin was examined in washed germinated conidia suspended in buffer; the level of N-acetylkynurenin-synthesizing activity was inversely related to the concentration of nicotinamide in the germination medium. The addition of large amounts of nicotinamide to suspensions of germinated conidia did not affect their N-acetylkynurenin-synthesizing activity. Formamidase activity, kynurenin-acetylating activity, and gross tryptophan metabolism in germinated conidia was not influenced by the concentration of nicotinamide in the germination medium. The results obtained indicate that the site of inhibition by nicotinamide is the first step in the pathway, the tryptophan pyrrolase reaction. The data are interpreted as nicotinamide or a product thereof, such as nicotinamide adenine dinucleotide, acting as a repressor of the formation of tryptophan pyrrolase in N. crassa.     

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.     

Structural basis for nicotinamide inhibition and base exchange in Sir2 enzymes

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called "C pocket" binding site for the nicotinamide group of NAD(+). These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.     

Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii

1. Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethylbenzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3--0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1'-14C]riboflavin as substrate the specific radioactivity of 5,6-dimethylbenzimidazole is higher than the specific radioactivity of flavin--adenine dinucleotide and lower than the specific radioactivie substrate for the formation of 5,6-dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed.     

Evidence for a physiologically active nicotinamide phosphoribosyltransferase in cultured human fibroblasts

Nicotinamide phosphoribosyltransferase, (EC 2.4.2.12) was examined in extracts of diploid human fibroblasts grown in culture. The enzyme was found to have an apparent Km for nicotinamide of 1.6 × 10^?6M, to be specific for nicotinamide, stimulated by adenosine triphosphate (ATP) and inhibited by nicotinamide adenine dinucleotide (NAD). In these respects it is very similar to rat liver nicotinamide phosphoribosyltransferase but not like the enzyme previously observed in human tissue extracts which had a Km for nicotinamide of approximately 0.1 M and was insensitive to ATP. Discovery of this enzyme activity supports previous studies using radiolabeled nicotinamide which show that human fibroblasts can incorporate nicotinamide into NAD directly through nicotinamide mononucleotide.     

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5��-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD⁺, which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD⁺ metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5′-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5′-nucleotidase, and Sdt1, initially classified as a pyrimidine 5′-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD⁺. Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD⁺ metabolism.     

Role of NAD(+) in the deacetylase activity of the SIR2-like proteins

In this report we describe the role of NAD(+) in the deacetylation reaction catalyzed by the SIR2 family of enzymes. We first show that the products of the reaction detected by HPLC analysis are ADP-ribose, nicotinamide, and a deacetylated peptide substrate. These products are in a 1:1:1 molar ratio, indicating that deacetylation involves the hydrolysis of one NAD(+) to ADP-ribose and nicotinamide for each acetyl group removed. Three results suggest that deacetylation requires an enzyme-ADP-ribose intermediate. First, the enzyme can promote an NAD(+) if nicotinamide exchange reaction that depends on an acetylated substrate. Second, a non-hydrolyzable NAD(+) analog is a competitive inhibitor of the enzyme, and, third, nicotinamide shows product inhibition of deacetylase activity.     

Enzymatic Synthesis of l-Carnitine by Reduction of an Achiral Precursor: the Problem of Reduced Nicotinamide Adenine Dinucleotide Recycling

Synthesis of l-carnitine has been carried out by the enzymatic reduction of the carbonyl group of the achiral precursor 3-dehydrocarnitine with the oxidized nicotinamide adenine dinucleotide-linked carnitine dehydrogenase. Various enzymatic or chemical systems have been tested to regenerate the reduced nicotinamide adenine dinucleotide oxidized in the reduction of 3-dehydrocarnitine. Because of the instability of this compound in aqueous solutions, it was added by continuous feeding as a rate-limiting constituent in the reaction mixture. Under these conditions, conversion yields of 95% were achieved with the glucose plus glucose dehydrogenase system. A total number of 530 reduced nicotinamide adenine dinucleotide recyclings was obtained with this system for a production of 45 g of l-carnitine per liter. The stabilities of the oxidized nicotinamide adenine dinucleotide and the reduced nicotinamide adenine dinucleotide have been determined at various pH values. In view of these results, several possible strategies for enzymatic syntheses with the reduced nicotinamide adenine dinucleotide as a regenerable coenzyme are discussed.     

Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells

The presence of 50 mM nicotinamide together with 100 milliunits/ml of TSH in the incubation medium prevented the decline in human thyroid cell cAMP from maximum, stimulated levels (15-30 min) that occurs when the cells are exposed to TSH alone. Nicotinamide in the absence of TSH did not increase thyroid cell cAMP content. TSH desensitization, and its prevention by nicotinamide, occurred in the presence or absence of 3-isobutyl-methylxanthine. 1-Methyl nicotinamide and N'-methyl nicotinamide similarly prevented TSH desensitization. Recovery from TSH desensitization was prolonged and incomplete after 72 h. The presence of 50 mM nicotinamide hastened recovery from desensitization. Desensitization of the cAMP response to 10(6) M prostaglandin E1 and 1 mM adenosine was unaffected by nicotinamide. Other inhibitors of poly(ADP-ribose) polymerase activity, 5-bromouridine, 5-bromo-2'-deoxyuridine, and thymidine (all at 50 mM) completely or partially prevented TSH desensitization. Pyridoxine (50 mM) similarly prevented this phenomenon. As with dog thyroid cells, 10(-4) M cycloheximide blocked TSH desensitization. The combination of 10(-4) M cycloheximide and 50 mM nicotinamide had a synergistic effect in augmenting the thyroid cell cAMP response to TSH stimulation.     

Nicotinamide and 3-aminobenzamide interfere with receptor-mediated transmembrane signaling in murine cytotoxic T cells: independence of Golgi reorientation from calcium mobilization and inositol phosphate generation.

The two competitive inhibitors of ADP-ribosylation, nicotinamide and 3-aminobenzamide, have been reported to interfere with TNF-induced cell apoptosis, and there is evidence that they inhibit killer-induced target cell lysis as well. There are very few drugs known to specifically interfere with target apoptosis induced by killer cells. We therefore sought to explore the effects these inhibitors have on CTL-mediated cell lysis. Here we show that TcR-mediated transmembrane signaling in CTL, measured by Ca2+ mobilization and generation of inositol phosphates, is inhibited by nicotinamide. The possibility that all cell functions are suppressed by the drug is excluded by the finding that constitutive secretion of BLT serine esterase is not inhibited, whereas stimulated secretion of this enzyme is suppressed. We also show that nicotinamide does not interfere with CTL target cell binding or reorientation of the Golgi apparatus toward the target binding site. It is concluded that nicotinamide inhibits transmembrane signaling in CTL and thereby interferes with delivery of the lethal hit to targets.     

Inhibiting effects of nicotinamide on urethane-induced malformations and tumors in mice

The antipellagratic vitamin, nicotinamide, significantly suppressed urethane-induced malformations, when it was given intraperitoneally to pregnant JCL:ICR mice immediately after a single subcutaneous injection of urethane (1.0 mg/g) on the 9th day of gestation. The level of inhibition increased with the doses of nicotinamide: 33.0, 55.8, and 70.0% at doses of 0.1, 0.3, and 0.5 mg/g, respectively. Polydactyly and tail anomalies were markedly suppressed by the post-treatment with nicotinamide, while cleft palates were less effectively suppressed. Nicotinamide was still effective, when it was given during the period of 24-48 h after urethane treatment. Furthermore, dietary administration of nicotinamide also reduced urethane-induced malformations. The level of inhibition was 39.4 and 61.1% at 0.5 and 1.0% of nicotinamide in the diet, respectively. Higher doses of nicotinamide (3 and 5% in diet) also inhibited urethane-induced malformations, but not so effectively as lower doses. The inhibiting effects of nicotinamide on the spontaneous incidence of cleft lips and palates in CL/Fr mice were significant at a low dose (0.5% in diet), but not at a higher dose (1.0%). When [carbonyl-14C]nicotinamide was given to pregnant mice, nicotinamide and small amounts of nicotinamide adenine dinucleotide (NAD+), but not nicotinic acid, were detected chromatographically in the fetus and placenta, indicating that nicotinamide or NAD+ acts directly on the fetus to suppress urethane-induced malformations. A preliminary study revealed that urethane-induced lung tumorigenesis in JCL:ICR mice was also inhibited by post-treatment with nicotinamide in the diet. The level of inhibition was proportional to the dose of nicotinamide, that is, 35.0 and 62.8% at 1.0 and 2.5% of nicotinamide in the diet, respectively.     

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.     

Nicotinamide Attenuates Focal Ischemic Brain Injury in Rats: With Special Reference to Changes in Nicotinamide and NAD+ Levels in Ischemic Core and Penumbra

We investigated the neuroprotective action of nicotinamide in focal ischemia. Male spontaneously hypertensive rats (5–7 months old) were subjected to photothrombotic occlusion of the right distal middle cerebral artery (MCA). Either nicotinamide (125 or 250 mg/kg) or vehicle was injected IV before MCA occlusion. Changes in the cerebral blood flow (CBF) were monitored using laser-Doppler flowmetry, and infarct volumes were determined with TTC staining 3 days after MCA occlusion. In another set of experiments, the brain nicotinamide and nicotinamide adenine dinucleotide (NAD⁺) levels were analyzed by HPLC using the frozen samples dissected from the regions corresponding to the ischemic core and penumbra. In the 250-mg/kg nicotinamide group, the ischemic CBF was significantly increased compared to that the untreated group, and the infarct volumes were substantially attenuated (–36%). On the other hand, the ischemic CBF in the 125 mg/kg nicotinamide group was not significantly different from the untreated CBF, however, the infarct volumes were substantially attenuated (–38%). Cerebral ischemia per se did not affect the concentrations of nicotinamide and NAD⁺ both in the penumbra and ischemic core. In the nicotinamide groups, the brain nicotinamide levels increased significantly in all areas examined, and brain NAD⁺ levels increased in the penumbra but not in the ischemic core. Increased brain levels of nicotinamide are considered to be primarily important for neuroprotection against ischemia, and the protective action may be partly mediated through the increased NAD⁺ in the penumbra.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

Pyridine nucleotide metabolism in mammalian cells in culture

The biosynthesis of pyridine nucleotides has been examined in a number of mammalian cell lines in culture. In all lines examined, nicotinamide is incorporated by a biochemical pathway distinct from the Preiss-Handler pathway for nicotinic acid. In at least the human cell line D98/AH2, there is no detectable endogenous synthesis of the pyridine ring from tryptophan. Although most cell lines examined (hamster BHK 21/13, mouse L929 and human D98/AH2) use either nicotinic acid or nicotinamide as a precursor for DPN and TPN, two mouse cell lines, 3T3-4E and LM CIID, are unable to utilize nicotinic acid as a source of the pyridine ring. If nicotinic acid is present in the medium, substantial amounts of intracellular desamido DPN accumulate suggesting that the last step (desamido DPN→DPN) is limiting in the Preiss-Handler pathway. With nicotinamide, the only compound which accumulates in substantial amounts apart from DPN and TPN is nicotinamide ribose; there is no detectable NMN. The results of pulse-labeling experiments suggest that nicotinamide ribose may be an intermediate in the nicotinamide pathway. Following growth of D98/AH2 cells in high concentrations of niacin, biosynthesis of DPN from nicotinamide was completely inhibited for at least six hours. The converse experiment revealed no inhibition of niacin incorporation. This observation suggests that a niacin pathway intermediate, which present evidence indicates is desamido-DPN. can inhibit nicotinamide utilization. Newly synthesized DPN turns over with a half-life of two hours in azaserine-treated D98/AH2 cells. In the absence of azaserine, the nicotinamide moiety of newly synthesized DPN is lost from D98/AH2 cells to the medium with a half-life of eight hours. About 80% of the nicotinamide is lost to medium as nicotinamide ribose.