Nicotinate Phosphoribosyltransferase Research Articles

18] Determining NAD synthesis in erythrocytes

This chapter describes methods for the study of nicotinamide adenine dinucleotide (NAD) synthesis in human erythrocytes, developed according to two approaches— the determination of intracellular concentrations of pyridine compounds and the rate of conversion of radiolabeled precursors in intact cells, and enzyme activity assays in crude lysates. All determinations are performed by methods involving high-performance liquid chromatography (HPLC). Different methods of obtaining protein-free extracts suitable for HPLC processing are examined. Acid or alkaline extraction provides better results than acetonitrile extraction or boiling, which cause oxidation of NADH or breakdown of NAD. The activities of several enzymes are measured in crude lysates made from fresh or, when suitable, stored erythrocytes: nicotinate phosphoribosyltransferase (NA–PRT), nicotinamide phosphoribosyltransferase (NAm–PRT), nicotinamide-mononucleotide and nicotinate-mononucleotide adenylyltransferase (NMN–AT and NAMN–AT), and NAD synthetase (NAD-s). All assay reactions are started by lysate addition. Hemoglobin content is measured in lysates by the cyanmethemoglobin method and used to quantify enzyme specific activity.

NAD + biosynthesis and metabolic fluxes of tryptophan in hepatocytes isolated from rats fed a clofibrate-containing diet

Hepatocytes were isolated from rats fed a diet with or without 0.25% clofibrate, and NAD + synthesis by the hepatocytes was determined using either [ carboxyl- 14C]nicotinic acid or [5- 3H]tryptophan. NAD + and total pyridine nucleotides synthesized from [ 14C]nicotinic acid by the clofibrate-treated cells were not significantly different from those synthesized by the control cells when expressed on the basis of nanomoles per hour per milligram of DNA. On the contrary, NAD + synthesized from [ 3H]tryptophan was significantly higher in the clofibrate-treated cells (158% of the control cells) on the basis of nanomoles per hour per milligram of DNA. Clofibrate was inhibitory to tryptophan metabolism as a whole, affecting the glutarate pathway more (decreased to 37% of control) than the kynureninase flux (decreased to 64% of control). As a result, the quinolinate-NAD flux, estimated as the difference in the amounts of tryptophan metabolized by the two metabolic pathways, increased in the clofibrate-treated hepatocytes. The increase in quinolinate during the incubation was 8 times more in the clofibrate-treated cells than in the control cells, which confirmed alteration in the metabolic fluxes of tryptophan in the clofibrate-treated cells. Hepatic quinolinate phosphoribosyltransferase (EC 2.4.2.19) activity increased with dietary clofibrate and returned to the control level 1 week after removing clofibrate from the diet. Nicotinate phosphoribosyltransferase (EC 2.4.2.11) and NAD + glycohydrolase (EC 3.2.2.5) activities remained unchanged with dietary clofibrate.

A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase.

The pncB gene of Salmonella typhimurium was used to develop an overexpression system for nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11), which forms nicotinate mononucleotide (NAMN) and PPi from nicotinate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAPRTase hydrolyzes ATP in 1:1 molar stoichiometry to NAMN synthesis. Hydrolysis of ATP alters the ratio of products/substrates for the reaction nicotinate + PRPP <--> NAMN + PPi from its equilibrium value of 0.67 to a steady-state value of 1100. The energy for the maintenance of this ratio must come from ATP hydrolysis. However, in contrast to other ATP-utilizing enzymes, when all ATP is hydrolyzed the unfavorable product/substrate ratio collapses. ATP/ADP exchange results suggest that the overall reaction involves a phosphoenzyme (E-P) arising from E.ATP. Km values for nicotinate and PRPP each decreased by 200-fold when ATP was present to phosphorylate the enzyme. PPi stimulated the ATPase activity of the enzyme to Vmax values, suggesting that PPi formation during catalysis provides a trigger for cleavage of the putative E-P in the overall reaction and regenerates the low affinity form of the enzyme. A model is presented in which alternation of high and low affinity forms of NAPRTase provides a "steady-state" coupling between ATP hydrolysis and NAMN formation.

Cloning and nucleic acid sequence of the Salmonella typhimurium pncB gene and structure of nicotinate phosphoribosyltransferase

The pncB gene of Salmonella typhimurium, encoding nicotinate phosphoribosyltransferase (NAPRTase), was cloned on a 4.7-kb Sau3A fragment. The gene contains a 1,200-bp open reading frame coding for a 400-residue protein. Amino acid sequencing of the amino-terminal and two interior peptides of the purified protein confirmed the deduced sequence and revealed that the amino-terminal methionine residue was removed, giving a 399-residue mature protein of Mr 45,512. No signal sequence was observed in the predicted NAPRTase primary structure, suggesting that the enzyme is not periplasmic. The protein does not demonstrate clear sequence similarity to the other seven phosphoribosyltransferases of known primary structure and frustrates attempts to define a consensus 5-phosphoribosyl-1-pyrophosphate-binding region. The NAPRTase reaction is ATP stimulated, and the protein contains a carboxy-terminal sequence diagnostic of an ATP-binding site. An inverted repeat of the sequence TAAACAA observed in the proposed promoter region of pncB is also present in the promoter of nadA, which, like pncB, is also regulated by the NadR (NadI) repressor. The sequence may thus define an NadR repressor-binding site.

Tissue distribution of the enzymes concerned with the biosynthesis of NAD in rats.

There are three NAD biosynthetic pathways: the nicotinic acid-NAD, nicotinamide-NAD, and quinolinic acid (derived from tryptophan)-NAD pathways. To discover the main pathways of NAD biosyntheses in various tissues of the rat, the tissue distribution of nicotinamidase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase, nicotinamide phosphoribosyl-transferase, nicotinamide mononucleotide adenylyltransferase, and NAD+ synthetase were investigated. All of the tissues could synthesize NAD from nicotinamide, judging from that the activities of nicotinamide phosphoribosyltransferase and NMN adenylyltransferase detected in all of the tissues. From nicotinic acid, only liver, kidneys, and heart could. Liver and kidney can also synthesize NAD de novo from quinolinic acid.

The biosynthesis of nicotinamide adenine dinucleotide. Part XIX. Nicotinate phosphoribosyltransferase from hog liver: Regulatory effect of ATP at a physiological concentrations of 5-phosphoribosyl-1-pyrophosphate.

Nicotinate phosphoribosyltransferase (NPRTase, EC 2.4.2.11) purified from hog liver was stimulated by orthophosphate. This stimulation was independent of ATP which activated this enzyme by increasing the velocity and lowering the Km values for nicotinic acid and 5-phosphoribosyl-1-pyrophosphate (PRPP). This independent stimulation by orthophosphate and activation by ATP were true of hog liver or rat liver homogenate as well as the purified hog liver NPRTase, showing that there may be some physiological role of these compounds in vivo. The Ka value for ATP was about 1.2mM. At a physiological concentration of PRPP, it was mainly used by quinolinate phosphoribosyltransferase (QPRTase, EC 2.4.2.19) in the reconstituted assay conditions of NPRTase and QPRTase if the ATP concentration was lower. But if the ATP concentration was higher, PRPP was used by both QPRTase and NPRTase. Therefore, ATP was found to play a very important role for the biosynthesis of NAD via the salvage pathway.

The biosynthesis of nicotinamide adenine dinucleotide. Part XVIII. Purification and some properties of nicotinate phosphoribosyltransferase from hog liver.

Purification of nicotinate phosphoribosyltransferase (NPRTase, EC 2.4.2.11) from hog liver and some of its properties were investigated. Purified enzyme was found to be homogeneous by the criterion of polyacrylamide gel disc electrophoresis and to have a molecular weight of about 120,000. The subunit molecular weight was found to be 64,000, using SDS polyacrylamide gel disc electrophoresis. The optimum pH and isoelectric point of the enzyme were 7.3~7.4 and 4.8, respectively. Mn2+, Co2+, and Mg2+ were effective in meeting the NPRTase requirement for a divalent cation. ATP had a stimulative effect on the enzyme activity in the presence of Mg2+, on the other hand, in the presence of Mn2+, ATP had an inhibitory effect on the enzyme activity. Of the purine nucleotides examined, only ATP stimulated NPRTase activity, and GTP did not. On the contrary, all other nucleotides inhibited the activity. Nicotinate mononucleotide, which is a reaction product, inhibited NPRTase activity. Among the nicotinic acid analogue...

Kinetic analysis of nicotinate phosphoribosyltransferase from yeast using high pressure liquid chromatography.

A new procedure has been designed for the purification of nicotinate phosphoribosyltransferase and orotate phosphoribosyltransferase from the same baker's yeast extract. Using purified nicotinate phosphoribosyltransferase, the enzyme-catalyzed formation of nicotinate mononucleotide was analyzed using a new high pressure liquid chromatographic assay (Hanna, L., and Sloan, D. L. (1980) Anal. Biochem. 103, 230-234). Initial velocity measurements and product inhibition studies, with pyrophosphate, were performed. In addition, this assay procedure was used to demonstrate that purified nicotinate phosphoribosyltransferase possesses an ATPase activity in the presence of either product (pyrophosphate or nicotinate mononucleotide (NaMN] but in the absence of 5-phosphoribosyl alpha-1-pyrophosphate (P-Rib-PP). Moreover, exchanges of radioactivity between specific substrate/product pairs [( 14C]nicotinate/NaMN and [32P]PPi/P-Rib-PP) in the absence of other substrates were not observed when these pairs were incubated with nicotinate phosphoribosyltransferase, and binding of [14C] nicotinate to nicotinate phosphoribosyltransferase was not detected in the presence of ATP. In contrast, an exchange of label between ATP and [14C]ADP was characterized in the absence of other substrates and in the presence of either P-Rib-PP or PPi. These results indicate that nicotinate phosphoribosyltransferase proceeds through the use of an ordered Uni Uni Bi Ter Ping Pong kinetic mechanism during which ATP reacts with nicotinate phosphoribosyltransferase to form ADP and a previously described phosphorylated enzyme (Kosaka, A., Spivey, H. O., and Gholson, R. K. (1977) Arch. Biochem. Biophys. 179, 334-341). Thereafter, P-Rib-PP and nicotinate bind in order to the active site, to produce PPi and NaMN which are released in a random order followed by Pi. The Km values for ATP, P-Rib-PP, and nicotinate were calculated to be 70 +/- 10, 24 +/- 3, and 23 +/- 4 microM, respectively, whereas a value for Ki(PRPP) of 5 +/- 1 microM was determined.

Probable mechanisms of regulation of the utilization of dietary tryptophan, nicotinamide and nicotinic acid as precursors of nicotinamide nucleotides in the rat.

1. The regulation of the utilization of dietary tryptophan, nicotinamide and nicotinic acid as precursors of the nicotinamide nucleotides has been studied in groups of rats fed on diets providing only one of these precursors at a time, in amounts adequate to meet their requirements for nucleotide synthesis.2. The concentration of nicotinamide nucleotides in the liver of rats receiving a high-tryptophan diet was 56% higher than in animals fed on a diet providing a minimum amount of tryptophan, together with nicotinic acid or nicotinamide. The excretion ofN1-methyl nicotinamidc was three times higher in the tryptophan-fed animals than in the other two groups.3. The concentration of quinolinic acid in the liver was significantly higher in animals receiving the high-tryptophan diet than in the other two groups; that of nicotinic acid was highest in those animals receiving the nicotinic-acid-containing diet. The concentration of nicotinamide was highest in the livers of those animals receiving the high-tryptophan diet, and lowest in those receiving the nicotinic-acid-contaning diet.4. The values of the Michaelis constantKmof nicotinamide deamidase (nicotinamide amidohydrolase.EC3.5,1,19) and nicotinamide phosphoribosylltransferase (nicotinamide nucleotide: pyrophosphate phosphoribosyltransferase,EC2.4.2.12) were approximately equal, and approximately one-tenth of the concentration of nicotinamide was in the liver. This suggests that both these enzymes normally function at their maximum rate, and a change in the availability of nicotinamide would not affect the rate of its incorporation into nucleotides.5. The maximum rate of reaction (Vmax) of nicotinamide deamidase was twice that of nicotinamide phosphoribosyltransferase; this suggests that unless compartmental or other factors are involved, the major route of nicotinamide utilization will be by way of deamidation.6. TheKmof nicotinate phosphoribosyltransferase (nicotinate nucleotide: pyrophosphate phosphoribosyltransferase,EC2.4.2.11) was less than twice the concentration of nicotinic acid in the liver, so that a change in the availability of nicotinic acid might be expected to lead to a small change in the rate of its utilization.7. TheKmof quinolinate phosphoribosyltransferase (nicotinate nucleotide: pyrophosphate phosphoribosyltransferase (carboxylating)EC2.4.2.19) was approximately 100 times greater than the concentration of quinolinic acid in the liver, so that any change in the availability of quinolinic acid would be expected to lead to a considerable change in the rate of its utilization. TheVmaxof quinolinate phosphoribosyltransferase was relatively low, so that under conditions of high tryptophan flux, some accumulation of quinolinic acid might be expected. This was observed in animals receiving the high-tryptophan diet.8. It is concluded that it is unlikely that the utilization of quinolinic acid, arising from tryptophan. for the synthesis of nicotinamide nucleotides is regulated, but that control over tissue concentrations of nucleotides is achieved by hydrolysis of NAD to nicotinamide. Incorporation of nicotinamide into nucleotides seems to be strictly limited by the activity of the enzymes involved.

A high-pressure liquid chromatography procedure for monitoring nicotinate phosphoribosyltransferase activity

A high-pressure liquid chromatography (hplc) procedure has been designed to monitor the reaction catalyzed by nicotinate phosphoribosyltransferase (N-PRTase). Two substrates (ATP and nicotinate) and two products of this reaction (nicotinate mononucleotide and ADP) can be resolved at pH 8 on a Waters μBondapak C 18 column, and the concentration of each reactant in the isocratic eluant (25 m m (NH 4) 3PO 4) can be determined spectroscopically at 254 nm. This separation method allows an analysis of the disappearance of substrates and the appearance of products simultaneously during the time course of the enzyme-catalyzed reaction. We have employed this new N-PRTase assay system: (1) to prove that a 1:1 stoichiometry exists between the enzymatic ATP hydrolysis and nicotinate mononucleotide formation and (2) to reveal for the first time an inhibition of the overall reaction by phosphoribosyl α-1-pyrophosphate at relatively low ATP concentrations.

Dietary tryptophan level and the enzymes of tryptophan NAD pathway.

1. Dietary tryptophan was found to regulate the activities of tryptophan oxygenase (EC 1.13.1.12) and quinolinate phosphoribosyltransferase (EC 2.4.2a) in liver. 2. With increasing tryptophan concentration in the diet containing 100 g protein/kg, tryptophan oxygenase activity increased while that of quinolinate phosphoribosyltransferase decreased. The response of these enzymes to dietary tryptophan at lower dietary protein level (25 g/kg) was not significant. 3. Liver nicotinate phosphoribosyltransferase (EC 2.4.2.11) activity and kidney picolinate carboxylase (EC 4.1.1.45) activity were unaltered with different tryptophan concentrations in the diet. 4. The response of various biochemical measurements was dependent on the tryptophan intake and the changes were marked below and above the requirement level of tryptophan. 5. It is suggested that the urinary excretion of quinolinic acid and N'-methylnicotinamide may be useful in assessing the tryptophan nutritional status and its requirement.

Periplasmic localization of nicotinate phosphoribosyltransferase in Escherichia coli

Nicotinate phosphoribosyltransferase (NAPRTase) in Escherichia coli mediates the formation of nicotinate mononucleotide, a direct precursor of nicotinamide adenine dinucleotide (NAD), from nicotinate and 5-phosphoribosyl-1-pyrophosphate. Specifically, NAPRTase contributes to NAD synthesis by utilizing intracellular nicotinate formed from NAD degradation products, which are recycled by NAD cycle enzymes and exogenous nicotinate when it is available. In previous studies, it has been tacitly assumed that almost all NAD cycle enzymes are localized in the cytoplasm of E. coli. The results of this investigation provide evidence that NAPRTase is a periplasmic (extracytoplasmic) enzyme. The osmotic shock of exponential-phase cells of E. coli K-12 and ML 308-225 resulted in the release of 63 to 72% and 42 to 48%, respectively, of the NAPRTase into the shock medium. In addition, when exponential cells of strains K-12 and ML 308-225 were converted into spheroplasts, 75 to 84% and 54 to 68%, respectively, of the enzyme was released into the spheroplast medium. Since previous estimates of the effective levels of NAPRTase present in putative repressed and derepressed E. coli cells appeared to be very low, a more convenient and accurate alternative method for the evaluation of NAPRTase in whole cells was developed. The results show that NAPRTase is subject only to a modest degree of enzyme repression. In addition, no evidence was found for the presence of a protein or low-molecular-weight inhibitor of the enzyme in repressed cells.

Effect of Diet Restriction on Some Key Enzymes of Tryptophan-NAD Pathway in Rats

Dietary intake of rats was restricted by feeding varying amounts of a 20% protein diet. After 6 weeks of feeding, some key enzymes of the tryptophan and nicotinic acid-NAD pathway, liver nicotinamide nucleotide concentration, and urinary metabolites of tryptophan and nicotinic acid were studied. With an increase in diet restriction, liver tryptophan oxygenase (EC 1.13.1.12) activity increased. Quinolinate phosphoribosyltransferase (EC 2.4.2.a) activity, on the other hand, was found to decrease with moderate diet restriction up to 50% restriction, but increased again with more severe diet restriction in rats fed 25% of ad libitum intake. Liver nicotinate phosphoribosyltransferase (EC 2.4.2.11) activity was also observed to decrease with moderate diet restriction and did not further change when the restriction was severe while picolinate carboxylase (EC 4.1.1.45) activity increased significantly only in severe diet restriction. In rats fed 25% of ad libitum intake, urinary quinolinic acid excretion was low whereas N'-methylnicotinamide excretion was elevated. Alterations in the enzyme activities accompanied by changes in the levels of urinary metabolites, observed in the present study, suggest that the potential efficiency of conversion of tryptophan to nicotinamide nucleotides is not constant and is influenced by dietary intake.

Effect of dietary protein level on some key enzymes of the tryptophan-NAD pathway

1. Six groups of rats were given diets containing protein at three levels (50, 100 and 200 g/kg), with and without nicotinic acid. After 4 weeks on these diets some key enzymes of the tryptophan and nicotinic acid-NAD pathway, liver nicotinamide nucleotide concentration, and urinary metabolites of tryptophan and nicotinic acid were studied. 2. Liver nicotinamide nucleotide levels were lower in rats given the diet with 50 g protein/kg as compared to those in rats given diets with 100 and 200 g protein/kg. The addition of nicotinic acid to the diet resulted in a significant increase in the levels of nicotinamide nucleotides only in rats given 50 g protein/kg diet but not in those given either 100 or 200 g protein/kg diet. 3. Liver tryptophan oxygenase (EC 1.13.1.12) activity increased with increasing dietary protein level. Niconitic acid in the diet had no effect on its activity. 4. Quinolinate phosphoribosyltransferase (EC 2.4.2.a) activity in liver was inversely related to dietary protein level, and nicotinic acid in the diet had no effect on its activity. 5. Liver nicotinate phosphoribosyltransferase (EC 2.4.2.11) activity and kidney picolinate carboxylase (EC 4.1.1.45) activity were not altered either by dietary protein level or nicotinic acid in the diet. 6. The addition of nicotinic acid to the diet resulted in increased excretion of N'-methylnicotinamide at all dietary protein levels. 7. The inverse relationship between protein level in the diet and liver quinolinate phosphoribosyltransferase activity, the rate-limiting enzyme of the tryptophan-NAD pathway suggests that the efficiency of conversion of tryptophan to NAD is related to protein level in the diet, the efficiency decreasing with an increase in the level of dietary protein.

Yeast nicotinic acid phosphoribosyltransferase: Studies of reaction paths, phosphoenzyme, and Mg 2+ effects

Initial velocity and ATP-ADP isotope exchange kinetic data on yeast nicotinate phosphoribosyltransferase indicate a sequential steady-state mechanism m which ATP adds first, followed by a random order of addition of the substrates PP-ribose-P and nicotinic acid. Reaction flux through the alternative ping-pong pathway, evidenced first by ATP-ADP isotope exchange in the absence of nicotinic acid and PP-ribose-P substrates, is reduced by low concentrations of either, or both, of these substrates. Both Mg 2+-complexed and -free PP-ribose-P are enzymatically active. Phosphorylated enzyme, prepared with [γ- 32P]ATP and separated by gel chromatography, retains 32P i until incubated with both substrates, PP-ribose-P and nicotinic acid, which completely discharge the 32P i.

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.

Biosynthesis of NAD in Haemophilus haemoglobinophilus

Haemophilus haemoglobinophilus, a species of Haemophilus which is not dependent upon NAD for growth, was investigated with regard to the pathway of biosynthesis synthesis of this cofactor. Cell-free extracts synthesized NAD directly from nicotinamide without prior deamidation via the following reactions: Nicotinamide+5-phosphoribosyl 1-prrophosphate+ATP → NMN NMN+ATP → NAD Of the enzymes known to be operative in the pathway of NAD biosynthesis from nicotinic acid (nicotinate phosphoribosyltransferase, nicotinate adeninedinucleotide pyrophosphorylase and NAD synthetase) only nicotinate phosphoribosyltransferase activity was demonstrable. Cell free extracts of H. haemoglobinophilus did not degrade NAD and nicotinamidase activity was absent. Quinolic acid was not converted to NAD. The results obtained provided evidence that this organism cannot recycle NAD or synthetize it via the de novo pathway. Direct synthesis from nicotinamide via NMN appears to be the sole pathway of NAD formation in this organism.

Effect of leucine on enzymes of the tryptophan–niacin metabolic pathway in rat liver and kidney

Dietary excess of leucine affects tryptophan-niacin metabolism adversely and has thus been implicated in the etiology of pellagra. To understand the biochemical basis of leucine-induced changes in tryptophan-niacin metabolism the effect of leucine on enzymes of tryptophan-niacin metabolism was investigated. Excess of leucine in the diet had no effect on rat liver 3-hydroxyanthranilate oxygenase and nicotinate phosphoribosyltransferase but significantly decreased the activity of quinolinate phosphoribosyltransferase of rat liver and kidney. The activities of tryptophan oxygenase in liver and picolinate carboxylase in kidney were significantly higher in leucine-fed animals than in the controls. Also, oxidation of [U-(14)C]tryptophan in vivo was higher in leucine-fed animals. Increased picolinate carboxylase and decreased quinolinate phosphoribosyltransferase activities would result in a decrease in NAD formation from dietary tryptophan. Lowered NAD formation from tryptophan particularly when the niacin concentrations in the diet are marginal would result in a state of conditioned niacin deficiency.

Nicotinate Phosphoribosyltransferase of Human Erythrocytes: PURIFICATION AND PROPERTIES

Abstract Nicotinate phosphoribosyltransferase from human erythrocytes has been purified approximately 30,000-fold. The purified enzyme migrates as a single protein band on disc electrophoresis at pH 7.9 and 9.5. The protein forms an inactive precipitate in salt concentrations below 50 mm. Addition of 25% glycerol protects against this precipitation. ATP stimulates the enzymatic reaction but is not an obligatory substrate. When present, ATP is hydrolyzed to ADP and Pi. This hydrolysis is stoichiometric with nicotinate mononucleotide formation. In the presence of saturating concentrations of ATP, the apparent Km values for nicotinate and 5-phosphoribosyl 1-pyrophosphate are decreased from 24 to 0.5 µm and 100 to 5 µm, respectively. The enzyme is not an ATPase in the absence of any of the other substrates. Its molecular weight is 86,000 by gel filtration in the absence or presence of 1.0 mm MgATP.

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.