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.     

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.     

Nucleoside salvage pathway for NAD biosynthesis in Salmonella typhimurium

A previously undescribed nucleoside salvage pathway for NAD biosynthesis is defined in Salmonella typhimurium. Since neither nicotinamide nor nicotinic acid is an intermediate in this pathway, this second pyridine nucleotide salvage pathway is distinct from the classical Preiss-Handler pathway. The evidence indicates that the pathway is from nicotinamide ribonucleoside to nicotinamide mononucleotide (NMN) and then to nicotinic acid mononucleotide, followed by nicotinic acid adenine dinucleotide and NAD. The utilization of exogenous NMN for NAD biosynthesis has been reexamined, and in vivo evidence is provided that the intact NMN molecule traverses the membrane.     

Utilization and metabolism of NAD by Haemophilus parainfluenzae

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.     

Pyridine nucleotide cycle and trigonelline (N-methylnicotinic acid) synthesis in developing leaves and fruits of Coffea arabica

We examined the biosynthesis of trigonelline in leaves and fruits of Arabica coffee ( Coffea arabica ) plants. [³H]Quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide and [¹⁴C]nicotinic acid, which are degradation products of NAD, were converted to trigonelline and pyridine nucleotides. These tracer experiments suggest that the pyridine nucleotide cycle, nicotinamide → nicotinic acid → nicotinic acid mononucleotide (NaMN) → nicotinic acid adenine dinucleotide (NaAD) → NAD → nicotinamide mononucleotide (NMN) → nicotinamide, operates in coffee plants, and trigonelline is synthesized from nicotinic acid formed in the cycle. Trigonelline accumulated up to 18 µmol per leaf in developed young leaves, and then decreased with age. Although the biosynthetic activity of trigonelline from exogenously supplied [¹⁴C]nicotinamide was observed in aged leaves, the endogenous supply of nicotinamide may be limited, reducing the contents in these leaves. Trigonelline is synthesized and accumulated in fruits during development. The trigonelline synthesis in pericarps is much higher than that in seeds, but its content in seeds is higher than pericaps, so that some of the trigonelline synthesized in the pericarps may be transported to seeds. Trigonelline in seeds may be utilized during germination, as its content decreases. Trigonelline synthesis from [¹⁴C]nicotinamide was also found in Theobroma cacao plants, but instead of trigonelline, nicotinic acid-glucoside was synthesized from [¹⁴C]nicotinamide in Camellia sinensis plants.     

Effects of Vitamin B6 Deficiency on the Conversion Ratio of Tryptophan to Niacin

To investigate how vitamin B₆ (B₆) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B₆, the control diet without nicotinic acid, and the control diet without nicotinic acid and B₆, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone3- carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B₆-deficient groups, when compared with the B₆-containing groups. These results indicate that the rats fed with the B₆-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B₆-free diet than in the B₆-containing diet under the niacin-free conditions.     

Simultaneous measurement of nicotinamide and its catabolites,nicotinamide N-oxide,N1-methyl-2-pyridone-5-carboxamide,and N1-methyl-4-pyridone-3-carboxamide,in mice urine

Nicotinamide N-oxide is a major nicotinamide catabolite in mice but not in humans and rats. A high-performance liquid chromatographic method for the simultaneous measurement of nicotinamide, nicotinamide N-oxide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide in mice urine was developed by modifying the mobile phase of a reported method for measurement of nicotinamide N-oxide.     

Some effects of hydrolytic enzymes on coupled and uncoupled electron flow in chloroplasts

Digestion of spinach chloroplasts with pancreatic lipase or trypsin effectively uncoupled electron transport. Continued digestion led to inhibition of saturated rates of Hill reaction activity and a decrease in quantum yield. Irradiation with ultraviolet light decreased the quantum yield and inhibited Hill activity, but did not uncouple. Ascorbate-dichlorophenol-indophenol-mediated reduction of nicotinamide adenine dinucleotide phosphate was not appreciably inhibited by treatment with either of the enzymes or by ultraviolet irradiation.     

Effects of excess nicotinamide administration on the urinary excretion of nicotinamide N-oxide and nicotinuric acid by rats

We investigated a useful chemical index for an excessive nicotinamide intake and how this excessive nicotinamide intake affects the tryptophan-nicotinamide metabolism in rats. Weaning rats were fed on a tryptophan-limited and nicotinic acid-free diet containing no, 0.003%, 0.1%, 0.2%, or 0.3% nicotinamide for 21 days. Urine samples were collected on the last day and analyzed the intermediates and metabolites on the tryptophan-nicotinamide pathway. Nicotinamide N-oxide, nicotinic acid and nicotinuric acid, metabolites of nicotinamide, were detected when nicotinamide at more than 0.1% had been taken. An intake of nicotinamide of more than 0.1% increased the urinary excretion of quinolinic acid, an intermediate on the pathway. Nicotinamide N-oxide and nicotinuric acid increased with increasing dietary concentration of nicotinamide. These results show that the measurements of nicotinamide N-oxide and nicotinuric acid in urine would be useful indices for an excessive nicotinamide intake.     

The urinary excretory ratio of nicotinamide catabolites was associated with the conversion ratio of tryptophan to nicotinamide in growing rats fed a niacin-free 20% casein diet

Weaning rats were fed a niacin-free 20% casein diet. Twenty-four-h-urine samples were collected, and nicotinamide and its catabolites were measured. A correlation was found between the urinary excretory ratio of nicotinamide catabolites (N(1)-methyl-2-pyridone-5-carboxamide + N(1)-methyl-4-pyridone-3-carboxamide)/N(1)-methylnicotinamide and the tryptophan-nicotinamide conversion ratio during growing period of the rats. This indicates the possibility that the conversion ratio can be deduced from the excretory ratio.     

The Urinary Excretory Ratio of Nicotinamide Catabolites Was Associated with the Conversion Ratio of Tryptophan to Nicotinamide in Growing Rats Fed a Niacin-Free 20% Casein Diet

Weaning rats were fed a niacin-free 20% casein diet. Twenty-four-h-urine samples were collected, and nicotinamide and its catabolites were measured. A correlation was found between the urinary excretory ratio of nicotinamide catabolites (N ¹-methyl-2-pyridone-5-carboxamide + N ¹-methyl-4-pyridone-3-carboxamide)/N ¹-methylnicotinamide and the tryptophan-nicotinamide conversion ratio during growing period of the rats. This indicates the possibility that the conversion ratio can be deduced from the excretory ratio.     

Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [³H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD → nicotinamide mononucleotide → nicotinamide riboside → nicotinic acid riboside → nicotinic acid mononucleotide → nicotinic acid adenine dinucleotide → NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-¹⁴C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-¹⁴C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.     

Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.

Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic studies of pyridine nucleotide metabolism in human culture cells

More than 80% of the intracellular pyridine metabolite pool of human culture cells is trapped by OsO₄ fixation. The fixed pyridine metabolites fully exchange with nicotinamide and nicotinic acid but not with nicotinamide adenine dinucleotide. Yet, chromatography of the exchanged compounds reveals that NAD and NADP constitute more than 95%. of the fixed material. Although the mechanism of OsO₄ fixation is not fully understood, such fixation has permitted the autoradiographic detection of intracellular pyridine metabolites. Cells of the human cell line, D98/AH2, synthesize pyridine nucleotides during all phases of the cell cycle at rates which do not vary by more than six-fold. There is no difference in the apparent concentration of pyridine metabolites between nucleus and cytoplasm after ten minute or three day pulses with ³H-nicotinic acid. The ³H-labeled pyridine ring is lost from D98/AH2 cells upon transfer to unlabeled medium. In general, the rate of loss is uniform among cells in the population. However, in a small proportion of cells there is little or no loss. Non-dividing cells lose the pyridine ring at approximately the same rate as dividing cells, yet the intracellular concentration of pyridine metabolites is 50% greater in non-dividing cells.     

Autoradiographic studies of nicotinic acid utilization in human-mouse heterokaryons and inhibition of utilization in newly-formed hybrid cells

Although most mammalian cell lines can utilize either nicotinic acid or nicotinamide for the biosynthesis of nicotinamide adenine dinucleotide (NAD), thymidine kinase-deficient, mouse 3T3–4F cells are unable to utilize nicotinic acid. When 3T3–4E cells were fused with human D98/AH2 cells, autoradiography showed that the resultant heterokaryons synthesized NAD from nicotinic acid at rates comparable to the human parental cell. The rate of nicotinic acid utilization in heterokaryons remained unchanged over the fourday period of study following cell fusion. In contrast to the results observed with heterokaryons, nicotinic acid utilization was markedly reduced in hybrid cells. Of 100 hybrid clones examined at four or five days following cell fusion, 60 utilized nicotinic acid at rates less than one tenth that of the parental human cell. Similar results were observed in hybrid clones at nine or ten days following fusion. Uniformly high rates of NAD biosynthesis were observed in hybrid clones with nicotinamide as the precursor. This excludes the possibility that the reduction in nicotinic acid utilization in hybrid cells is due to a general metabolic dysfunction. The biochemical mechanism by which nicotinic acid utilization is markedly reduced has not been determined with certainty, however, several observations suggest genetic suppression.     

Effects of fatty liver induced by niacin-free diet with orotic acid on the metabolism of tryptophan to niacin in rats

The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20% casein diet containing 0% (control diet) or 1% orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.     

Effect of Adding Methionine and Threonine to a Protein-free Diet on the Metabolism of Nicotinamide

We have been attempting to confirm the hypothesis that the excretion ratio of nicotinamide metabolites, [N¹-methyl-2-pyridone-5-carboxamide (2-Py) + N¹-methyl-4-pyridone-3-carboxamide (4-Py)]/N¹-methylnicotinamide (MNA), reflects the adequacy of amino acid nutrition, but not of niacin nutrition. It is known that methionine and threonine supplementation to a protein-free diet reduced body weight loss. In this paper, we investigated whether rats fed with a protein free-diet supplemented with methionine and threonine would result in this excretion ratio being increased or not. The body weight loss was markedly reduced by the supplementation with both amino acids, as has been reported, and under the conditions, the excretion ratio significantly increased. The activity of 4-Py-forming MNA oxidase, which controls the change in excretion ratio, also increased significantly. From the present results and our previous results, it was proved that the excretion ratio of nicotinamide metabolites, (2-Py +4-Py)/MNA, reflects the adequacy of amino acid nutrition, but not of niacin nutrition.     

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.     

Thiosulfate- and Sulfide-Dependent Pyridine Nucleotide Reduction and Gluconeogenesis in Intact Thiobacillus neapolitanus

Nicotinamide adenine dinucleotide phosphate (reduced form) is formed more rapidly after the addition of thiosulfate to suspensions of intact Thiobacillus neapolitanus in the absence of CO(2) than nicotinamide adenine dinucleotide (reduced form). Measurement of acid-stable metabolites shows this phenomenon to be the result of rapid reoxidation of nicotinamide adenine dinucleotide (reduced form) by 3-phosphoglyceric acid and other oxidized intermediates, which are converted to triose and hexose phosphates, and that, in reality, the rate of nicotinamide adenine dinucleotide (oxidized form) reduction exceeds that of nicotinamide adenine dinucleotide phosphate (oxidized form) by approximately 4.5-fold. The overall rate of pyridine nucleotide reduction by thiosulfate (264 nmol per min per mg of protein) is in excess of that rate needed to sustain growth. Pyridine nucleotide reduction, adenosine triphosphate synthesis, and carbohydrate synthesis are prevented by the uncoupler m-Cl-Carbonylcyanide phenylhydrazone. Sodium amytal inhibits pyridine nucleotide reduction and carbohydrate synthesis are prevented by the uncoupler m-Cl-carbonylcyanide observations are reproduced when sulfide serves as the substrate. The rate of pyridine nucleotide anaerobic reduction with endogenous substrates or thiosulfate is less than 1% of the aerobic rate with thiosulfate. We conclude that the principal, if not the only, pathway of pyridine nucleotide reduction proceeds through an energy-dependent and amytal-sensitive step when either thiosulfate or sulfide is used as the substrate.