Nicotinamide nucleotide levels

D. E. F. Harrison and B. Chance, Fluorimetric technique for monitoring changes in the level of reduced nicotinamide nucleotides in continuous cultures of microorganisms, Appl. Microbiol. 19, 446-450 (1970). 

Nicotinamide nucleotide (NAD, NADP, NADH, NADPH) levels (Table 4.6) have been measured only in one cestode, H. diminuta (42), which probably reflects the difficulty of carrying out such an analysis. In this... 

Table 4.6. The levels of nicotinamide nucleotides in Hymenolepis diminuta compared with Fasciola hepatica and Ascaris... 

Barrett, J. Beis, I. (1973). Nicotinamide and adenosine nucleotide levels in Ascaris lumbricoides, Hymenolepis diminuta and Fasciola hepadca. International Journal for Parasitology, 3 271-3. 

Any intracellular nicotinamide which is produced from NAD, either directly or indirectly (from NMN), must be deamidated to nicotinic acid before it can be resynthesized into NAD. When nicotinamide deamidase levels faU, intracellular nicotinamide is not efficiently recycled, and a significant fraction of nicotinamide produced by pyridine nucleotide cycles is excreted into the medium. Thus, lowering nicotinamide deamidase levels shunts the normal pyridine nucleotide cycle to cause excretion of nicotinamide. 

The rise in liver DPN in the mouse is correlated with the concentration of nicotinamide injected, as illustrated in Fig. 4. High concentrations (2000 mg/kg) of the amide are inhibitory to the synthesis of the DPN. The maximum increase is around tenfold and usually occurs from 8 to 10 hr following the administration of the nicotinamide the DPN level then returns to normal at the end of about 20 to 24 hr after the injection. It is of considerable interest to note that the pyridine nucleotide level always drops to the normal base level and not below the original value. 

From the evidence described above it appears that both nicotinamide and nicotinic acid are convoted to nicotinic acid- DPN. This would suggest that the deamidation of nicotiiiainide occurs during the conversion of the amide to DPN. Deamidation of nicotinamide has been shown to take place in bacteria (see Section IV), but no dear evidence for this reaction has yet been found in mammalian tissues. Nicotinic add mononucleotide also has not been found in liver following injection of nicotinic add or nicotinamide. Further work is essential before it can be concluded that nicotinamide is converted to nicotinic acid as the free base, or that the conversion is at a nucleoside or nucleotide level. 

The study of bioenergetics involves the study of (1) the processes by which reduced nicotinamide and flavin nucleotides, generated primarily from the oxidation of carbohydrates (Chap. 11) and lipids (Chap. 13), are oxidized ultimately by molecular oxygen via the mitochondrial electron-transport chain, and (2) the mechanism by which this oxidation is coupled to ATP synthesis. The synthesis of ATP in this way is referred to as oxidative phosphorylation, in contrast to phosphorylation of ADP via soluble enzymes. The latter involves intermediate phosphate derivatives of the substrate and is known as substrate-level phosphorylation (Chap. 11). 

Nicotinamide adenine dinucleotide (NAD and its reduced form dihydronicotinamide adenine dinucleotide (NADH)) as well as nicotinamide adenine dinucleotide phosphate (NADP and its reduced form nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)) are essential co-factors for many dehydrogenases. The presence of NAD was first demonstrated in P. gallinaceum (Speck and Evans, 1945) and later studies showed increased levels of these pyridine nucleotides in P. lophurae, P. berghei and P. falciparum. Trager (1977) reported that high levels of... 

An alternative explanation for the accumulation of DNA strand breaks with increasing time of incubation with MBA and the continued effects of MBA at times well after the minimum number of strand breaks was attained at 2 h of culture, would be a nonspecific effect of the inhibitor on DNA synthesis rather than that on ADP-ribosylation-mediated repair. One such possibiUty consistent with the data of Fig. 1, would be that MBA was itself directly or indirectly introducing breaks into the DNA. This hypothesis was excluded by experiments in which the effect of MBA was measured in cultures in which ADP-ribosylation had already been maximally inhibited by alternative inhibitors such as 3-aminobenzamide or high concentrations of nicotinamide, or heat shock [11] (data not shown). No additional breaks were introduced by MBA under these conditions. Furthermore, there was no effect of MBA on cellular levels of purine or pyrimidine nucleotides, and MBA treatment resulted in only 4% cell death. Table 2 shows that the inhibitory effects of MBA on ADP-ribosylation, and on cell proliferation were completely reversible. In addition, the data in Table 3 show a direct proportionality between concentration of MBA on the one hand and inhibition of ADP-ribosylation in permeabilized cells, increased number of DNA strand breaks and decreased cell proliferation at 48 h on the other hand. This, and the data cited above, permit us to conclude that MBA acts on this system by inhibiting the poly(ADP-ribosylation) reaction,... 

Why might it be desirable to coordinately lower the levels of nicotinamide deamidase The 8-fold depression in nicotinamide deamidase activity causes excretion of nicotinamide xthR mutants have shown to be "feeders" for nicotinamide auxotrophs, indicating that these strains continuously excrete nicotinamide into the medium. This is presumably a consequence of the pyridine nucleotide cycle, shown in Fig. 2. Nicotinamide deamidase is not only an enzyme for the salvage of exogenous pyridine, but it is part of a NAD recycling pathway, i.e., a "pyridine nucleotide cycle"... 

Fig. 2. Intracellular pyridine nucleotide cycles in enteric bacteria. The breakdown and resynthesis of NAD occurs in bacteria by the metabolic steps shown above. All abbreviations are as in Fig. 1, with the addition of NMN, nicotinamide mononucleotide. The metabolic step catalyzed by nicotinamide deamidase, the levels of which are reduced by anxt/iR mutation (see text) is shown by the bold arrow.

Although nicotinamide is a vitamin, it can also be synthesized from tryptophan. Therefore, the NAD levels of the organism depend on both nicotinamide and tryptophan intake. The pathway of NAD biosynthesis starting from tryptophan is discussed in detail later. NADP is much less abundant in the cell than NAD. The biosynthesis of NADP probably involves the reaction of NAD with ATP in the presence of a nucleotide pyrophosphorylase. 

A considerable advance in elaborating the pathway of pyridine coenzyme synthesis resulted from the contributions of Preiss and Handler (118, 1B6-1B8). Before discussing the experiments of these investigators, it may be of value to review some of the earlier work on the metabolism of nicotinic acid and nicotinamide in erythrocytes. It has been known for some time that nicotinic acid taken orally results in a significant increase in the pyridine nucleotide content of human red blood cells (1B9, ISO). When equal concentrations of nicotinamide were given under identical conditions, there was no effect on the erythrocyte DPN level. Isolated erythrocytes have also been found to show a rise in DPN when incubated with nicotinic acid and not with nicotinamide. However, incubation with nicotinamide leads to a marked accumulation of nicotinamide mononucleotide (ISl). In this connection, it is of importance to point out that the level of the DPN pyrophosphorylase is extremely low in the red blood cell (ISB). 

Through the study of Roth el al. with carboxyl C -labeled niacin, it has been estimated that the turnover time of niacin in tissues is from 4 to 8 days 19ff). More recent data, however, indicate that in liver the half-life of the pyridine nucleotides may be considerably shorter 197). The observation that the injection of nicotinamide into mice can produce large increases in the levels of liver DPN has provided a useful means of studying the in vivo metabolism of the P3uidine coenzymes. The results obtained with this method of approach are presented in this section. 

Fio. 8. Effect of reserpine upon the level of diphosphopyridine nucleotide in mouse liver. The experiment was conducted as described in the text. Open triangles, data obtained from untreated control animals and from animals that had received only reserpine solid squares, data for animals that had received nicotinamide and nicotinamide-and-vehicle (7). Data from mice that had received injections of reserpine 4 hr prior to the injection of nicotinamide are indicated by the following symbols solid triangle (10 mg/kg), solid circle (4 mg/kg), open circle (1.6 mg/kg). Each experimental point represents the average value obtained from three animals. From Burton et al. (tl8). 

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