NAD Pyrophosphorylase

NAD pyrophosphorylase catalyzes a reversible reaction in which the nucleotidyl moiety of ATP is transferred to nicotinamide mononucleotide to form NAD and pyrophosphate. The enzyme activity has been suggested as a target for cancer chemotherapy and is thought to be critical for cell survival. 

NAD was separated from other nucleotides by chromatography on a Waters Partisil 10-SAX column (8 mm x 100 mm). The column was equilibrated with 5 mM ammonium phosphate buffer (pH 2.9). After injection, the same solvent was used for 10 minutes at a flow rate of 1 mL/min, and then for 2 minutes at 1.5 mL/min. A mobile phase was then switched to 0.65 M ammonium phosphate (pH 3.7) for 8 minutes at a flow rate of 2 mL/min, after which the column was reequilibrated with the initial solvent. Fractions from the NAD area were collected and counted by scintillation spectrometry. 

The assay mixture in a total volume of 50 fiL contained 50 mM glycylglycine buffer (pH 7.6), 6 mM ATP, 4 mM [4-3H]nicotinamide mononucleotide (2 mCi/mmol, 8 /uCi/mL), 60 mM MgG2, 20 mM nicotinamide, and enzyme extract. The mixture was incubated at 37°C for 10 or 20 minutes before the reaction was stopped by the addition of 50 /u.L of 20% trichloroacetic acid. The samples were cooled on ice before 10 minutes and centrifuged, and the supernates were neutralized with 0.5 M tri-n-octylamine in Freon before analysis by HPLC. 

The assay was used to determine NAD pyrophosphorylase activity in extracts prepared from human chronic myelogenous leukemia cells, and in extracts prepared from rat tissues. 

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Key AcK, acetate kinase AdK, adenylate kinase NAD-PP, NAD pyrophosphorylase PPase, pyrophosphorylase NADK, NAD kinase r-5-P, ribose-5-phosphate rA-5-P, ribosylamine-5-phosphate NMN, nicotinamide mononucleotide AcP, acetyl phosphate FPi, pyrophosphate and NDC, N J(2,4-dinitrophenyl)-3-carbamoylpyridinium chloride. 

Greenberg, E., Preiss, J. E., VanBoldrick, M., and Preiss, J. 1983. Biosynthesis of bacterial glycogen Activator specificity of the ADPglucose pyrophosphorylase of Rhodopseudomo-nads. Arch. Biochem. Biophys. 220, 594-604. 

Bacterial ADP-ribosylating toxins and non-enzymatic NAD hydrolysis 291 Nucleoside and AMP hydrolases 294 Phosphorylase and pyrophosphorylase reactions 297 DNA and RNA 301 Conclusions and future directions 306 The immediate future 306 Crucial questions 307 Acknowledgments 308 References 308... 

Pyridine nucleotide metabolism has an essential role in regulating multiple and diverse aspects of cellular metabolism ranging from carbohydrate utilization to DNA repair processes. As shown in Fig. 1, most cells convert nicotinamide to pyridine nucleotides via NMN pyrophosphorylase followed by NMN ATP adenylyltransferase. The resultant product, NAD, is used in oxidation-reduction reactions leading ultimately to the synthesis of ATP. It is also important as a co-factor for dehydrogenase enzymes that provide essential components for cell growth such as IMP dehydrogenase, whose activity is required to provide guanine nucleotides. 

The exquisite sensitivity of resting lymphocytes to DNA damage and poly(ADP-ribose) polymerase activation may be due, in part, to a decreased capacity of the cells to synthesize NAD. Human lymphocytes possess an intact pyridine nucleotide cycle (10). The cells synthesize NAD from either nicotinamide or nicotinic acid, and they release nicotinamide as a by-product of ADP-ribosylation reactions. NMN pyrophosphorylase is the rate-limiting enzyme in NAD biosynthesis from nicotinamide, and the content of this enzyme in unstimulated human lymphocytes is quite low (9). We have examined the rate of NAD turnover in resting lymphocytes in order to quantitate the contribution of ADP-ribosylation to the overall consumption of NAD (10). In addition, we present here preliminary results of in vitro biochemical studies performed on malignant lymphocytes from patients with chronic lymphocytic leukemia (CLL), who were treated with 2-chlorodeoxyadenosine. 

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. 

The third hypothesis has found some support from the work of Pardee [96] and his collaborators. These investigators studied in Escherichia coli the activity of nicotinic acid, mononucleotide pyrophosphorylase, nicotinic acid dinucleotide pyrophosphorylase, NAD... 

On the basis of these findings, the investigators concluded that pyrophosphorylase activity controls the rate of NAD biosynthesis, and, therefore, its concentration in the bacterial cell and that the activity of that enzyme is regulated by repressive genes. 

The breakdown pathways for NAD and NADP were discussed in the section on electron transport. Studies in Pardee s laboratory have shed some new light on the control of NAD synthesis in E. coli (see Fig. 4-10). Pardee was able to demonstrate that in the sequence of reactions, the NAD mononucleotide pyrophosphorylase reaction is the rate-limiting step. The levels of pyrophosphorylase are low in the wild type of E. coli, but they are even lower in mutants requiring nicotinic acid for growth when the bacteria... 

Nicotinic acid mononucleotide reacts with ATP in the presence of nicotinic acid adenine dinucleotide pyrophosphorylase, a magnesium-dependent enzyme, to yield the deamido derivative of NAD (see Fig. 4-11). Deamido NAD, in the presence of ATP, glutamine, Mg, K", and an NAD synthetase, is converted to NAD. In this reaction, the amino group of glutamine is transferred to the carboxyl group of the nicotinic acid moiety of deamido NAD. Yet the nicotinamide moiety of NAD synthetase is found in liver supernatant and, as may be expected, it is inhibited by azaserine. 

The conversion of nicotinamide to nicotinic acid requires the activities of a deaminase and a ribonucleotide pyrophosphorylase. Although a detailed description of mechanisms controlling NAD coenzyme synthesis in mammalian tissues is not available, it has been suggested that the regulatory restraints are... 

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