The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei

1. The activities of NMN adenylyltransferase and an enzyme that synthesizes poly (ADP-ribose) from NAD were investigated in the various classes of rat liver nuclei fractionated by zonal centrifugation. 2. The highest specific activities of these two nuclear enzymes occur in different classes of nuclei. In very young and in mature rats it was shown that a correlation exists between DNA synthesis and NMN adenylyltransferase activity, but in rats of intermediate age this correlation is less evident. The highest activities of the enzyme that catalyses formation of poly (ADP-ribose) are in the nuclei involved in the synthesis of RNA. 3. The significance of these results in relation to NAD metabolism is discussed.     

Evidence for an inhibitory effect exerted by yeast NMN adenylyltransferase on poly(ADP-ribose) polymerase activity

We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.     

The activities of nicotinamide mononucleotied adenylyltransferase and of nicotinamide–adenine dinucleotide kinase in the livers of rats subjected to different hormonal treatments

1. The activities of NMN adenylyltransferase and of NAD(+) kinase have been measured in the livers of adrenalectomized or alloxan-diabetic rats and in the livers of rats treated with glucagon, pituitary growth hormone or thyroxine. 2. The activities of these enzymes have been compared with the effects of the same treatments on the nicotinamide nucleotide concentrations in the liver. 3. Alloxandiabetes (+37%) and thyroxine (+27%) both increased the activity of NMN adenylyltransferase. The other treatments were without effect on this enzyme. 4. Only thyroxine increased the activity of NAD(+) kinase significantly (+26%) although both adrenalectomy and glucagon tended to increase its activity. 5. The activity of NAD(+) glycohydrolase was measured in the livers of diabetic rats, and in the livers of rats treated with either growth hormone or thyroxine. Of these treatments, only growth hormone altered the enzyme activity (+26%, calculated on a total hepatic activity basis). 6. Female rats had a greater hepatic NAD(+)-kinase activity than males but there was no sex difference with respect to NMN adenylyltransferase. 7. The lack of correlation between the maximum potential activity of these three enzymes and the known changes of the nicotinamide nucleotides in each of the hormone conditions is discussed.     

The concentration and biosynthesis of nicotinamide nucleotides in the livers of rats treated with carcinogens

1. The oxidoreduction state and concentration of both NAD and NADP as well as the maximum potential activities of NMN adenylyltransferase and NAD(+) kinase have been measured in the livers of rats treated for 14-28 days with 4-dimethylamino-3'-methylazobenzene, 4-dimethylamino-4'-fluoroazobenzene, alpha-naphthyl isothiocyanate or ethionine and in primary hepatomas induced by 4-dimethylamino-3'-methylazobenzene. 2. The total NAD and total NADP both decreased in the livers of rats treated with either azo-dyes or alpha-naphthyl isothiocyanate but not in those treated with ethionine. The activities of NMN adenylyltransferase and NAD(+) kinase did not alter appreciably after such treatments. 3. In the primary hepatomas the concentrations of both NAD and NADP fell drastically and the activities of NMN adenylyltransferase and NAD(+) kinase fell to about 50% of the control activities. 4. No correlation could be established between the concentrations of the nucleotides and the activities of the enzymes synthesizing them. It appears, however, that a relationship exists between the NAD content of the tissue and the amount of NADP present. 5. The results are discussed with respect to the control of NAD and NADP synthesis by ATP. At the concentrations of NAD normally present in the cell it is suggested that NAD may be a rate-limiting substrate in NADP synthesis.     

Metabolism of NAD+ in the nuclei of human placenta

It has long been known that the major function of NAD+ is as an electron carrier in various biological oxidation-reduction systems. From many papers it is evident that NAD+ is involved as substrate in ADP-ribosylation reactions. We have focused our attention on two chromatin enzymes: NMN-adenylyltransferase that catalyzes reversible synthesis of NAD+ utilizing ATP and NMN, and poly(ADP-ribose)polymerase that covalently modifies nucleosomal proteins through poly ADP-ribosylation reactions. Here we provided evidence of these activities in a system of isolated nuclei from human placenta. The data presented in this report show that purified nuclei might be useful to study the nuclear location of these enzymes and their reciprocal interactions.     

Synechocystis sp. slr0787 protein is a novel bifunctional enzyme endowed with both nicotinamide mononucleotide adenylyltransferase and 'Nudix' hydrolase activities

Synechocystis sp. slr0787 open reading frame encodes a 339 residue polypeptide with a predicted molecular mass of 38.5 kDa. Its deduced amino acid sequence shows extensive homology with known separate sequences of proteins from the thermophilic archaeon Methanococcus jannaschii. The N-terminal domain is highly homologous to the archaeal NMN adenylyltransferase, which catalyzes NAD synthesis from NMN and ATP. The C-terminal domain shares homology with the archaeal ADP-ribose pyrophosphatase, a member of the 'Nudix' hydrolase family. The slr0787 gene has been cloned into a T7-based vector for expression in Escherichia coli cells. The recombinant protein has been purified to homogeneity and demonstrated to possess both NMN adenylyltransferase and ADP-ribose pyrophosphatase activities. Both activities have been characterized and compared to their archaeal counterparts.     

Changes in the activities of enzymes of the biosynthetic pathway of the nicotinamide nucleotides in rat mammary gland during the lactation cycle

1. The activities of NMN pyrophosphorylase, NMN adenylyltransferase and NAD kinase in the mammary glands of rats at different stages of pregnancy, lactation and involution were measured. 2. NMN pyrophosphorylase has a low activity early in pregnancy, but its activity increases at parturition and in early lactation to reach a maximum at the tenth day of lactation, after which it remains constant until it declines abruptly in involution. 3. NMN adenylyltransferase is already quite active by the tenth day of pregnancy and its activity does not rise further in the second half of gestation. After a sharp rise in activity at parturition, the activity of the enzyme declines slowly throughout the period of lactation and, more sharply, in involution. 4. NAD kinase has a low activity for most of pregnancy, but its activity rises at parturition to a value at 2 days of lactation that is maintained until the tenth day. Between the tenth and fifteenth days of lactation the activity almost doubles, but falls sharply in mammary involution. 5. The relation of the activities of these enzymes to the rates of synthesis of NAD and NADP is discussed.     

Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrial NAD generation

NAD is a vital redox carrier, and its degradation is a key element of important regulatory pathways. NAD-mediated functions are compartmentalized and have to be fueled by specific biosynthetic routes. However, little is known about the different pathways, their subcellular distribution, and regulation in human cells. In particular, the route(s) to generate mitochondrial NAD, the largest subcellular pool, is still unknown. To visualize organellar NAD changes in cells, we targeted poly(ADP-ribose) polymerase activity into the mitochondrial matrix. This activity synthesized immunodetectable poly(ADP-ribose) depending on mitochondrial NAD availability. Based on this novel detector system, detailed subcellular enzyme localizations, and pharmacological inhibitors, we identified extracellular NAD precursors, their cytosolic conversions, and the pathway of mitochondrial NAD generation. Our results demonstrate that, besides nicotinamide and nicotinic acid, only the corresponding nucleosides readily enter the cells. Nucleotides (e.g. NAD and NMN) undergo extracellular degradation resulting in the formation of permeable precursors. These precursors can all be converted to cytosolic and mitochondrial NAD. For mitochondrial NAD synthesis, precursors are converted to NMN in the cytosol. When taken up into the organelles, NMN (together with ATP) serves as substrate of NMNAT3 to form NAD. NMNAT3 was conclusively localized to the mitochondrial matrix and is the only known enzyme of NAD synthesis residing within these organelles. We thus present a comprehensive dissection of mammalian NAD biosynthesis, the groundwork to understand regulation of NAD-mediated processes, and the organismal homeostasis of this fundamental molecule.     

Identification of the archaeal NMN adenylyltransferase gene

Increasing evidence on the importance of fluctuations in NAD+ levels in the living cell is accumulating. Therefore a deeper knowledge on the regulation of coenzyme synthesis and recycling is required. In this context the study of NMN adenylyltransferase (EC 2.7.7. 1), a key enzyme in the NAD+ biosynthetic pathway, assumes a remarkable relevance. We have previously purified to homogeneity and characterized the protein from the thermophilic archaeon Sulfolobus solfataricus. The determination of partial sequence of the S. solfataricus enzyme, together with the recent availability of the genome sequence of the archaeon Methanococcus jannaschii allowed us, based on sequence similarity, to identify the M. jannaschii NMN adenylyltransferase gene. As far as we know from literature, this is the first report on the NMN adenylyltransferase gene.     

Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

Induction of the pyridine nucleotide synthesis pathway in mitogen-stimulated human T-lymphocytes

Mitogen stimulation of purified human T-lymphocytes with the phorbol ester 12-O-tetradecanoyl, phorbol-13-acetate (TPA) and a monoclonal antibody to the T3 cell surface antigen caused a 6-11-fold increase in cellular levels of poly(ADP-ribose) polymerase, a 6-20-fold amplification of cellular NAD+ levels and a 3-21-fold increase in NADP+ levels. Treatment of the cells with a combination of the two mitogenic signals also caused a 5-20-fold increase in NMN pyrophosphorylase activity, a 3-14-fold increase in ATP-NMN adenylyl transferase activity, and a 5-13-fold increase in NAD kinase activity. This is the first report showing induction of these three enzymes as part of the mitogenic response in purified human T-lymphocytes. Maximum increases in activity of each of these three enzymes required the combined presence of TPA and monoclonal antibody to human T-cell T3 antigen anti-T3. Analysis of the relative enzyme levels indicates that NMN pyrophosphorylase is the rate-limiting enzyme for NAD synthesis and NAD kinase is the rate-limiting enzyme for NADP synthesis.     

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.     

The biosynthesis of nicotinamide adenine dinucleotide during early stages of frog embryonic development of haploid and diploid embryos.

1. Concentration of NAD during embryonic development of haploid and diploid embryos of frog was followed. NAD content in haploid embryonic forms is twice that in diploid embryos. 2. The variation of the NMN adenylyltransferase activity in the oocytes and during the first states of embryonic development as surveyed in the nuclear soluble fraction and the nuclear insoluble fraction (chromatin). 3. The enzyme activity in the soluble fraction is low during embryonic development and shows higher values in haploid embryos. 4. In the nonfertilized mature oocytes, the NMN adenylyltransferase activity is sixfold higher in the insoluble chromatin fraction than in the soluble fraction. 5. The evolution of the NMN adenylyltransferase in the insoluble chromatin fraction also shows higher values in haploid embryos, as compared with diploid forms.     

Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle.

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase alpha and thymidine kinase. When DNA synthesis and the activity of DNA polymerase alpha are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.     

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.     

Identification of a novel human nicotinamide mononucleotide adenylyltransferase

The enzyme nicotinamide mononucleotide adenylyltransferase is an ubiquitous enzyme catalyzing an essential step in NAD (NADP) biosynthetic pathway. In human cells, the nuclear enzyme, which we will now call NMNAT-1, has been the only known enzyme of this type for over 10 years. Here we describe the cloning and expression of a human cDNA encoding a novel 34.4kDa protein, that shares significant homology with the 31.9kDa NMNAT-1. We propose to call this enzyme NMNAT-2. Purified recombinant NMNAT-2 is endowed with NMN and nicotinic acid mononucleotide adenylyltransferase activities, but differs from NMNAT-1 with regard to chromosomal and cellular localization, tissue-specificity of expression, and molecular properties, supporting the idea that the two enzymes might play distinct physiological roles in NAD homeostasis.     

Enzymological properties of poly(ADP-ribose)polymerase: characterization of automodification sites and NADase activity.

We have characterized the effect of poly(ADP-ribose) polymerase automodification on the enzyme's activities, which include poly(ADP-ribose) synthesis and NADase activity. The apparent Km of the enzyme for NAD+ during polymer synthesis is higher than the one measured for alternate NADase activity. Furthermore, we have found that there are 28 automodification sites, in contrast to the 15 sites (postulated to be on the 15 glutamic acids) reported to be present in the automodification domain. For the first time, we show that some of these acceptor sites are outside the reported automodification domain (15 kDa); we demonstrate automodification in the NAD+ binding domain (55.2 kDa) and the DNA binding domain (42.5 kDa). We have analyzed the relationship between the number of sites modified on poly(ADP-ribose) polymerase and its effect on the polymerization activity and its alternate NADase activity. Automodification greatly altered both enzyme activities, decreasing both polymer synthesis and alternate NADase activity.     

Protection by picolinamide,a novel inhibitor of poly (ADP-ribose) synthetase,against both streptozotocin-induced depression of proinsulin synthesis and reduction of NAD content in pancreatic islets

Picolinamide, 2-pyridinecarboxylic acid amide, was found to be a strong inhibitor of poly (ADP-ribose) synthetase of nuclei from rat pancreatic islet cells. Another experiment using isolated pancreatic islets of rats showed that picolinamide protects against streptozotocin-induced depression of proinsulin synthesis as well as against streptozotocin-induced reduction of NAD content. The protection by picolinamide against the NAD depression was considered to be due to the blockage of an increased degradation of NAD mediated by a streptozotocin-induced increase in poly (ADP-ribose) synthetase activity. A possible mechanism of streptozotocin diabetes and its prevention is discussed.