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1.
The synthesis of nicotinamide–adenine dinucleotide and poly(adenosine diphosphate ribose) in various classes of rat liver nuclei
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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. 相似文献
2.
The metabolism of nicotinamide-adenine dinucleotide in various classes of rat liver nuclei. 总被引:1,自引:0,他引:1
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. 相似文献
3.
S Dragos M Radu N Anca 《Comparative biochemistry and physiology. B, Comparative biochemistry》1979,62(4):335-338
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. 相似文献
4.
Two different approaches were used to define the intracellular localization in mouse L929 cells of two deoxyribonucleotide biosynthetic enzymes: ribonucleoside diphosphate reductase (EC1.17.4.1) and thymidylate synthase (EC2.1.1.45). The first involved treatment with saponins, which render the plasma membrane permeable to proteins without disrupting intracellular organelles. Under conditions where nuclear DNA synthesis and the activity of the nuclear enzyme NMN adenylyltransferase were unaffected, the entire cellular complements of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, and of ribonucleotide reductase and thymidylate synthase were released at the same rate and with similar dependence on saponin concentration. The second approach involved centrifugal enucleation of cells treated with cytochalasin B (CB) and measurement of the distribution of enzyme activities in the resulting cytoplast and karyoplast fractions. Whereas most NMN adenylyltransferase activity remained with the karyoplasts, glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were almost exclusively associated with the enucleated cytoplasts. These results indicate that, under conditions where nuclear DNA synthesis is apparently unperturbed, the intracellular distribution of the deoxyribonucleotide biosynthetic enzymes studied is the same as that of glucose-6-phosphate dehydrogenase, a typical cytosol enzyme, and clearly differs from that of NMN adenylyltransferase, a nuclear enzyme. 相似文献
5.
Tianjun Zhou Oleg Kurnasov Diana R Tomchick Derk D Binns Nick V Grishin Victor E Marquez Andrei L Osterman Hong Zhang 《The Journal of biological chemistry》2002,277(15):13148-13154
Nicotinamide/nicotinate mononucleotide (NMN/ NaMN)adenylyltransferase (NMNAT) is an indispensable enzyme in the biosynthesis of NAD(+) and NADP(+). Human NMNAT displays unique dual substrate specificity toward both NMN and NaMN, thus flexible in participating in both de novo and salvage pathways of NAD synthesis. Human NMNAT also catalyzes the rate-limiting step of the metabolic conversion of the anticancer agent tiazofurin to its active form tiazofurin adenine dinucleotide (TAD). The tiazofurin resistance is mainly associated with the low NMNAT activity in the cell. We have solved the crystal structures of human NMNAT in complex with NAD, deamido-NAD, and a non-hydrolyzable TAD analogue beta-CH(2)-TAD. These complex structures delineate the broad substrate specificity of the enzyme toward both NMN and NaMN and reveal the structural mechanism for adenylation of tiazofurin nucleotide. The crystal structure of human NMNAT also shows that it forms a barrel-like hexamer with the predicted nuclear localization signal sequence located on the outside surface of the barrel, supporting its functional role of interacting with the nuclear transporting proteins. The results from the analytical ultracentrifugation studies are consistent with the formation of a hexamer in solution under certain conditions. 相似文献
6.
M Emanuelli F Carnevali M Lorenzi N Raffaelli A Amici S Ruggieri G Magni 《FEBS letters》1999,455(1-2):13-17
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. 相似文献
7.
8.
The activities of nicotinamide mononucleotied adenylyltransferase and of nicotinamide–adenine dinucleotide kinase in the livers of rats subjected to different hormonal treatments
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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. 相似文献
9.
S Ruggieri L Gregori P Natalini A Vita M Emanuelli N Raffaelli G Magni 《Biochemistry》1990,29(10):2501-2506
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. 相似文献
10.
E Balducci M Emanuelli N Raffaelli G Magni 《Bollettino della Società italiana di biologia sperimentale》1989,65(11):1059-1066
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. 相似文献
11.
Raffaelli N Sorci L Amici A Emanuelli M Mazzola F Magni G 《Biochemical and biophysical research communications》2002,297(4):835-840
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. 相似文献
12.
M Emanuelli P Natalini N Raffaelli S Ruggieri A Vita G Magni 《Archives of biochemistry and biophysics》1992,298(1):29-34
Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity. 相似文献
13.
Changes in the activities of enzymes of the biosynthetic pathway of the nicotinamide nucleotides in rat mammary gland during the lactation cycle 总被引:1,自引:1,他引:0
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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. 相似文献
14.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,676(1):13-18
NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly catalyzes the synthesis of NAD from ATP and NMN. In this paper, we describe a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C18 reversed-phase (RP) column. The activity was measured by separating in less than 3 min the substrates (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22°C. NAD was directly quantitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfully applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) cells. 相似文献
15.
ISOLATION OF CELL NUCLEI FROM THE MAMMALIAN CEREBRAL CORTEX AND THEIR ASSORTMENT ON A MORPHOLOGICAL BASIS 总被引:7,自引:1,他引:6
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An aqueous method is described for the isolation of highly purified nuclei from the cerebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through dense sucrose solutions. The nuclear preparation retained the activity of ATP:NMN adenylyltransferase. Recoveries of DNA in the P4I, P4II, PL and PS preparations were 30, 43, 8, and 7%, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragments, capillaries, and cytoplasmic elements. Biochemical purity of samples was verified by the absence of several cytoplasmic enzyme activities. In the electron microscope, the majority of nuclei showed well-preserved nuclear membranes, with nuclear pores, and were provided with a finely textured nucleoplasm. Occasional contaminants were elements of endoplasmic reticulum and of the endothelium. Assortment of nuclei on a morphological basis showed that 55–65% and 47–53% of nuclei in the P4I and P4II preparations, respectively, consisted of neuronal nuclei. In the PL preparation, the population of neuronal nuclei ranged between 72 and 83%, while 94–99% of the nuclei in the PS preparation consisted of smaller nuclei, most likely of oligodendroglial origin. 相似文献
16.
1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+). 相似文献
17.
The concentration and biosynthesis of nicotinamide nucleotides in the livers of rats treated with carcinogens 总被引:2,自引:2,他引:0
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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. 相似文献
18.
Raffaelli Nadia Emanuelli Monica Pisani Francesca M. Amici Adolfo Lorenzi Teresa Ruggueru Silverio Magni Giulio 《Molecular and cellular biochemistry》1999,193(1-2):99-102
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. 相似文献
19.
Emanuelli M Amici A Carnevali F Pierella F Raffaelli N Magni G 《Protein expression and purification》2003,27(2):357-364
The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD(+). On the basis of a remarkable structural similarity with previously described Saccharomyces cerevisiae NMNAT (yNMNAT-1), the YGR010-encoded protein was identified as a second isoform of yeast NMNAT (yNMNAT-2). The YGR010 gene was isolated, cloned into a T7-based vector, and successfully expressed in Escherichia coli BL21 cells, yielding high level of NMN adenylyltransferase activity. The purification procedure reported in this paper, consisting of two chromatographic steps, allowed the isolation of 3mg of electrophoretically homogeneous yNMNAT-2 from 1 liter of E. coli culture. Under SDS/PAGE, the recombinant protein resulted in a single polypeptide of 46 kDa, in agreement with the molecular mass of the hypothetical protein encoded by YGR010 gene. The N-terminal sequence of the purified recombinant yNMNAT-2 exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant yNMNAT-2 are reported and compared with those already known for yNMNAT-1. 相似文献
20.
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. 相似文献