Assay Methods for Nicotinamide Mononucleotide Adenylyltransferase of Wide Applicability

Balducci, Enrico; Emanuelli, Monica; Raffaelli, Nadia; Ruggieri, Silverio; Amici, Adolfo; Magni, Giulio; Orsomando, Giuseppe; Polzonetti, Valeria; Natalini, Paolo

doi:10.1006/abio.1995.1315

Cited by 80 publications

(70 citation statements)

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“…Nmnat activity was calculated after HPLC identification and quantification of the product (NAD þ ). 33 One unit of enzyme was defined as the amount capable of producing 1 mmol of NAD þ per minute at 371C. Pairwise statistical analyses (unpaired Student's t-test) were performed using Prism.…”

Section: Methodsmentioning

confidence: 99%

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

et al. 2006

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The slow Wallerian degeneration protein (Wld S ), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld S mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld S mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld S , and lentiviral overexpressed enzyme-dead Wld S still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld S at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld S requires more N-terminal sequences of the protein.

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Section: Methodsmentioning

confidence: 99%

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

et al. 2006

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“…100~I of the neutralized supernatant was injected into a high performance liquid chromatography system 10 Dvp-uv-vis photodiode array detector (Shimadzu, Duisburg, Germany) using a 250 x 4.6 mm inner diameter Supelcosil® LC-18 5 urn reversed phase column. Elution conditions were as previously described (7). Enzyme activities were tested by measuring the amount of N'-methylnicotinamide produced, as determined by the peak areas of the separated compound with 1 U activity representing the formation of 1 nmol N'-methylnicotinamide per hour of incubation at 37°C.…”

Section: Nnmt Enzyme Assaymentioning

confidence: 99%

Inhibiting Proliferation in KB Cancer Cells by RNA Interference-Mediated Knockdown of Nicotinamide N-Methyltransferase Expression

Pozzi

Mazzotta²,

Muzio

et al. 2011

Int J Immunopathol Pharmacol

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The enzyme Nicotinamide N-methyltransferase (NNMT) catalyzes the methylation of nicotinamide and other pyridines, playing a pivotal role in the biotransformation and detoxification of many drugs and xenobiotic compounds. Several tumours have been associated with abnormal NNMT expression, however its role in tumour development remains largely unknown. In this study we investigated expression levels of Nicotinamide N-methyltransferase in a cancer cell line and we evaluated the effect of shRNA-mediated silencing of NNMT on cell proliferation. Cancer cells were examined for NNMT expression by semiquantitative RT-PCR and Western blot analysis. A HPLC-based catalytic assay was performed to assess enzyme activity. Cells were transfected with four shRNA plasmids against NNMT and control cells were treated with transfection reagent only (mock). The efficiency of gene silencing was detected by Real-Time PCR and Western blot analysis. MTT cell proliferation assay and the soft agar colony formation assay were then applied to investigate the functional changes in cancerous cell. NNMT mRNA was detected in cancer cells, showing a very high expression level. In keeping with the results of RT-PCR analysis, the protein level and NNMT enzyme activity were particularly high in KB cells. ShRNA vectors targeted against NNMT efficiently suppressed gene expression, showing inhibition observed at both the mRNA and protein levels. Down-regulation of NNMT significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The present data support the hypothesis that the enzyme plays a role in tumour expansion and its inhibition could represent a possible molecular approach to the treatment of cancer.Tumour arises as a result of gene mutations and abnormal expression that endow the cell with many specific functional capabilities, such as cell proliferation, invasion and metastasis (l). The cellular and biochemical factors that underline cancer dissemination are poorly understood. Treatment of

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“…NMNAT-and NaMNAT-coupled Assay-The hsPNAT-3 activity with both substrates NMN and NaMN was analyzed using a continuous assay coupling NAD formation to alcohol dehydrogenase-catalyzed conversion of NAD to NADH as previously described (39,40). Briefly, the assay was performed in UV-transparent plastic cuvettes in the six-cuvette autosampler of a DU-640 spectrophotometer (Beckman, Fullerton, CA) at 37°C.…”

Section: Materials-the Poly(a)mentioning

confidence: 99%

Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

Zhang

Kurnasov

Karthikeyan

et al. 2003

Journal of Biological Chemistry

126

148

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Pyridine dinucleotides (NAD and NADP) are ubiquitous cofactors involved in hundreds of redox reactions essential for the energy transduction and metabolism in all living cells. In addition, NAD also serves as a substrate for ADP-ribosylation of a number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing regulation, and for cyclic ADP ribose (cADPR)-dependent Ca 2؉ signaling. Pyridine nucleotide adenylyltransferase (PNAT) is an indispensable central enzyme in the NAD biosynthesis pathways catalyzing the condensation of pyridine mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or NaAD). Here we report the identification and structural characterization of a novel human PNAT (hsPNAT-3) that is located in the cytoplasm and mitochondria. Its subcellular localization and tissue distribution are distinct from the previously identified human nuclear PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the catalytic mechanism of the enzyme. The characterization of the cytosolic human PNAT-3 provided compelling evidence that the final steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and mitochondria, potentially contributing to their NAD/NADP pool. The coenzymes NADϩ (H) 1 and NADP ϩ (H) have been known for many decades as the major hydrogen donor or acceptor in hundreds of metabolic redox reactions throughout the cell. Together these nucleotides have a direct impact on virtually every cellular metabolic pathway. Additionally, NAD serves as a substrate for the covalent modification of nuclear proteins by ADP-ribosylation, a process involved in DNA repair and the regulation of genomic instability (1-3). Recently, many new exciting functions have been discovered for this long-known molecule. These include its role as co-substrate in Sir2-mediated histone deacetylation involved in gene silencing regulation and in increasing the lifespan of species ranging from yeast, to worm, to certain mammals (4, 5). Moreover, several derivatives of NAD and NADP were found to be potent intracellular calcium-mobilizing agents and are involved in a variety of Ca 2ϩ -signaling pathways (6 -8). These recent developments brought a significant amount of additional interest to the investigation of cellular NAD biosynthesis and regulation.NMN and/or NaMN adenylyltransferase (NMNAT and/or NaMNAT, collectively named pyridine nucleotide adenylyltransferase, or PNAT) is an indispensable enzyme catalyzing the central step of all NAD biosynthesis pathways (9, 10). It links the AMP moiety of ATP with the nicotinamide mononucleotide (NMN, or its deamidated form NaMN) to form the dinucleotide product NAD (or deamido-NAD, NaAD). A practical aspect of human PNAT function is that it catalyzes the rate-limiting step in the metabolic conversion of the anticancer agent tiazofurin to its active form TAD (tiazofurin adenine dinucleotide, an NAD analog) (11). The development of ti...

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Assay Methods for Nicotinamide Mononucleotide Adenylyltransferase of Wide Applicability

Cited by 80 publications

References 0 publications

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

NAD+ and axon degeneration revisited: Nmnat1 cannot substitute for WldS to delay Wallerian degeneration

Inhibiting Proliferation in KB Cancer Cells by RNA Interference-Mediated Knockdown of Nicotinamide N-Methyltransferase Expression

Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

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