The Journal of Neuroscience, August 16, 2006, ():
Stimulation of Nicotinamide Adenine Dinucleotide Biosynthetic Pathways Delays Axonal Degeneration after Axotomy
J. Neurosci. Sasaki et al.
26: 8484
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Figure S1. NAD biosynthetic pathway. Predicted mammalian NAD biosynthesis is based on studies from yeast and prokaryotes (Magni et al., 2004). Abbreviations used: QPRT, quinolinate phosphoribosyltransferase; NaPRT, nicotinic acid phosphoribosyltransferase; NmPRT, nicotinamide phosphoribosyltransferase; Nrk, nicotinamide riboside kinase; Nmnat, nicotinamide mononucleotide adenylyltransferase; QNS, NAD synthetase. Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S (2004) Enzymology of NAD+ homeostasis in man. Cell Mol Life Sci 61:19-34.
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Figure S2. The hexahistidine tagged NAD biosynthetic enzymes are catalytically active. The enzymatic activity of the indicated enymes was examined qualitatively by incubating the purifed enzymes with their cognate substrate and monitoring product formation using HPLC (see Methods). In each case, reactions were analyzed in the absence (top) or presence (bottom) of the enzyme to be tested A, QPRT activity was assessed by conversion of Qa to NaMN followed by the conversion of NaMN to NaAD by Nmnat1. NaAD was observed in the presence of QPRT (bottom) but not in its absence (top). B, NaPRT activity was assessed by monitoring the production of NaMN from Na. NaMN was only observed in the presence of NaPRT (bottom). C, NmPRT activity was examined by monitoring NMN production from Nam. NMN was only observed in the presence of NmPRT (bottom). D, Nrk1 and Nrk2 activities were assessed by monitoring the phosphorylation of NmR to produce NMN. NMN was only observed in the presence of Nrk1 (middle) or Nrk2 (bottom).
Materials and methods QPRT, NaPRT, NmPRT, Nrk1, and Nrk2 enzymatic activity measurement. The cDNAs for QPRT, NaPRT, NmPRT, Nrk1 and Nrk2 were cloned into the BamHI site of the pET3a bacterial expression vector (Novagen, San Diego, CA ). BL21(DE3)pLysS cells were transformed with these constructs and protein expression was induced by growth in 1mM IPTG according manufacturer’s instructions (Stratagene, Garden Grove, CA). The enzymes were purified using Nickel affinity chromatography according to manufacturer’s instructions (Sigma). The catalytic activity of QPRT, NaPRT, and NmPRT was examined by incubating 10 μg of purified protein with the appropriate substrates 4 mM Qa for QPRT, 5 mM Na for NaPRT, and 10 mM Nam for NmPRT in buffer containing 50 mM Tris-HCl (pH7.5), 10 mM MgCl2, 0.4 mM 5-Phospho-D-ribose 1-diphosphate (PRPP; Sigma P8296). Nrk activity was measured by incubation with 1 mM NmR in buffer containing 20 mM Tris-HCl (pH7.5), 5 mM 2-Mercaptoethanol, 1 mM ATP, 5m M MgCl2. All reactions were carried out for 30 min at 37°C, stopped by addition of 1 M HClO4 and incubation for 5 min at 4°C. The reactants were spun at 14,000 g in a microcentrifuge and neutralized with K2CO3.
For QPRT measurement, 20 ul of reacted sample was treated with Nmnat1 by incubating with 15 μg of recombinant Nmnat1 in buffer containing 30 mM HEPES (pH7.4), 12 mM MgCl2, and 1 mM ATP for 30 min at 37°C. All control reactions were performed under the same conditions in the absence of the requisite enzyme. All neutralized reactants were cleared by centrifugation and diluted with 3 volumes of buffer containing 50 mM K2HPO4 and 50 mM KH2PO4 (pH 7.0). An aliquot of each reactant solution was analyzed by HPLC using LC-18T reverse phase column at flow rate of 1 ml/min and the absorbance at 254 nm was recorded. Each elution peak was compared with standards (obtained from Sigma) to determine its identity.
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Figure S3. Enzymatic activities of wild type and mutant Nmnat1 and Nmnat3. The enzymatic activities of Nmnat1, cytNmnat1, Nmnat3, and nucNmnat3 were examined as described bellow. For each enzyme, triplicate reactions were averaged and normalized to activity of Nmnat1. Control reactions were performed without recombinant protein.
Materials and methods Nmnat protein overexpression and enzymatic assay. HEK293T cells were transfected with Nmnat1, cytNmnat1, Nmnat3, or nucNmnat3 expression plasmids. Cell lysates were prepared and Nmnat proteins were purified using Nickel affinity chromatography according to manufacturer’s instructions (Sigma). Proteins (0.2 μg) were assayed in 100 μl of reaction buffer containing 28 mM HEPES (pH 7.4), 10 mM MgCl2, 46 mM ethyl alcohol, 16 mM semicarbazide-HCl (pH 7.4), 0.5 unit of alcohol dehydrogenase (Sigma), 1 mM ATP, 1 mM NMN, and 5 μl solution consisting of MTS and phenazine methosulphate (CellTiter 96 AQueous cell proliferation assay: Promega). Reactions were carried out for 1 hour at 37°C. In this reaction, NMN is converted to NAD by Nmnat and then to NADH by alcohol dehydrogenase. The NADH reduces the MTS. The absorbance at 490 nm, which is specific for reduced MTS, is monitored to determine the relative amount of NADH (Cuzzocrea et al., 1999). Cuzzocrea S, Costantino G, Mazzon E, Caputi AP. (1999) Beneficial effects of raxofelast (IRFI 016), a new hydrophilic vitamin E-like antioxidant, in carrageenan-induced pleurisy. Br J Pharmacol 126:407-414.
