Abstract
Nicotinamide adenine dinucleotide (NAD+) is an essential redox cofactor and signaling molecule that controls the activity of enzymes involved in metabolism, DNA repair, and cellular survival, such as the PARPs, CD38, and the sirtuins. Here, we describe three methods for measuring the activity of these enzymes: the etheno-NAD+ assay measures NAD+ hydrolase activity using an NAD+ analog to produce a fluorescent product that is measured in real time; the PNC1 assay converts a native product of NAD+ hydrolysis, nicotinamide, into a quantitative fluorescent readout; and liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to characterize the entire NAD+ metabolome in a sample. These methods will enable new insights into the roles that NAD+ and the enzymes that utilize it play in health and disease.
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Acknowledgments
N.B. is the recipient of the Australian Research Council Discovery Early Career Research Award at the University of New South Wales, Sydney, Australia.
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Schultz, M.B., Lu, Y., Braidy, N., Sinclair, D.A. (2018). Assays for NAD+-Dependent Reactions and NAD+ Metabolites. In: Chang, P. (eds) ADP-ribosylation and NAD+ Utilizing Enzymes. Methods in Molecular Biology, vol 1813. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8588-3_6
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DOI: https://doi.org/10.1007/978-1-4939-8588-3_6
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