Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD+ biosynthetic machinery in mammalian cells

• Published in: The FEBS journal Vol. 281; no. 22; pp. 5104 - 5119
• Main Authors: Zamporlini, Federica, Ruggieri, Silverio, Mazzola, Francesca, Amici, Adolfo, Orsomando, Giuseppe, Raffaelli, Nadia
• Format: Journal Article
• Language: English
• Published: England Published by Blackwell Pub. on behalf of the Federation of European Biochemical Societies 01-11-2014; Blackwell Publishing Ltd
• Subjects: Animals; Bioassays; Biosynthesis; Biosynthetic Pathways; Cell Line; Cell-Free System; Cellular biology; Enzyme Assays; Enzymes; Fluorometry; gene expression regulation; human cell lines; Humans; Liver - enzymology; metabolic diseases; Metabolic disorders; Mice; Mice, Inbred C57BL; NAD - biosynthesis; NAD - chemistry; Nicotinamide Mononucleotide - analogs & derivatives; Nicotinamide Mononucleotide - chemistry; nicotinamide phosphoribosyltransferase; nicotinamide riboside kinase; Nicotinamide-Nucleotide Adenylyltransferase - chemistry; Nicotinamide-Nucleotide Adenylyltransferase - physiology; nicotinate phosphoribosyltransferase; Organ Specificity; Pentosyltransferases - chemistry; Pentosyltransferases - physiology; Phosphotransferases (Alcohol Group Acceptor) - chemistry; Phosphotransferases (Alcohol Group Acceptor) - physiology; quinolinate phosphoribosyltransferase; screening; tissue; NAD+ biosynthesis; nicotinamide phosphoribosyltransferase; nicotinamide riboside kinase; nicotinate phosphoribosyltransferase; quinolinate phosphoribosyltransferase
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.13050

The redox coenzyme NAD⁺is also a rate‐limiting co‐substrate for several enzymes that consume the molecule, thus rendering its continuous re‐synthesis indispensable. NAD⁺biosynthesis has emerged as a therapeutic target due to the relevance of NAD⁺‐consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD⁺biosynthesis with tissue/cell‐specific efficiencies, probably reflecting differential expression of the corresponding rate‐limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD⁺levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD⁺availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole‐cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell‐specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD⁺precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.