title: Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.
abstract: 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.
authors.affiliation: Department of Agricultural, Food and Environmental Sciences, Università Politecnica delle Marche, Ancona, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Zamporlini
authors.forename: Silverio
authors.initials: S
authors.lastname: Ruggieri
authors.forename: Francesca
authors.initials: F
authors.lastname: Mazzola
authors.forename: Adolfo
authors.initials: A
authors.lastname: Amici
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Orsomando
authors.forename: Nadia
authors.initials: N
authors.lastname: Raffaelli
mesh_terms: D000818:Animals; D053898:Biosynthetic Pathways; D002460:Cell Line; D002474:Cell-Free System; D057075:Enzyme Assays; D005470:Fluorometry; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D008810:Mice, Inbred C57BL; D009243:NAD / Q000096:biosynthesis* / Q000737:chemistry; D009537:Nicotinamide Mononucleotide / Q000031:analogs & derivatives / Q000737:chemistry; D009612:Nicotinamide-Nucleotide Adenylyltransferase / Q000737:chemistry / Q000502:physiology; D009928:Organ Specificity; D010430:Pentosyltransferases / Q000737:chemistry / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000502:physiology
chemical_list: D009243:NAD; D009537:Nicotinamide Mononucleotide; C002953:nicotinate mononucleotide; D010430:Pentosyltransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C061783:nicotinamide riboside kinase; D009612:Nicotinamide-Nucleotide Adenylyltransferase; C021864:nicotinate phosphoribosyltransferase
keywords: NAD+ biosynthesis; nicotinamide phosphoribosyltransferase; nicotinamide riboside kinase; nicotinate phosphoribosyltransferase; quinolinate phosphoribosyltransferase