A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.
Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to alpha-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of k(cat) and K(m) values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC(50) values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.
Smith
Brian C
BC
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Hallows
William C
WC
Denu
John M
JM
D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002457:Cell Extracts; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004798:Enzymes / Q000032:analysis / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009536:Niacinamide / Q000096:biosynthesis*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017385:Sequence Homology; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013997:Time Factors
D002457:Cell Extracts; D004791:Enzyme Inhibitors; D004798:Enzymes; D010455:Peptides; D009243:NAD; D009536:Niacinamide; D009249:NADP; D005969:Glutamate Dehydrogenase; D051997:ADP-ribosyl Cyclase 1; D037761:Sirtuins