A continuous microplate assay for sirtuins and nicotinamide-producing enzymes

Abstract

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 α-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340nm. 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 kcat and Km values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC50 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.