title: A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase.
abstract: NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.
authors.affiliation: Millennium Pharmaceuticals, Inc., Cambaridge, MA 02139, USA. michael.bembenek@mpi.com
authors.forename: Michael E
authors.initials: ME
authors.lastname: Bembenek
authors.forename: Eric
authors.initials: E
authors.lastname: Kuhn
authors.forename: William D
authors.initials: WD
authors.lastname: Mallender
authors.forename: Lester
authors.initials: L
authors.lastname: Pullen
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Thomas
authors.initials: T
authors.lastname: Parsons
mesh_terms: D019732:Amide Synthases / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006801:Humans; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D019732:Amide Synthases; C038479:NAD+ synthase