Juliano Alves 1 , Jacquelyn Hennek 1,2 , Mentioned A. Goueli 1,3 and Hicham Zegzouti 1, 2Promega Corporation, R D Department, 2800 Woods Hollow Road, Madison, WI 53719, USA; laurie.engel@promega (L.E.); juliano.alves@promega (J.A.); jhennek@HDAC4 Inhibitor Purity & Documentation exactsciences (J.H.); said.goueli@promega (S.A.G.) Exact Sciences Corporation, 5505 Endeavor Lane, Madison, WI 53719, USA Division of Pathology and Laboratory Medicine, University of Wisconsin College of Medicine and Public Health, Madison, WI 53719, USA Correspondence: hicham.zegzouti@promegaCitation: Engel, L.; Alves, J.; Hennek, J.; Goueli, S.A.; Zegzouti, H. Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors. Molecules 2021, 26, 6230. doi.org/10.3390/ moleculesAbstract: Standard glycosyltransferase (GT) activity assays are certainly not easily configured for fast detection nor for higher throughput screening simply iNOS Activator manufacturer because they depend on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a standard glycosyltransferase biochemical reaction, two goods are generated, a glycosylated product plus a nucleotide released from the sugar donor substrate. For that reason, an assay that detects the nucleotide may be universal to monitor the activity of diverse glycosyltransferases in vitro. Right here we describe 3 homogeneous and bioluminescent glycosyltransferase activity assays determined by UDP, GDP, CMP, and UMP detection. Each of those assays are performed inside a one-step detection that relies on converting the nucleotide solution to ATP, then to bioluminescence applying firefly luciferase. These assays are hugely sensitive, robust and resistant to chemical interference. Different applications of those assays are presented, including studies around the specificity of sugar transfer by diverse GTs along with the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. In addition, their utility in screening for specific GT inhibitors and also the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large variety of GTs and may have a considerable effect on diverse regions of Glycobiology research. Key phrases: nucleotide assays; bioluminescence; sugar substrate; fucosyltransferase; OGT; inhibitorAcademic Editor: Stefan Janecek Received: 16 September 2021 Accepted: 12 October 2021 Published: 15 October1. Introduction Glycosyltransferases (GT) represent a sizable family of enzymes that belong to a welldefined enzymatic network that orchestrates the formation and upkeep of complicated carbohydrate structures located abundantly in all living organisms [1]. Employing activated sugars as donor substrates, glycosyltransferases transfer the sugar moiety to an array of acceptor substrates of numerous chemical natures, including proteins, lipids, sugars, nucleic acids, and modest molecules [2]. The most typical donor substrates utilised by glycosyltransferases are nucleotide-activated sugars, for instance UDP-, GDP-, and CMP-sugars, but they may also use lipid sugar phosphates (e.g., dolichol phosphate sugar), and unsubstituted phosphates. Glycosyltransferases that use nucleotide-activated sugars are called Leloir enzymes, in honor of your 1970 chemistry Nobel Prize winner Luis F. Leloir, who discovered the initial sugar nucleotide [3]. As a result of the significance of your numerous oligosaccharide structures to cell f
