UMP/CMP-Glo Glycosyltransferase Assay
The UMP/CMP-Glo Glycosyltransferase Assay is a bioluminescent assay for detecting the activity ofglycosyltransferases that use CMP-, CDP- or UDP-sugars as donor substrates and release CMP or UMP. Glycosylating reactions catalyzed by glycosyltransferases (GTs) are central to many biological processes, including cell-cell interactions, cell signaling and bacterial cell wall biosynthesis. Because of the importance of this class of enzymes, there is a need for biochemical assays to monitor their activity, their mode of regulation, and to search for their selective and potent inhibitors. Phosphoglycosyltransferases (PGTs) transfer the phospho-sugar from a nucleotide diphosphate sugar donor (e.g., UDP-diNAcBac, UDP-GlcNAc) to an acceptor molecule such as polyprenol-phosphate or polysaccharide-phosphate. In a PGT reaction, the UMP moiety is released as a product. Sialyltransferases (STs) transfer the sugar from a nucleotide monophate-sugar donor (e.g., CMP-sialic acid) to an acceptor molecule. In a sialyltransferase reaction, the CMP moiety is released as a product. Therefore, an assay that detects UMP or CMP as products of these reactions would be suitable for monitoring the activity of all the UMP- or CMP-releasing glycosyltransferases.The UMP/CMP-Glo Glycosyltransferase Assay is a homogeneous, one-step-reagent-addition method to rapidly detect UMP or CMP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of UMP/CMP Detection Reagent is added to simultaneously convert the UMP or CMP product to ATP and generate light in a luciferase reaction. The light generated is detected using a luminometer. Luminescence can be correlated to UMP or CMP concentration by using a UMP or CMP standard curve. The light output is proportional to the concentration of UMP or CMP from low nM to 50microM. The assay is easy to use and highly sensitive, two features that are desirable and essential for measuring the activity of different UMP- or CMP releasing glycosyltransferases such as sialyltransferases or PGTs. Therefore, the UMP/CMP detection assay requires less glycosyltransferase enzyme in the reactions. This assay is fast and simple (Figure 5). The UMP/CMP-Glo Assay is performed in a single well of a multiwell plate and can be used to detect glycosyltransferase activity in as little as a 5microl reaction.