Utility of AMP Detection System for Monitoring the Activities of Diverse Enzyme Reactions

Abstract

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are disregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. Here, we demonstrate the versatility of an enzyme coupled assay that quantifies the amount of AMP produced by any enzymatic reactions regardless of its substrates. We successfully implemented it to enzyme reactions that use ATP as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP generating enzymes that do not require ATP as substrate, including phosphodiesterases (cAMP) and E.coli DNA ligases (NAD+). In a further elaboration of the AMP‐Glo platform, we coupled it to E.coli DNA ligase, enabling measurement of NAD+ and enzymes that use NAD+ like mono‐ and poly‐ADP‐ribosyltransferases. Sulfotransferases use 3′‐Phosphoadenosine‐5′‐phosphosulfate (PAPS) as the universal sulfo‐group donor and Phosphoadenosine‐5′‐phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgiresident PAP‐specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP‐Glo system we can measure the activities of sulfotransferases. Thus by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP‐Glo assay.Support or Funding InformationThe research was supported by Promega Corp.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.