title: A continuous spectrophotometric assay for monitoring adenosine 5'-monophosphate production.
abstract: A number of biologically important enzymes release adenosine 5'-monophosphate (AMP) as a product, including aminoacyl-tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5'-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5'-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD(+)) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center in Shreveport, Shreveport, LA 71130, USA. Electronic address: efirst@lsuhsc.edu.
authors.forename: Eric A
authors.initials: EA
authors.lastname: First
mesh_terms: D000659:AMP Deaminase / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007168:IMP Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*
chemical_list: D009243:NAD; D000249:Adenosine Monophosphate; D007168:IMP Dehydrogenase; D000659:AMP Deaminase
keywords: AMP; AMP deaminase; Aminoacyl–tRNA synthetase; High-throughput assay; IMP dehydrogenase; cAMP phosphodiesterase