Said A. Goueli1,2, Kevin Hsiao1, and Hicham Zegzouti1
1Cell Signaling, Research and Development, Promega Corporation and 2University of Wisconsin School of Medicine and Public Health, Madison, WI
Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. ATP generating enzymes are usually involved in anabolic processes, while ATP consuming enzymes are involved in catabolite processes.
We have developed biochemical assays that monitor the concentrations of ATP, ADP and AMP in a biochemical reaction. For instance ATP depletion dependent assays and ADP generating assays monitor the activity of kinases and ATPases. These universal assays can measure the activity of various enzymes with no modification of the native substrate and the ability to use diverse substrates such as proteins, peptides, lipids, sugars, etc. The AMP generating reactions such as ubiquitin ligases, aa aminoacyl t-RNA synthetases, DNA ligases, cAMP-dependent phosphodiesterases, etc. can be also monitored with high sensitivity and reproducibility. We will show data using these various technologies in monitoring the various adenine nucleotide concentrations in biochemical reactions.