Development and validation of a transcreener assay for detection of AMP- and GMP-producing enzymes.
Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3',5'-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC(50) ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z' values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin- activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes.
Staeben
Matt
M
BellBrook Labs, Madison, Wisconsin, USA. matt.staeben@bellbrooklabs.com
Kleman-Leyer
Karen M
KM
Kopp
Andrew L
AL
Westermeyer
Thane A
TA
Lowery
Robert G
RG
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000101:Acetyl-CoA C-Acetyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000096:biosynthesis*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000465:Algorithms; D001667:Binding, Competitive / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005454:Fluorescence Polarization; D006157:Guanosine Monophosphate / Q000096:biosynthesis*; D006801:Humans; D007202:Indicators and Reagents; D009243:NAD / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D044764:Ubiquitin-Activating Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009243:NAD; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000101:Acetyl-CoA C-Acetyltransferase; C086376:UBE2H protein, human; D044763:Ubiquitin-Conjugating Enzymes; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D044764:Ubiquitin-Activating Enzymes; D006152:Cyclic GMP