A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin.
Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [alpha-(32)P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K(M)), the v(0)(max) value of ATP turnover, and the IC(50) values for three inhibitors of EF by this newly developed fluorescent method.
Spangler
Corinna M
CM
Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
Spangler
Christian
C
Göttle
Martin
M
Shen
Yuequan
Y
Tang
Wei-Jen
WJ
Seifert
Roland
R
Schäferling
Michael
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D037361:Adenylate Cyclase Toxin / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism*; D001408:Bacillus anthracis / Q000201:enzymology; D002138:Calibration; D002147:Calmodulin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D009643:Norfloxacin / Q000378:metabolism*; D011874:Radiometry; D015203:Reproducibility of Results; D013725:Terbium / Q000378:metabolism*; D014757:Viper Venoms / Q000378:metabolism* / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D002147:Calmodulin; D014757:Viper Venoms; C416405:edema factor; D013725:Terbium; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D009643:Norfloxacin