A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin |
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Authors: | Spangler Corinna M Spangler Christian Göttle Martin Shen Yuequan Tang Wei-Jen Seifert Roland Schäferling Michael |
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Institution: | a Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany b Department of Pharmacology and Toxicology, University of Regensburg, D-93040 Regensburg, Germany c Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, 300071 Tianjin, People’s Republic of China d Ben May Department of Cancer Research, University of Chicago, Chicago, IL 60637, USA |
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Abstract: | 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 α-32P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (KM), the v0max value of ATP turnover, and the IC50 values for three inhibitors of EF by this newly developed fluorescent method. |
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Keywords: | Adenylyl cyclase Edema factor Fluorescence assay Terbium norfloxacin High-throughput screening |
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