Spangler, Corinna M. and Spangler, Christian and Goettle, Martin and Shen, Yuequan and Tang, Wei-Jen and Seifert, Roland and Schaeferling, Michael (2008) A fluorimetric assay for real-time monitoring of adenylyl cyclase activity based on terbium norfloxacin. ANALYTICAL BIOCHEMISTRY, 381 (1). pp. 86-93. ISSN 0003-2697,
Full text not available from this repository. (Request a copy)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 [alpha-(32)P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K(M)), the vo value of ATP turnover, and the IC(50) values for three inhibitors of EF by this newly developed fluorescent method. (c) 2008 Elsevier Inc. All rights reserved.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | CYCLIC-AMP; INHIBITION; ISOFORMS; CALMODULIN; BINDING; ANTHRAX; DRUG; CAMP; adenylyl cyclase; edema factor; fluorescence assay; terbium norfloxacin; high-throughput screening |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy > Pharmacology and Toxicology (Prof. Schlossmann, formerly Prof. Seifert) Chemistry and Pharmacy > Institut für Analytische Chemie, Chemo- und Biosensorik > Chemo- und Biosensorik (Prof. Antje J. Bäumner, formerly Prof. Wolfbeis) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 21 Oct 2020 12:56 |
| Last Modified: | 21 Oct 2020 12:56 |
| URI: | https://pred.uni-regensburg.de/id/eprint/30189 |
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