A Direct Pyrophosphatase-coupled Assay Provides New Insights into the Activation of the Secreted Adenylate Cyclase from Bordetella pertussis by Calmodulin
Autor: | Paul D. Lawrence, F. Yasmin Kazi, Mathis O. Riehle, Anthony J. Lawrence, Barbara M. Orr, John Young, Nicholas C. Price, James Sinclair, Julia MacDonald-Fyall, Roger Parton, John G. Coote |
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Rok vydání: | 2002 |
Předmět: |
Bordetella pertussis
Time Factors Calmodulin chemistry.chemical_element Calcium Biochemistry Cyclase Pyrophosphate Catalysis chemistry.chemical_compound Bacterial Proteins Escherichia coli Protein Precursors Pyrophosphatases Molecular Biology chemistry.chemical_classification Pyrophosphatase Dose-Response Relationship Drug biology Chemistry Temperature Cell Biology cyaA biology.organism_classification Recombinant Proteins Protein Structure Tertiary Electrophysiology Kinetics Enzyme Calibration biology.protein Adenylate Cyclase Toxin Adenylyl Cyclases Protein Binding |
Zdroj: | Journal of Biological Chemistry. 277:22289-22296 |
ISSN: | 0021-9258 |
Popis: | Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC 4.6.1.1) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k on by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated. |
Databáze: | OpenAIRE |
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