MRC National Institute for Medical Research, London
Science for Health
A novel way to measure ADP
18 February 2010
Adenosine diphosphate (ADP) is a molecule that plays an important role in energy transfer in cells. It is generated in various fundamental biological reactions, catalyzed by two large classes of enzymes, ATPases and kinases. Hydrolysis of adenosine triphosphate (ATP) to ADP and inorganic phosphate provides the energy for a large number of cellular processes, mediated by ATPases. Kinases are important both in metabolic pathways and in protein phosphorylation, which plays a central role in cellular signal transduction. Notably, protein kinases are the second most important target for drug screening, following G-protein coupled receptors. Methods to assay the ADP product are, therefore, of great importance to study these enzymatic reactions. ADP-specific sensors provide a generic method to measure the activity of any ATPase or kinase and potentially provide methods of screening these enzymes as drug targets.
Martin Webb (pictured right) and Simon Kunzelman, in NIMR's Division of Physical Biochemistry, have developed a new sensor for ADP, based on a protein scaffold of a bacterial actin homologue, ParM, which binds ADP. ADP binding was coupled to a fluorescence signal by labelling the protein with two rhodamines. Various mutations were introduced to provide a scaffold with the required characteristics, particularly low affinity for ATP.
ADP is an important and widespread product of key reactions in the cell but, surprisingly, there are relatively few methods to measure it. This new method provides a real-time, fluorescent assay that can be carried out at relatively high wavelengths with a photostable fluorophore. Furthermore there is high discrimination against ATP. The assay can be done at low concentrations of the sensor, but in the presence of high concentrations of ATP.
Martin Webb
Original article
The research findings are published in full in: .
Kunzelmann, S., and Webb, M. R. (2010)
A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM.
ACS Chemical Biology epub ahead of print. Publisher abstract
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