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Adenosine kinase (ADK) regulates extracellular concentration of adenosine, an important “metabokine” with neuroprotective, analgesic and anti-inflammatory properties. ADK inhibitors have been shown to provide potent antinociceptive, anti-inflammatory and anticonvulsant activity in animal model suggesting their potential utility for pain, inflammation, epilepsy, and possibly other CNS and PNS diseases associated with cellular trauma and inflammation.
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PRECICE® ADK Screening Assay Kit
PRECICE® ADK Screening Assay Kit is based on the use of inosine as a substrate of ADK and a coupled reaction involving a highly active IMPDH (Inosine Monophosphate Dehydrogenase) for a direct measurement of the inosine monophosphate (IMP) formed by ADK.
In the presence of inosine and ATP, ADK catalyses the phosphorylation of inosine to form IMP and ADP. IMP is then immediately oxidized to Xanthosine Monophosphate (XMP) by a highly active IMPDH in the presence of NAD, leading to NADH2 formation.
This coupling reaction is immediate when IMPDH activity is much higher than ADK activity in the assay. The enzymatic activity of ADK, which corresponds to the formation kinetics of IMP, is then stoichiometrically and directly monitored by the formation kinetics of NADH2. The velocity of NADH2 formation is measured with a spectrophotometer at 340nm (molar extinction coefficient of NADH2 at 340nm = 6220 M-1.cm-1).
Validation
The use of PRECICE® ADK Screening Assay Kit for the screening of ADK inhibitors and IC50 measurements was validated with compound A-134974, a known ADK inhibitor (Sigma-Aldrich, under license from Abbott Laboratories).
Z'-Factor was calculated from 44 negative (no ADK inhibition) and positive (inhibition of ADK with 500nM A-134974) and measured as 0.68, which is an excellent level of performance for a screening assay.
All data points represent the mean (+/-SD) of 8 wells (2 wells - duplicate - on 4 independent plates). IC50 was determined by fitting a 4-parameter non-linear curve to the data with least-square method. IMPDH activity assays were run to confirm that IMPDH is not inhibited by compound A-134974 (data not shown).
| Kit Content: | • Bacterial recombinant IMPDH
• IMPDH Reconstitution Buffer |
• Human recombinant ADK
• ADK Reconstitution Buffer |
• NAD
• ATP |
• Inosine
• HKM Buffer(10x) |
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