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PRECICE® dCK Assay Kit is a very simple and fast assay designed to measure Deoxycytidine Kinase activity in vitro. It can be used for the screening of dCK inhibitors. In this assay, dCK activity is continuously monitored at 340nm through the coupling of the synthesis of monophoshorylated deoxyinosine, catalyzed by dCK, and the oxidation of dIMP by a recombinant IMP-dehydrogenase (IMPDH).
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NOVOCIB's PRECICE® dCK Assay Kit is designed to measure the activity of dCK (deoxycytidine kinase) in vitro.
Human deoxycytidine kinase (dCK, EC 2.7.1.74) plays a key role in the salvage pathway of deoxynucleotides synthesis providing resting cells with deoxynucleotides for DNA repair and mitochondrial DNA synthesis. The enzyme has a broad substrate specificity and provides the phosphorylation of both purine and pyrimidine deoxynucleosides (e.g. deoxyadenosine (dA), deoxyguanosine (dG)) and deoxycytidine (dC) and pyrimidine ribonucleoside, cytidine (C)). The enzyme can utilize both ATP and UTP as phosphate donor with UTP as a preferred substrate.
PRECICE® dCK Assay Kit can be used for the screening of dCK inhibitors (see also our PRECICE® dCK Inhibiton Screening Service).
Principle
PRECICE® dCK Assay Kit is based on the use of deoxyinosine (dIR) as a substrate of dCK and a coupled reaction involving a highly active IMPDH (Inosine Monophosphate Dehydrogenase, bacterial recombinant, Ref. #E-Nov7) for a direct measurement of the deoxyinosine monophosphate (dIMP) formed by dCK.
In the presence of dIR and ATP, dCK catalyses the phosphorylation of dIR to form dIMP and ADP.The dIMP formed is then oxidized to deoxyxanthosine monophosphate (dXMP) by IMPDH in the presence of NAD, leading to NADH2 formation.
This coupling reaction is immediate when IMPDH activity is much higher than dCK activity in the assay. The enzymatic activity of dCK, which corresponds to the formation kinetics of dIMP, 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).
Enzymatic characterization of Human recombinant dCK (Ref. E-Nov3)
NOVOCIB's Human recombinant dCK activity was characterized using dIR as a substrate. Assays were performed in 100mM Tris-HCl Buffer, pH8, KCl 100mM, Mg Acetate 12mM, ATP 2mM, NAD 2mM, DTT 4mM and IMPDH at ≈25mU/ml (NovoCIB, Ref. #E-Nov7) and with various concentrations of dIR.
Figures 1 and 2 show the kinetics of formation of NADH2 as a function of dIR concentration and the corresponding Lineweaver-Burk plots, respectively. KM for IR was estimated at 1.46mM under our assay conditions.
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Human deoxycytidine kinase (dCK, EC 2.7.1.74) plays a key role in the salvage pathway of deoxynucleotides synthesis providing resting cells with deoxynucleotides for DNA repair and mitochondrial DNA synthesis. The enzyme has a broad substrate specificity and provides the phosphorylation of both purine and pyrimidine deoxynucleosides (e.g. deoxyadenosine (dA), deoxyguanosine (dG)) and deoxycytidine (dC) and pyrimidine ribonucleoside, cytidine (C)). The enzyme can utilize both ATP and UTP as phosphate donor with UTP as a preferred substrate.
PRECICE® dCK Assay Kit can be used for the screening of dCK inhibitors (see also our PRECICE® dCK Inhibiton Screening Service).
Principle
PRECICE® dCK Assay Kit is based on the use of deoxyinosine (dIR) as a substrate of dCK and a coupled reaction involving a highly active IMPDH (Inosine Monophosphate Dehydrogenase, bacterial recombinant, Ref. #E-Nov7) for a direct measurement of the deoxyinosine monophosphate (dIMP) formed by dCK.
In the presence of dIR and ATP, dCK catalyses the phosphorylation of dIR to form dIMP and ADP.The dIMP formed is then oxidized to deoxyxanthosine monophosphate (dXMP) by IMPDH in the presence of NAD, leading to NADH2 formation.
This coupling reaction is immediate when IMPDH activity is much higher than dCK activity in the assay. The enzymatic activity of dCK, which corresponds to the formation kinetics of dIMP, 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).
Enzymatic characterization of Human recombinant dCK (Ref. E-Nov3)
NOVOCIB's Human recombinant dCK activity was characterized using dIR as a substrate. Assays were performed in 100mM Tris-HCl Buffer, pH8, KCl 100mM, Mg Acetate 12mM, ATP 2mM, NAD 2mM, DTT 4mM and IMPDH at ≈25mU/ml (NovoCIB, Ref. #E-Nov7) and with various concentrations of dIR.
Figures 1 and 2 show the kinetics of formation of NADH2 as a function of dIR concentration and the corresponding Lineweaver-Burk plots, respectively. KM for IR was estimated at 1.46mM under our assay conditions.
| Figure 1
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| Figure 2
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