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ADK Screening Assay Kit
A non-isotopic and high-performance assay (Z'-factor = 0.68) for a fast and simple measurement of adenosine kinase (ADK) activity in vitro.

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Dr Larissa Balakireva, CEO & Founder of NovoCIB, was awarded with the Trophy of
"Femmes en Or 2011, Femme de l'Innovation"
in September 2011
.


NOVOCIB is certified by the French Ministry of Research as a "Research Tax Credit"
(Crédit Impôt Recherche - CIR) company.
To know more:
• French Ministry of Research (in French)
• Press Release

Phosphorylation of Nucleoside Analogues by Deoxycytidine Kinase

Aim: Characterization of substrate properties (Km and Vmax) of new nucleoside analogues for human deoxycytidine kinase in comparison with the properties of known nucleoside analogues (e.g. aracytidine, gemcitabine, cladribine and lamivudine).

Substrate NovoCIB Published data  
Km (µM) Vmax
(µmol/mg/min)		 Relative Vmax
(% of dCR)		 Km (µM) Vmax
(µmol/mg/min)		 Ref
Deoxycytidine 0.577 0.026 100 0.16 0.033 Recombinant,
Johansson Karlsson 1995
1.3 0.069 Recombinant,
Usova & Eriksson, 1997
0.57 0.004 Partially purified,
Someya H. et al 2003
Gemcitabine (dFdC) 42.71 0.325 1250  
Deoxyadenosine 150.5 1.08 4153 115   Recombinant,
Sabini E. et al 2008
480 1.5 Recombinant,
Johansson Karlsson 1995
Aracytidine (AraC) 6.81 0.224 862 15 0.009 Partially purified,
Someya H. et al 2003
Cladribine 56.5 0.285 1096 89 0.126 Recombinant,
Usova & Eriksson, 1997
24 0.76 Recombinant,
Johansson Karlsson 1995

Deoxycytidine kinase (dCK) enzyme: The dCK used in the assays is a human recombinant dCK, cloned from human cells, expressed in E. coli, produced and purified by NOVOCIB (click here for details). The enzyme purity is controlled by SDS-PAGE. Protein concentration is measured by Bradford method (Bio-Rad). dCK enzymatic activity (≥ 0.025 unit/mg protein) is systematically controlled before performing any assay.
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Kinetics Analysis: Enzymatic activity of deoxycytidine kinase with particular nucleoside substrate is measured continuously by spectrophotometric assays in a coupled lactate dehydrogenase/pyruvate kinase system. Assays are carried out at 37°C, at 50mM Tris-HCl pH7,6; 50mM KCl, 10mM MgCl2, 5mM ATP, 0,1mM NADH, 1mM phosphoenolpyruvate, 1mM DTT, PK 10U/ml, LDH 15U/ml, 0,9µM dCK. The nucleosides, nucleotides, LDH and PK are purchased from Sigma-Aldrich. Reaction is followed in an iEMS Reader MF (Labsystems) microtiter plate reader at 340nm. Assays are performed in duplicate (2 wells per compound and per concentration). Triplicates are available upon request. Km and Vmax are calculated from spectroscopic data using Michaelis-Menten equation.
A confirmation by HPLC analysis of formation of monophosphorylated forms is available upon request.
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References (with links to PubMed)
1. M. Johansson and A. Karlsson (1995): Differences in kinetic properties of pure recombinant human and mouse deoxycytidine kinase Biochem. Pharmacol. 50(2), 163-168
2. E. V. Usova and S. Eriksson (1997): The effects of high salt concentrations on the regulation of the substrate specificity of human recombinant deoxycytidine kinase Eur. J. Biochem. 248(3), 762-766
3. H. Someya et al. (2003): Phosphorylation of 4'-thio-beta-D-arabinofuranosylcytosine and its analogs by human deoxycytidine kinase J. Pharmacol. Exp. Ther. 304(3), 1314-1322
4. E. Sabini et al. (2008): Structural basis for substrate promiscuity of dCK J. Mol. Biol. 378(3), 607-621
Download this document "dCK nucleoside phosphorylation assay" 
To evaluate Nucleoside Analogues as potential IMPDH inhibitors, see also our
Nucleoside Kinase - IMPDH coupled assay
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To know more about our catalogue of Nucleoside kinases.


Related Links
• AK Phosphorylation
• CMK Phosphorylation
• dCK-CMK Phosphorylation 		• Nucleoside Kinases
• Phosphorylation of Nucleoside Analogues
  in Whole Cell Assay
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