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Phosphorylation of Nucleoside Analogues by Adenosine Kinase
Aim: Characterization of substrate properties (Km and Vmax) of new nucleoside analogues for human adenosine kinase in comparison with properties of known nucleoside analogues (e.g. ribavirine, tubercidine or mizoribine).
Adenosine Kinase (AK) enzyme: The AK used in the assays is a human recombinant AK, 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). AK enzymatic activity (≥ 0.030 unit/mg protein) is systematically controlled before performing any assay.
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| Substrate | NovoCIB | Published data | |||
| Km (µM) | Kcat (min-1) | Km (µM) | Kcat (min-1) | Ref | |
| Adenosine | 11 | 1.5 | 3.2 | 13 | (1) |
| 0.15 | (2) | ||||
| Ribavirin | 328 | 1.9 | 540 | 1.8 | (1) |
| Deoxyadenosine | 295 | 3.4 | 360 | (2) | |
| Tubercidine | 12 | 2.2 | |||
| Inosine | 1758 | 2.6 | |||
Adenosine Kinase (AK) enzyme: The AK used in the assays is a human recombinant AK, 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). AK enzymatic activity (≥ 0.030 unit/mg protein) is systematically controlled before performing any assay.
The phosphorylation of ribavirin by adenosine kinase was confirmed by HPLC analysis as illustrated by Ribavirine-MP formation (red) from ribavirine (blue). |
 Kinetics Analysis: Enzymatic activity of adenosine 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, 5mM MgCl2, 2,5mM ATP, 0,1mM NADH, 1mM phosphoenolpyruvate, 1mM DTT, PK-LDH (5U/ml each), 0,85µM AK. 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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