ADK/Adenosine Kinase Protein, Human (HEK293)

ADK is an enzyme that catalyzes the transfer of gamma-phosphate from adenosine triphosphate (ATP) to adenosine (Ado) to form adenosine monophosphate (AMP). ADK acts as a potential regulator of extracellular adenosine and intracellular adenine nucleotide concentrations. ADK is a therapeutic target for controlling obesity and non-alcoholic fatty liver disease. ADK/Adenosine Kinase Protein, Human (HEK293) is the recombinant human-derived ADK/Adenosine Kinase protein, expressed by HEK293 , with tag free.

Adenosine kinase is an enzyme that catalyzes the transfer of γ-phosphate from adenosine triphosphate (ATP) to adenosine (Ado) to form adenosine monophosphate (AMP). Catalyze the phosphorylation of purine nucleoside adenosine at the 5 'position in an ATP-dependent manner. ADK acts as a potential regulator of extracellular adenosine and intracellular adenine nucleotide concentrations. ADK promotes excess fat deposition and liver inflammation by inhibiting fatty acid oxidation in hepatocytes and producing hepatocellular proinflammatory mediators, which is a therapeutic target for controlling obesity and non-alcoholic fatty liver disease^[1][2][3].

Measured by its ability to phosphorylate Adenosine that incubate at room temperature for 60 minutes. The specific activity is 32.58 pmo/min/μg.

Materials
Assay buffer (10×): 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl₂, 100 mM CaCl₂, pH 7.0
ADK/Adenosine Kinase Protein, Human (HEK293) (HY-P75530)
Substrates: Adenosine, ATP
Detection Kit: Kinase-Lumi^TM Enhanced Chemiluminescent Kinase Activity Detection Kit
Standard: ATP

Procedure
1. Dilute the 10× assay Buffer to 1× with ddH₂O.
2. Dilute ATP to 0, 0.024414063, 0.048828125, 0.09765625, 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, and 50 μM with 1× assay buffer.
3. Add 50 μL of ATP solution at different concentrations to each well, then add 50 μL of kinase detection reagent.
4. Incubate at room temperature for 10 min.
5. Measure the RLU values using a microplate reader with chemiluminescence detection.
6. Obtain the standard curve equation with the measured RLU values as the ordinate and the amount of ATP as the abscissa.
7. Dilute Human ADK protein to 50 μg/mL with 1× assay buffer.
8. Dilute ATP to 90 μM with 1× Detection Buffer and mix well, then dilute adenosine to 2 mM and mix.
9. Add 25 μL of 50 μg/mL protein solution to a 96-well plate, then add 25 μL of the mixed solution; for blank wells, add 25 μL of 1× assay buffer, then add 25 μL of the mixed solution.
10. Incubate at room temperature (22-25°C) for 60 minutes.
11. After incubation, add 50 μL of kinase detection reagent to each well.
12. Incubate at room temperature for 10 min.
13. Measure the RLU values using a microplate reader.
14. Calculate the specific activity:

Human

HEK293

Tag Free

AAH03568 (M1-H345)

132  [NCBI]

Full Length

Approximately 38.74 kDa

• 
  Greater than 95% as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM Tris, 300 mM NaCl pH 8.0, 0.5 mM PMSF, 0.5 mM TCEP, 20% glycerol.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. Li H, et al. Hepatocyte Adenosine Kinase Promotes Excessive Fat Deposition and Liver Inflammation. Gastroenterology. 2023 Jan;164(1):134-146.  [Content Brief]

  [2]. Bjursell MK, et al. Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function. Am J Hum Genet. 2011 Oct 7;89(4):507-15.  [Content Brief]

  [3]. McNally T, et al. Cloning and expression of the adenosine kinase gene from rat and human tissues. Biochem Biophys Res Commun. 1997 Feb 24;231(3):645-50.  [Content Brief]