Adenylate Kinase 1/AK1 Protein, Rat (His)

The adenylate kinase 1 (AK1) protein plays a crucial role in cellular energy homeostasis by maintaining the balance of adenylate nucleotides by catalyzing the reversible transfer of terminal phosphate groups between ATP and AMP.Adenylate Kinase 1/AK1 Protein, Rat (His) is the recombinant rat-derived Adenylate Kinase 1/AK1 protein, expressed by E.coli , with N-His labeled tag.

Adenylate Kinase 1 (AK1) is a versatile enzyme that catalyzes the reversible transfer of the terminal phosphate group between ATP and AMP, a critical step in cellular energy homeostasis. Additionally, AK1 exhibits nucleoside diphosphate kinase activity, facilitating the production of various nucleoside triphosphates (ATP, CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP) from their corresponding diphosphate substrates, using either ATP or GTP as a phosphate donor. This enzymatic flexibility highlights its essential role in nucleotide biosynthesis. Furthermore, AK1 demonstrates a lower-rate catalysis of the synthesis of thiamine triphosphate (ThTP) from thiamine diphosphate (ThDP) and ADP, indicating its involvement in thiamine metabolism. The multifaceted functions of AK1 underscore its importance in maintaining cellular energy levels and participating in diverse nucleotide and cofactor biosynthetic pathways.

Specific activity is 933.598 units/mg. One unit will convert ATP + AMP to 2.0 µmoles of ADP per minute at pH 7.5 at 37 °C.

Materials
Kinase-Lumi^TM Enhanced Chemiluminescent Kinase Assay Kit
Assay buffer: 40 mM Tris, 20 mM MgCl₂, 0.1 mg/mL BSA, pH 7.4
Adenylate Kinase 1/AK1 Protein, Rat (His)（HY-P74427）
Substrate: AMP, ATP
Standard: ATP

Assay Procedure
Standard Curve Preparation
1. Dilute ATP (standard) in assay buffer to the following concentrations: 0, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, 50 μM.
2. Add 50 μL of each standard concentration to a well and add 50 μL of Kinase-Lumi? Enhanced Chemiluminescent Kinase Assay Reagent.
3. Incubate at room temperature in the dark for 10 minutes.
4. Using a microplate reader, measure the RLU value under luminescence.
5. Plot the measured RLU on the x-axis and the standard concentration on the y-axis to generate a standard curve and obtain the standard curve equation.

Protein Activity Assay
1. Dilute AK1 to 10 μg/mL in assay buffer.
2. Dilute ATP to 40 μM (provided in the kit) and AMP to 40 μM in assay buffer to prepare the reaction mixture.
3. Add 25 μL of 10 μg/mL AK1 to a 96-well plate, then add 25 μL of the ATP-AMP mixture to start the reaction; for the blank, add 25 μL of assay buffer and 25 μL of the ATP-AK1 mixture.
4. Incubate at 37°C in the dark for 50 minutes.
5. After incubation, add 50 μL of Kinase-Lumi^TM Enhanced Chemiluminescent Kinase Assay Reagent to each well.
6. Incubate at room temperature for 10 minutes.
7. Using a microplate reader, measure the RLU value under luminescence.
8. Calculate the specific activity:

Rat

E. coli

N-6*His

P39069 (M1-K194)

24183  [NCBI]

Full Length

MEDKLKKAKIIFVVGGPGSGKGTQCEKIVQKYGYTHLSTGDLLRAEVSSGSSRGKMLSSIMEKGELVPLETVLDMLRDAMLAKVDSSNGFLIDGYPREVKQGEEFERKIAQPTLLLYVDAGPETMTQRLLKRGETSGRVDDNEETIKKRLETYYKATEPVISFYDKRGIVRKVNAEGSVDTVFSQVCTYLDSLK

Approximately 23 kDa

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

Solution.

Supplied as a 0.22 μm filtered solution of PBS, 100 mM arginine, pH 7.1, 20% glycerol.

<1 EU/μg, determined by LAL method.

N/A.

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.