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Adenosine kinase from bovine adrenal medulla
European Journal of Biochemistry
|September 2, 1985
Summary
This study details the purification and kinetic analysis of bovine adrenal medulla adenosine kinase. The enzyme exhibits specific inhibition patterns, suggesting an ordered bi-bi reaction mechanism with adenosine as the initial substrate.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Adenosine kinase plays a crucial role in purine metabolism.
- Understanding its kinetic properties is vital for comprehending cellular adenosine regulation.
Purpose of the Study:
- To purify and characterize adenosine kinase from bovine adrenal medulla.
- To elucidate the enzyme's kinetic mechanism and substrate specificity.
Main Methods:
- Purification using ammonium sulfate precipitation, gel filtration, and affinity chromatography.
- Kinetic analysis including Michaelis constant determination and inhibitor studies.
- Determination of relative molecular mass via gel filtration.
Main Results:
- Adenosine kinase was purified 1600-fold with a relative molecular mass of approximately 42,000.
- Michaelis constants for adenosine and MgATP were determined (0.2 µM and 20 µM, respectively).
- The enzyme demonstrated broad specificity for purine nucleoside triphosphates and was inhibited by AMP, ADP, Mg2+, ATP, Ap4A, and Ap5A, with specific inhibition patterns.
Conclusions:
- Kinetic analysis suggests an ordered bi-bi mechanism with adenosine as the first substrate.
- Bis(adenylyl) polyphosphates (Ap4A, Ap5A) are potent competitive inhibitors versus MgATP.
- The phosphorylation of adenosine by this enzyme is not affected by vanadate ions.