Adenosine kinase from bovine adrenal medulla

Adenosine kinase from bovine adrenal medulla was purified 1600‐fold by using ammonium sulfate precipitation, gel filtration and affinity chromatography. Gel filtration yielded a relative molecular mass around 42000 and Michaelis constants were 0.2 μM for adenosine and 20 μM for MgATP. The enzyme showed a broad specificity for purine nucleoside triphosphate as phosphate donors. Both free Mg2+ and ATP were inhibitors. AMP was a competitive inhibitor with regard to adenosine and a non‐competitive inhibitor versus MgATP, while ADP was a uncompetitive inhibitor with regard to adenosine and a non‐competitive inhibitor versus MgATP. Adenosine kinase was strongly inhibited by the bis(adenylyl) polyphosphates Ap4A and Ap5A. These compounds inhibited the enzyme competitively versus MgATP (Ki= 0.06 μM for Ap4A and 0.4 μM for Ap5A) and uncompetitively with regard to adenosine. The results of the kinetic analysis suggest an ordered bi‐bi mechanism, adenosine being the first substrate. The phosphorylation of adenosine was unaffected in the presence of vanadate ions.