Purification and Properties of Adenosine Kinase from Rat Liver: Separation from Deoxyadenosine Kinase Activity

Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS- polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 μmol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources.

Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2′-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (~ 1.5%) under optimal conditions (pH 7.5) vs- 2′-deoxy- adenosine, the lowest yet reported for such a preparation, with a K_m of 670 μᴍ, as compared to 0.3 μᴍ for adenosine.

The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a of 70 μᴍ. Competitive inhibition was also exhibited by cordycepin (3′-deoxyadenosine) with a K_i of 150 μᴍ. A more potent competitive inhibitor was tubercidin, the K_i for which was 1.9 μᴍ.