Abstract

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) was partially purified by DEAE-cellulose column chromatography from Sarcoma 180 cells grown in vitro. This enzyme preparation, which was free of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) but contained adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3), was studied with respect to its kinetic properties and specificity for substrates and inhibitors.

At pH 7.0 and 35° the K_m for adenosine was 0.5 µM and the V_max was 63 mµmoles/mg of protein per minute. Strong substrate inhibition was observed at adenosine concentrations greater than 4 µM, 50% inhibition occurring at about 100 µM. The reaction required ATP (K_m = 200 µM) and Mg⁺⁺. The optimal Mg⁺⁺ concentrations were 0.1 and 0.25 mM at 0.5 and 2.5 mM ATP, respectively. Concentrations of Mg⁺⁺ higher than these were inhibitory, and Mn⁺⁺ substituted poorly for Mg⁺⁺.

Most of the N⁶-substituted adenosine analogues which were substrates of adenosine kinase inhibited the growth of S-180 cells in vitro. Among these were two compounds which are also known to be potent cytokinins in plant systems, namely, N⁶-furfuryl- and N⁶-(Δ²-isopentenyl)adenosine. The 5'-monophosphate of the latter compound was not phosphorylated further by adenylate kinase. The N⁶-substituted adenosines which were poorly or not at all phosphorylated by adenosine kinase were also poor inhibitors of S-180 cells in vitro. Several of these were potent inhibitors of the kinase, such as N⁶-phenyladenosine, which had a K_i value of 0.6 µM.