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 Km for adenosine was 0.5 µM and the Vmax 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 (Km = 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 Ki value of 0.6 µM.
ACKNOWLEDGMENT The authors wish to express their appreciation to Miss Dorris Sugg for her excellent assistance in these studies.
- Copyright ©, 1971, by Academic Press, Inc.
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|