Purification and properties of adenosine kinase from rat brain

Biochim Biophys Acta. 1980 Dec 4;616(2):199-207. doi: 10.1016/0005-2744(80)90138-2.

Abstract

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to apparent homogeneity from rat brain by (NH4)2SO4 fractionation, affinity chromatography on AMP-Sepharose 4B, gel filtration with Sephadex G-100, and DE-52 cellulose column chromatography. The yield was 56% of the initial activity with a final specific activity of 7.8 mumol/min per mg protein. The molecular weight was estimated as 38 000 by gel filtration with Sephadex G-100 and 41 000 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 20% that of adenosine phosphorylation. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad pH optimum at pH 7.5-8.5. The Km value for adenosine was 0.2 microM and the maximum activity was observed at 0.5 microM. At higher concentrations of adenosine, the activity was strongly inhibited. The Km value for ATP was 0.02 mM and that for Mg2+ was 0.1 mM. GTP, dGTP, dATP and UTP were also proved to be effective phosphate donors. Co2+ was as effective as Mg2+, and Ca2+, Mn2+ or Ni2+ showed about 50% of the activity for Mg2+. The kinase is quite unstable, but stable in the presence of a high concentration of salt; e.g., 0.15 M KCl.

Publication types

  • Comparative Study

MeSH terms

  • Adenosine Kinase / isolation & purification
  • Adenosine Kinase / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Animals
  • Brain / enzymology*
  • Deoxyadenosines
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Kinetics
  • Magnesium / pharmacology
  • Molecular Weight
  • Phosphotransferases / metabolism*
  • Rats
  • Substrate Specificity

Substances

  • Deoxyadenosines
  • Adenosine Triphosphate
  • Phosphotransferases
  • Adenosine Kinase
  • Magnesium