Plant adenosine kinase: purification and some properties of the enzyme from Lupinus luteus seeds.

Adenosine kinase (ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) from Lupinus luteus seeds has been obtained with good yield in almost homogeneous state by ammonium sulfate fractionation, chromatography on aminohexyl-Sepharose, and gel filtration. Active enzyme is a single polypeptide chain with a molecular weight of about 38,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel nitration. Estimated molecular activity is 156. The enzyme exhibits a strict requirement for divalent metal ions. Among several ions tested the following appeared to be active as cofactors: Co²⁺ ? Mn²⁺ > Mg²⁺ = Ca²⁺ ? Ni²⁺ > Ba²⁺. The optimal metal ion concentrations were as follows: Mn²⁺, 0.5 mm, Mg²⁺ and Ca²⁺, 1 mm, Co²⁺, 1.5 mm. The adenosine kinase shows optimum activity at pH 7.0–7.5. K_m values for adenosine and ATP are 1.5 × 10^?6 and 3 × 10^?4m, respectively. Lupin adenosine kinase is completely inhibited by antisulfhydryl reagents. ATP is the main phosphate donor and among other nucleoside triphosphates ITP, dATP, GTP, and XTP can substitute it but less effectively. Among the ribo- and deoxyribonucleosides occurring in nucleic acids adenosine is phosphorylated effectively and 2′-deoxyadenosine at a lower rate. Of other adenosine analogs tested all adenine d-nucleosides and purine derivative ribosides, besides those with a hydroxyl group at C-6, were found to be substrates for lupin adenosine kinase. Pyrimidine ribo- and deoxyribonucleosides were not phosphorylated.