Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Abstract

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.

Erk5 contributes to maintaining the balance of cellular nucleotide levels and erythropoiesis

“…Metabolic flux through the salvage pathway is regulated by thymidine kinase 1 (TK1), which phosphorylates thymidine, and deoxycytidine kinase, which phosphorylates deoxycytidine, deoxyadenosine and deoxyguanosine. 17,30,31 To test whether thymidine salvaging is increased in Erk5-depleted cells, we incubated cells with [ 3 H]-thymidine and analyzed cellular uptake and TK activity in cell extracts. Although [ 3 H]-thymidine uptake by shErk5 cells was slightly higher, TK activity in shErk5 and control cells was similar (supplementary material Fig.…”

Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA

“…113) is a nuclear coded enzyme localised to the mitochondria (mt), catalysing the phosphorylation of purine deoxynucleosides and their analogs, using a nucleoside triphosphate as phosphate donor. dGK activity is found in most tissues and the enzyme has been purified from skin, thymus, placenta, liver, brain and leukemic cells [1][2][3][4][5][6][7][8]. The active form of dGK is a dimer of 28-29 kDa subunits and dGuo, dAdo, dlno and several cytostatic analogs, e.g.…”

Characterization of the deoxycytidine kinase promoter in human lymphoblast cell lines.

“…deoxycytidine to its 5'-monophosphate and, despite a much higher Km for the purine deoxyribonucleosides, also plays a major role in phosphorylating 2 '-deoxyadenosine and 2 '-deoxyguanosine to their corresponding monophosphates (1)(2)(3)(4). dCK is also central in the activation of anticancer and antiviral agents such as cytosine arabinoside, fludarabine, 2 ',3 '-dideoxycytidine, and 2-chlorodeoxyadenosine (CdA) (5)(6)(7)(8)(9).…”