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.