EFFECTS OF MUTATIONAL LOSS OF NUCLEOSIDE KINASES ON DEOXYADENOSINE 5'-PHOSPHATE DEOXYADENOSINE SUBSTRATE CYCLE IN CULTURED CEM AND V79 CELLS

The functions of a deoxynucleoside kinase and a deoxynucleotidase can give rise to substrate cycles in which the two enzymes catalyze in opp osite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence sho wed that pyrimidine dNMP cycles occur in cultured cells and participat e in the regulation of the size of dNMP pools there by affecting the t ransport of deoxyribonucleosides across the cell membrane. Here, we ap ply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catab olic activity of a putative dAMP/deoxyadenosine cycle. A comparison of human CEM lymphoblasts and hamster V79 fibroblasts, including mutant cells lacking kinases for the phosphorylation of deoxyadenosine, shows a much lower deoxyadenosine excretion in CEM cells (0.05% of dATP syn thesized by reduction of ADP) as compared with V79 cells (4% of dATP). Mutational loss of deoxycytidine kinase increases these values to 0.3 % in CEM cells and to 10% in V79 cells. This strongly suggests the pre sence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additi onal loss of adenosine kinase only marginally affects deoxyadenosine e xcretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in sit u activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.