V. Bianchi et al., EFFECTS OF MUTATIONAL LOSS OF NUCLEOSIDE KINASES ON DEOXYADENOSINE 5'-PHOSPHATE DEOXYADENOSINE SUBSTRATE CYCLE IN CULTURED CEM AND V79 CELLS, The Journal of biological chemistry, 269(24), 1994, pp. 16677-16683
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.