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 opposite directions the irreversible interconversion of a deoxynucleoside 5'-monophosphate (dNMP) and its deoxynucleoside. Earlier evidence showed that pyrimidine dNMP cycles occur in cultured cells and participate in the regulation of the size of dNMP pools there by affecting the transport of deoxyribonucleosides across the cell membrane. Here, we apply an isotope flow method using labeled adenine as precursor of dAMP and DNA to quantify deoxyadenosine excretion as a measure of the catabolic 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 synthesized 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 presence of a dAMP/deoxyadenosine cycle in both CEM and V79 cells. Additional loss of adenosine kinase only marginally affects deoxyadenosine excretion in CEM cells. The small excretion of deoxyadenosine (also in the absence of both kinases) demonstrates that in CEM cells the in situ activity of the deoxynucleotidase affecting the dAMP/deoxyadenosine substrate cycle is very low and that the cycle has mainly an anabolic function there.
Bianchi
V
V
Department of Biology, University of Padova, Italy.
Ferraro
P
P
Borella
S
S
Bonvini
P
P
Reichard
P
P
D000218:Adamantane / Q000031:analogs & derivatives* / Q000378:metabolism; D000225:Adenine / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism; D000248:Adenosine Kinase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D002455:Cell Division; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D003839:Deoxyadenosines / Q000378:metabolism*; D003841:Deoxycytidine / Q000378:metabolism; D003842:Deoxycytidine Kinase / Q000235:genetics* / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D007700:Kinetics; D008214:Lymphocytes; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D014316:Tritium
D003839:Deoxyadenosines; D003854:Deoxyribonucleotides; D007042:Hypoxanthines; D003841:Deoxycytidine; D014316:Tritium; D019271:Hypoxanthine; C016358:2,4-diamino-5-adamantyl-6-methylpyrimidine; D004247:DNA; D000248:Adenosine Kinase; D003842:Deoxycytidine Kinase; D000225:Adenine; D000241:Adenosine; D000218:Adamantane; D013936:Thymidine