Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells
Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by the allosteric control of ribonucleotide reductase. The authors determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from (5-TH)cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain these results, the authors propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.
- Research Organization:
- Karolinska Institutet, Stockholm, Sweden
- OSTI ID:
- 6085902
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 16
- Country of Publication:
- United States
- Language:
- English
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NUCLEOTIDES
BIOCHEMISTRY
OXIDOREDUCTASES
TRITIUM COMPOUNDS
LABELLED POOL TECHNIQUES
CYTIDINE
DNA REPLICATION
FIBROBLASTS
HYDROXYUREA
KINETICS
METHOTREXATE
MICE
THYMIDINE
AMIDES
ANIMAL CELLS
ANIMALS
ANTIMETABOLITES
AZINES
CHEMISTRY
CONNECTIVE TISSUE CELLS
DRUGS
ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
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LABELLED COMPOUNDS
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NUCLEOSIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PYRIMIDINES
RIBOSIDES
RODENTS
SOMATIC CELLS
TRACER TECHNIQUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques