Cytosolic high K-m 5 '-nucleotidase and 5 '(3 ')-deoxyribonucleotidase in substrate cycles involved in nucleotide metabolism

5'-Nucleotidases are the catabolic members of the substrate cycles postulat ed to be involved in the regulation of intracellular deoxyribonucleoside tr iphosphate pools. Here, we attempt to identify the nature of the nucleotida ses. Earlier, we constructed various mammalian cell lines that can be induc ed to overproduce the high K-m 5'-nucleotidase (hkm-NT) or the 5'(3')-deoxy nucleotidase (dNT-1). Now we labeled control and induced human 293 cells an d hamster V79 cells with radioactive hypoxanthine or uridine and during a c hase measured quantitatively the metabolism of ribo- and deoxyribonucleotid es, DNA replication, and excretion of nucleosides into the medium. Overprod uction of hkm-NT greatly increased excretion of inosine and guanosine but d id not affect adenosine or deoxyribonucleosides. dNT-1 overproduction incre ased excretion of deoxycytidine, thymidine, and in particular deoxyuridine but also uridine and cytidine. We conclude that the hkm-NT is not involved in the regulation of deoxyribonucleotide pools but affects IMP and GTP pool s. dNT-1, instead, appears to be the catabolic arm of substrate cycles regu lating pyrimidine nucleotide pools.