CYTOSOLIC 5'-NUCLEOTIDASE NUCLEOSIDE PHOSPHOTRANSFERASE - A NUCLEOSIDE ANALOG ACTIVATING ENZYME

Nucleoside phosphotransferase acting on inosine and deoxyinosine has b een partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase act ing on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisph osphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are sub strates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GM P, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleo tidase/phosphotransferase substrate, 8-azaguanosine, was found to be v ery toxic for cultured fibroblasts (LD50 = 0.32 muM). Mutants resistan t to either 8-azaguanosine and the correspondent base 8-azaguanine wer e isolated and characterized. Our results indicated that the 8-azaguan osine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine res istant cells were lacking only the latter enzyme. Despite this observa tion, both mutants displayed 8-azaguanosine resistance, thus indicatin g that cytosolic 5'-nucleotidase is not essential for the activation o f this nucleoside analog.