CYTOSOLIC 5'-NUCLEOTIDASE NUCLEOSIDE PHOSPHOTRANSFERASE - A SINGLE ASSAY FOR A BIFUNCTIONAL ENZYME

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase has been purif ied from calf thymus. Since the same protein is able to catalyze both the hydrolysis and the interconversion of several nucleoside monophosp hates, it is necessary to study the effect of different metabolites an d assay conditions on both activities in order to elucidate their phys iological roles. We describe herein a method which allowed us to follo w both activities contemporaneously in the same assay mixture. The met hod takes advantage of the observation that deoxyGMP is both a good su bstrate for hydrolysis and a good phosphate donor for the phosphotrans ferase reaction, but its dephosphorylated product, deoxyguanosine, is not a phosphate acceptor. As a consequence, it is possible to follow b oth the deoxyguanosine production and the transfer of phosphate from d eoxyGMP to the best phosphate acceptor, inosine, during the reaction, applying a method for the chromatographic separation on HPLC of both s ubstrates (inosine and deoxyGMP) and both products (IMP and deoxyguano sine). The method was applied to the determination of the K(M) for ino sine.