Human high-K-m 5 '-nucleotidase - Effects of overexpression of the cloned cDNA in cultured human cells

5'-Nucleotidases participate, together with nucleoside kinases, in substrat e cycles involved in the regulation of deoxyribonucleotide metabolism. Thre e major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high-K-m nucl eotidases and 5'(3')-nucleotidases. Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y. & Inoue, S. (1994) Biochem . Biophys. Res. Commun. 205, 917-922) we cloned the complete cDNA of the hu man high-K-m nucleotidase into vectors suitable for transfection of Escheri chia coli or mammalian cells. After transfection, E. coli overproduced larg e amounts of the enzyme. Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein. Part of the enzyme, corresponding to approximate to 2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtai ned with a vector where the 3'-end of the nucleotidase coding sequence is l inked to the 5'-end of the green fluorescent protein coding sequence. Sever al green clones overproduced both mRNA and fusion protein. Two clones with 10-fold higher enzyme activity were analyzed further. The nucleotidase acti vity of cell extracts showed the same substrate specificity and allosteric regulation as the high-K-m enzyme. The growth rate of the two clones did no t differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate d ideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate poo ls were decreased slightly, suggesting participation of the enzyme in their regulation.