Human high-Km 5'-nucleotidase effects of overexpression of the cloned cDNA in cultured human cells.
5'-Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism. Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high-Km nucleotidases 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 human high-Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells. After transfection, E. coli overproduced large 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 approximately 2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtained with a vector where the 3'-end of the nucleotidase coding sequence is linked to the 5'-end of the green fluorescent protein coding sequence. Several green clones overproduced both mRNA and fusion protein. Two clones with 10-fold higher enzyme activity were analyzed further. The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high-Km enzyme. The growth rate of the two clones did not differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation.
Rampazzo
C
C
Department of Biology, University of Padova, Italy.
Gazziola
C
C
Ferraro
P
P
Gallinaro
L
L
Johansson
M
M
Reichard
P
P
Bianchi
V
V
D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D016049:Didanosine / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012263:Ribonucleosides / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D012263:Ribonucleosides; D049452:Green Fluorescent Proteins; D015720:5'-Nucleotidase; D016049:Didanosine