FIGURE 2.

Cytosolic 5′-nucleotidases CN-IA, CN-II, and CN-III generate nicotinic acid riboside in human cells. A and B, HEK293 (A) and HeLa S3 (B) cells were transiently transfected with vectors encoding FLAG-tagged 5′-NTs CN-IA, CN-II, CN-III, and Sdt1. The expression of FLAG-tagged 5′-NT was confirmed by immunoblotting using antibody to FLAG peptide. SOD2 served as a loading control (A). Subcellular distribution of FLAG-tagged proteins is shown in B. Cell nuclei were stained with DAPI. Scale bars, 10 μm. C, schematic representation of the experimental approach. 5′-NTs were expressed or co-expressed with NamPRT or NAPRT in human cells in the presence of Nam and NA. NR and NAR release from transfected cells to culture medium was analyzed by NMR spectroscopy. D, HEK293 (left panel) and HepG2 (right panel) cells were co-transfected with plasmid encoding NAPRT and vectors encoding the indicated 5′-NT or empty vector. 3 (for HEK293 cells) and 7 days (for HepG2 cells) after transfection, culture media from control and transfected cells were analyzed by NMR spectroscopy. ¹H NMR spectra show NAR release from cells transiently expressing NAPRT. Co-expression with 5′-NT significantly increased the extracellular level of NAR.