FIGURE 5.

1,N⁶-etheno-NAADP is metabolized by NAADP-degrading activity at neutral pH. Membrane protein from HeLa cells (0.05 μg/ml) was incubated with 100 nm 1,N⁶-etheno-NAADP (e-NAADP) at 37 °C and pH 7.2 and 9. Samples were deproteinized using 10-kDa cutoff filters (Vivaspin). Product formation was analyzed by HPLC. Shown are representative chromatograms from samples incubated with 100 nm 1,N⁶-etheno-NAADP for 0, 5, 15, 40, and 60 min. Peak areas for 1,N⁶-etheno-2-phospho-ADPR (e-ADPRP) and 1,N⁶-etheno-ADPR (e-ADPR) are considerably larger due to the higher fluorescence intensity upon cleavage of the intramolecular quencher, the nicotinic acid moiety. FU, fluorescence units.