Figure 4

Demonstration of the specificity of cADPR phosphohydrolase in complex nucleotidic mixtures. Mixtures of CDP-choline, cADPR, 2′,3′-cAMP, ADP-ribose, Ap2A, ADP and ATP, each at 0.1 mM concentration were incubated for 60 min at 37 °C in the absence or in the presence of 1 µg ml⁻¹ of the specific cADPR phosphohydrolase under otherwise standard conditions. The chromatograms were run with buffers at pH 8.5 as described in Methods, and monitored at 260 nm and 310 nm. The retention times of all the substrates used and of most potential products expected (5′-CMP from CDP-choline; 5′-AMP from ADP-ribose, Ap2A and ADP; 3′-AMP from 2′,3′-cAMP) were determined with samples of the standard compounds chromatographed individually. The pRibAMP produced by phosphohydrolysis of cADPR was characterised in previous work⁵¹.