Figure 3. Synthesis of NAADP in HSR membranes.

(A) HPLC chromatograms of a typical synthesis of NAADP in the absence (broken line) and presence of HSR membranes (solid line; 0.5 mg/ml). The HSR was incubated with 0.5 mM NADP and 10 mM nicotinic acid for 45 min at 37 °C. The pH of the mixture was adjusted to 4.3 with ethanoic acid. The peaks for nicotinic acid, NADP and NAADP were indicated by arrows. The insert depicts a magnification of the elution profile of NAADP in the absence (broken line) and presence of HSR (solid line). a.u., arbitrary units. (B) Substrate-dependence of the base-exchange reaction. The reaction was carried out under similar conditions to those in (A) with increasing concentrations of NADP. (C) Formation of NAADP was measured in the presence of increasing concentrations of nicotinic acid and 0.5 mM NADP. The pH was kept constant at 4.3. Results are means±S.E.M. of a representative experiment carried out in duplicate.