FIGURE 4.

[³²P-5N₃]NAADP photolabeling and binding display saturable kinetics. A, photolabeling of sea urchin egg homogenate performed at increasing concentrations of [³²P-5N₃]NAADP in the absence (−) or presence of (+) 1 μm NAADP. A phosphor image of acrylamide gel is shown. B, saturation kinetics of [³²P-5N₃]NAADP photolabeling. Total labeling (closed circles), nonspecific labeling (in the presence of 1 μm NAADP, closed squares), and specific labeling (open circles) were determined from densitometric analyses of phosphor images of experiments similar to the one shown in panel A (n = 3). DLU, digital light units. C, saturation kinetics of [³²P-5N₃]NAADP binding. The conditions for binding were the same as used for photoaffinity labeling. Total binding (closed circles), nonspecific binding (in the presence of 1 μm NAADP, closed squares), and specific binding (open circles) were determined by a conventional filtration binding assay.