title: Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.
abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.
authors: Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. walse001@umn.edu
authors: Timothy F
authors: TF
authors: Walseth
authors: Yaping
authors: Y
authors: Lin-Moshier
authors: Pooja
authors: P
authors: Jain
authors: Margarida
authors: M
authors: Ruas
authors: John
authors: J
authors: Parrington
authors: Antony
authors: A
authors: Galione
authors: Jonathan S
authors: JS
authors: Marchant
authors: James T
authors: JT
authors: Slama
mesh_terms: D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D010063:Ovum / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry; D011485:Protein Binding; D047270:Strongylocentrotus purpuratus / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D019920:Photoaffinity Labels; D009249:NADP; C024376:NAADP