Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg |
| |
Authors: | Walseth Timothy F Lin-Moshier Yaping Jain Pooja Ruas Margarida Parrington John Galione Antony Marchant Jonathan S Slama James T |
| |
Institution: | Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA. walse001@umn.edu |
| |
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. |
| |
Keywords: | Calcium Intracellular Release Calcium Signaling NAADP Nucleoside Nucleotide Analogs Photoaffinity Labeling Two-pore Channels |
本文献已被 PubMed 等数据库收录! |
|