Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.
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...
| Автори: | , , , , , , , |
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| Формат: | Journal article |
| Мова: | English |
| Опубліковано: |
2012
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| _version_ | 1826298089900605440 |
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| author | Walseth, T Lin-Moshier, Y Jain, P Ruas, M Parrington, J Galione, A Marchant, J Slama, J |
| author_facet | Walseth, T Lin-Moshier, Y Jain, P Ruas, M Parrington, J Galione, A Marchant, J Slama, J |
| author_sort | Walseth, T |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T04:41:31Z |
| format | Journal article |
| id | oxford-uuid:d1d15186-b948-4c6e-9e63-4a34fb29e3bb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:41:31Z |
| publishDate | 2012 |
| record_format | dspace |
| spelling | oxford-uuid:d1d15186-b948-4c6e-9e63-4a34fb29e3bb2022-03-27T07:59:29ZPhotoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:d1d15186-b948-4c6e-9e63-4a34fb29e3bbEnglishSymplectic Elements at Oxford2012Walseth, TLin-Moshier, YJain, PRuas, MParrington, JGalione, AMarchant, JSlama, JNicotinic 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. |
| spellingShingle | Walseth, T Lin-Moshier, Y Jain, P Ruas, M Parrington, J Galione, A Marchant, J Slama, J Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. |
| title | Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. |
| title_full | Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. |
| title_fullStr | Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. |
| title_full_unstemmed | Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. |
| title_short | Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. |
| title_sort | photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate naadp binding proteins in sea urchin egg |
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