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The synthesis and characterization of a clickable-photoactive NAADP analog active in human cells. Cell Calcium 2019 11;83:102060

Date

08/24/2019

Pubmed ID

31442840

Pubmed Central ID

PMC6774857

DOI

10.1016/j.ceca.2019.102060

Scopus ID

2-s2.0-85070876728   1 Citation

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing second messenger which triggers Ca2+ release in both sea urchin egg homogenates and in mammalian cells. The NAADP binding protein has not been identified and the regulation of NAADP mediated Ca2+ release remains controversial. To address this issue, we have synthesized an NAADP analog in which 3-azido-5-azidomethylbenzoic acid is attached to the amino group of 5-(3-aminopropyl)-NAADP to produce an NAADP analog which is both a photoaffinity label and clickable. This 'all-in-one-clickable' NAADP (AIOC-NAADP) elicited Ca2+ release when microinjected into cultured human SKBR3 cells at low concentrations. In contrast, it displayed little activity in sea urchin egg homogenates where very high concentrations were required to elicit Ca2+ release. In mammalian cell homogenates, incubation with low concentrations of [32P]AIOC-NAADP followed by irradiation with UV light resulted in labeling 23 kDa protein(s). Competition between [32P]AIOC-NAADP and increasing concentrations of NAADP demonstrated that the labeling was selective. We show that this label recognizes and selectively photodervatizes the 23 kDa NAADP binding protein(s) in cultured human cells identified in previous studies using [32P]5-N3-NAADP.

Author List

Asfaha TY, Gunaratne GS, Johns ME, Marchant JS, Walseth TF, Slama JT

Author

Jonathan S. Marchant PhD Chair, Professor in the Cell Biology, Neurobiology and Anatomy department at Medical College of Wisconsin




MESH terms used to index this publication - Major topics in bold

Animals
Benzoic Acid
Binding Sites
Calcium
Calcium Signaling
Cell Line, Tumor
Click Chemistry
Humans
NADP
Photoaffinity Labels
Protein Binding
Sea Urchins
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a