Inositol 1,4,5-trisphosphate receptors: Labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands

Robert J. Mourey, Virginia A. Estevez, James F. Marecek, Roxanne K Barrow, Glenn D. Prestwich, Solomon H Snyder

Research output: Contribution to journalArticle

Abstract

We have photolabeled the inositol 1,4,5-trisphosphate (IP3) receptor and probed the IP3 ligand binding site using two novel photoaffinity ligands, [125I](azidosalicyl)aminopropyl-IP3 ([125I]ASA-IP3) and [3H] (benzoyldihydrocinnamyl)aminopropyl-IP3 ([3H] BZDC-IP3). Both ligands have high affinity for the IP3 receptor and, when photoactivated, label the IP3 receptor protein with appropriate inositol phosphate selectivity. The high specific activity of [125I]ASA-IP3 allowed identification of a single photolabeling site within the IP3R by two-dimensional peptide analysis. Substantially higher levels of incorporation into the receptor are achieved with [3H]BZDC-IP3 (50-60% efficiency) than with [125I]ASA-IP3 (3%), facilitating the use of [3H] BZDC-IP3 as a better ligand for the high-efficiency labeling and purification of IP3R-labeled peptides. Peptides were generated from photolabeled IP3 receptor by trypsin digestion and purified by high-pressure liquid chromatography (HPLC). A single purified [3H] BZDC-IP3-labeled peptide, corresponding to IP3R amino acids 476-501, was sequenced and shown to match specific sequences in the N-terminal 20% of the IP3 receptor, an area suggested on the basis of mutagenesis studies to contain the IP3 recognition site.

Original languageEnglish (US)
Pages (from-to)1719-1726
Number of pages8
JournalBiochemistry®
Volume32
Issue number7
StatePublished - 1993

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Inositol 1,4,5-Trisphosphate Receptors
Inositol 1,4,5-Trisphosphate
Labeling
Binding Sites
Ligands
Peptides
High pressure liquid chromatography
Mutagenesis
Inositol Phosphates
Trypsin
Purification
Labels
Digestion
High Pressure Liquid Chromatography
Amino Acids
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Inositol 1,4,5-trisphosphate receptors : Labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands. / Mourey, Robert J.; Estevez, Virginia A.; Marecek, James F.; Barrow, Roxanne K; Prestwich, Glenn D.; Snyder, Solomon H.

In: Biochemistry®, Vol. 32, No. 7, 1993, p. 1719-1726.

Research output: Contribution to journalArticle

Mourey, Robert J. ; Estevez, Virginia A. ; Marecek, James F. ; Barrow, Roxanne K ; Prestwich, Glenn D. ; Snyder, Solomon H. / Inositol 1,4,5-trisphosphate receptors : Labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands. In: Biochemistry®. 1993 ; Vol. 32, No. 7. pp. 1719-1726.
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