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 language | English (US) |
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Pages (from-to) | 1719-1726 |
Number of pages | 8 |
Journal | Biochemistry |
Volume | 32 |
Issue number | 7 |
DOIs | |
State | Published - 1993 |
ASJC Scopus subject areas
- Biochemistry