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) |
|---|---|
| Pages (from-to) | 1719-1726 |
| Number of pages | 8 |
| Journal | Biochemistry® |
| Volume | 32 |
| Issue number | 7 |
| State | Published - 1993 |
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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 journal › Article
}
TY - JOUR
T1 - Inositol 1,4,5-trisphosphate receptors
T2 - Labeling the inositol 1,4,5-trisphosphate binding site with photoaffinity ligands
AU - Mourey, Robert J.
AU - Estevez, Virginia A.
AU - Marecek, James F.
AU - Barrow, Roxanne K
AU - Prestwich, Glenn D.
AU - Snyder, Solomon H
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0027450067&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0027450067&partnerID=8YFLogxK
M3 - Article
C2 - 8382518
AN - SCOPUS:0027450067
VL - 32
SP - 1719
EP - 1726
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 7
ER -