Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic Cholecystokinin receptor

Ulrich G. Klueppelberg, Herbert Y. Gaisano, Stephen P. Powers, Laurence J Miller

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85000-95000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85000-95000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp 30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85000-95000 protein. This confirms that the Mr = 85000-95000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe. This should provide a generally applicable substitution for photoaffinity labeling of molecules in which a Trp resides in a domain relevant for the characterization of a bimolecular interaction.

Original languageEnglish (US)
Pages (from-to)3463-3468
Number of pages6
JournalBiochemistry
Volume28
Issue number8
StatePublished - 1989

Fingerprint

Cholecystokinin Receptors
Labeling
Hormones
Peptides
Proteins
Halogenation
Cholecystokinin
Amylases
Mass Spectrometry
Membrane Proteins
High Pressure Liquid Chromatography
Temperature
Membranes
Mass spectrometry
Substitution reactions
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic Cholecystokinin receptor. / Klueppelberg, Ulrich G.; Gaisano, Herbert Y.; Powers, Stephen P.; Miller, Laurence J.

In: Biochemistry, Vol. 28, No. 8, 1989, p. 3463-3468.

Research output: Contribution to journalArticle

Klueppelberg, Ulrich G. ; Gaisano, Herbert Y. ; Powers, Stephen P. ; Miller, Laurence J. / Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic Cholecystokinin receptor. In: Biochemistry. 1989 ; Vol. 28, No. 8. pp. 3463-3468.
@article{a08d5219024b4510a96c79a2fe4795a3,
title = "Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic Cholecystokinin receptor",
abstract = "We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85000-95000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85000-95000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp 30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85000-95000 protein. This confirms that the Mr = 85000-95000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe. This should provide a generally applicable substitution for photoaffinity labeling of molecules in which a Trp resides in a domain relevant for the characterization of a bimolecular interaction.",
author = "Klueppelberg, {Ulrich G.} and Gaisano, {Herbert Y.} and Powers, {Stephen P.} and Miller, {Laurence J}",
year = "1989",
language = "English (US)",
volume = "28",
pages = "3463--3468",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "8",

}

TY - JOUR

T1 - Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic Cholecystokinin receptor

AU - Klueppelberg, Ulrich G.

AU - Gaisano, Herbert Y.

AU - Powers, Stephen P.

AU - Miller, Laurence J

PY - 1989

Y1 - 1989

N2 - We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85000-95000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85000-95000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp 30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85000-95000 protein. This confirms that the Mr = 85000-95000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe. This should provide a generally applicable substitution for photoaffinity labeling of molecules in which a Trp resides in a domain relevant for the characterization of a bimolecular interaction.

AB - We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85000-95000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85000-95000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp 30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85000-95000 protein. This confirms that the Mr = 85000-95000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe. This should provide a generally applicable substitution for photoaffinity labeling of molecules in which a Trp resides in a domain relevant for the characterization of a bimolecular interaction.

UR - http://www.scopus.com/inward/record.url?scp=0024604460&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024604460&partnerID=8YFLogxK

M3 - Article

VL - 28

SP - 3463

EP - 3468

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 8

ER -